Nanoparticle immunogenic compositions and vaccination methods

Abstract

Compositions and methods for inducing protective mucosal immunity against an antigen in a subject include administering to a mucosal tissue an effective amount of a vaccine composition comprising an antigen or a polynucleotide encoding the antigen bound or encapsulated in a carrier such as a poly(amine-co-ester) polymer in the form of particles (e.g., solid nanoparticles formed from PACE) or a PACE copolymer and / or blend. Typically, the subject has been previously exposed to the antigen, for example, by administering a priming antigen via a systemic or mucosal route of administration to the same subject. In some embodiments, the polynucleotide-based vaccine is a messenger RNA encoding a viral antigen, for example, a coronavirus spike protein sequence or a portion thereof. In a preferred embodiment, the vaccine composition is administered intranasally.

Description

[Technical Field]

[0001] This application claims the benefit of and priority to U.S. Patent Application No. 63 / 287,410, filed December 8, 2021, U.S. Patent Application No. 63 / 290,042, filed December 15, 2021, U.S. Patent Application No. 63 / 292,200, filed December 21, 2021, U.S. Patent Application No. 63 / 301,942, filed January 21, 2022, U.S. Patent Application No. 63 / 302,413, filed January 24, 2022, and U.S. Patent Application No. 63 / 418,744, filed October 24, 2022, which are hereby expressly incorporated by reference in their entireties.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under award K08AI163493 from the National Institutes of Health and the National Institute of Allergy and Infectious Diseases, and award 5UG3HL147352-03 from the National Institutes of Health. The government has certain rights in this invention.

[0003] Reference to sequence listing The Sequence Listing, entitled "YU_8277_PCT_ST26.xml," created on December 8, 2022, and submitted on December 8, 2022 as a text file having a size of 19,830 bytes, is hereby incorporated by reference in accordance with 37 CFR 1.834(c)(1).

[0004] FIELD OF THE INVENTION The field of the invention relates generally to compositions and methods for improved vaccines, particularly nucleic acid-based agents, for administration to induce an immune response. [Background technology]

[0005] Respiratory pathogens, such as pandemic influenza viruses and coronaviruses, represent a significant and recurring public health problem. Influenza-related illnesses account for millions of hospitalizations and over 35,000 deaths annually in the United States. Recently, coronaviruses have caused severe epidemic and pandemic respiratory illnesses in human populations, leading the World Health Organization (WHO) to declare severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a global pandemic in mid-March 2020.

[0006] The primary defense against these pathogens is mass vaccination of the population to create "herd immunity" based on protective antibody responses in the host. Currently, most approved vaccines and vaccine candidates in clinical development for respiratory viruses are administered intramuscularly. While such parenteral vaccines provide good systemic immunity by inducing virus-specific IgG and T cell responses in the circulation, they generate very few memory T cells and mucosal IgA in the respiratory tract, where infection is first established and later spreads.

[0007] Respiratory viruses enter the body through respiratory mucosa and then spread via respiratory droplets and aerosols. Therefore, vaccine strategies aimed at eliciting protective immune effector mechanisms at these sites are greatly needed to provide a first line of defense against respiratory virus invasion and dissemination and may serve as a transmission-blocking vaccine strategy. Strong mucosal cellular and humoral immune responses have the potential to induce sterilizing immunity by interfering with binding to and uptake across epithelial surfaces. However, significant hurdles remain to mucosal vaccine development. Summary of the Invention [Problem to be solved by the invention]

[0008] It is therefore an object of the present invention to provide improved vaccines and immunization strategies that can provide effective protection against respiratory pathogens, particularly coronaviruses such as SARS and COVID-19, and influenza viruses.

[0009] It is an object of the present invention to provide improved vaccine reagents and methods of use that can provide protective mucosal immunity against respiratory pathogens.

[0010] A further object of the present invention is to convert the systemic immunity induced by parenteral immunization into protective immunity against respiratory infections.

[0011] It is also an object of the present invention to provide reagents and methods for effective immunization against pandemic SARS and influenza viruses. [Means for solving the problem]

[0012] (Summary of the Invention) Immunogenic compositions and methods are provided for inducing a mucosal immune response and / or sustained mucosal immunity against respiratory viruses and / or boosting existing immunity to those viruses. In one embodiment, the composition comprises a viral antigen in a form suitable for mucosal delivery. The viral antigen can be a protein or peptide provided in / on a carrier or a polynucleotide encoding the viral antigen.

[0013] In preferred embodiments, the compositions comprise polynucleotide-based antigens conjugated or encapsulated within poly(amine-co-ester) polymers in the form of micellular particles or solid polymer nanoparticles, which are effective in inducing antigen-specific mucosal immune responses and / or long-lasting, protective mucosal immunity in a subject. In other embodiments, the nucleic acid is provided in a lysosome, lipid nanoparticle, lipid emulsion, or organogel formulation.

[0014] In some embodiments, the formulation is administered to boost or enhance existing immunity in a subject against infection by a virus. In some embodiments, the virus is one that causes a respiratory infection. In some embodiments, the virus is a coronavirus, influenza virus, herpes simplex virus, or any combination thereof.

[0015] A method for enhancing mucosal immunity to an antigen in a subject comprises administering to a mucosal tissue in the subject an effective amount of a vaccine composition comprising an antigen or a poly(amine-co-ester) polymer and a polynucleotide encoding the antigen. The subject has preferably been previously exposed to the antigen by, for example, administering a priming antigen that shares one or more antigenic sites with the antigen (or the antigen encoded by the polynucleotide) to the same subject via a systemic or mucosal administration route. Thus, in some embodiments, the subject's immune system has been "primed" by prior exposure to the antigen. In some embodiments, the prior exposure is a systemic exposure, e.g., prior administration of a vaccine to the subject via a systemic route, e.g., via intramuscular or intradermal administration. In other embodiments, the prior exposure is a mucosal exposure, e.g., prior administration of a mucosal vaccine to the subject, or prior natural infection at a mucosal site.

[0016] In some embodiments, prior exposure via systemic or mucosal routes of administration does not induce protective mucosal immunity. In some embodiments, prior exposure via systemic or mucosal routes of administration does not induce detectable numbers of CD8+ cells against the antigen in mucosal tissues. + Tissue resident memory (T RM ) cells, CD4 + Tissue resident memory (T RM ) cells and / or memory B cells. In some embodiments, prior exposure via systemic or mucosal routes of administration does not induce detectable mucosal IgA to the antigen. In preferred embodiments, the priming antigen comprises messenger RNA (mRNA) encoding the antigen bound or encapsulated within a lipid nanoparticle.

[0017] Exemplary mucosal tissues for administering the disclosed compositions include the lungs, nasal cavity, oral cavity, digestive system, vaginal, and rectal mucosa. In a preferred embodiment, the mucosal tissue is the lung and / or nasal mucosa. Suitable antigens are derived from or elicit a protective immune response against one or more respiratory viruses, such as orthomyxoviruses, paramyxoviruses, coronaviruses, adenoviruses, herpesviruses, and human bocaviruses.

[0018] The antigen is derived from or generates a protective immune response against one or more of influenza virus, parainfluenza virus, measles virus (measles morbillivirus), mumps virus, and respiratory syncytial virus (RSV), human metapneumovirus, severe acute respiratory syndrome (SARS) virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella-zoster virus (VZV). In preferred embodiments, the antigen is derived from influenza virus, SARS-Cov-2 virus, or RSV. Typically, the polynucleotide encoding the antigen is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule. In a preferred embodiment, the polynucleotide encoding the antigen is messenger RNA (mRNA) comprising an open reading frame encoding a coronavirus spike protein sequence or a portion thereof. In some embodiments, the coronavirus is a variant of SARS-CoV-2, such as SARS-CoV-2 B.1.1.7 (alpha variant), SARS-CoV-2 B.1.351 (beta variant), SARS-CoV-2 P.1 (gamma variant), SARS-CoV-2 B.1.617, SARS-CoV-2 B.1.617.1 (kappa variant), SARS-CoV-2 B.1.621 (mu variant), SARS-CoV-2 B.1.617.2 (delta variant), SARS-CoV-2 B.1.617.3, and SARS-CoV-2 B.1.1.529 (omicron variant).

[0019] An effective amount of the immunogenic composition induces a mucosal immune response specific to the antigen and / or long-lasting, protective mucosal immunity in a subject. In some embodiments, the immunogenic composition induces CD8 responses to the antigen in mucosal tissues. + Tissue resident memory (T RM ) cells, CD4 + Tissue resident memory (T RM In a preferred embodiment, the immunogenic composition is administered in an amount effective to induce increased numbers of CD8) cells and / or memory B cells compared to an immunogenic composition delivered in the absence of the poly(amine-co-ester) polymer via the same route of administration. + Tissue resident memory (T RM ) cells, CD4 + Tissue resident memory (T RM In some embodiments, the immunogenic composition is administered in an amount effective to induce mucosal immunoglobulin A (IgA) and / or immunoglobulin G (IgG) against the antigen. In preferred embodiments, the immunogenic composition induces increased amounts of mucosal IgA and / or mucosal IgG against the antigen compared to an immunogenic composition delivered in the absence of the poly(amine-co-ester) polymer via the same route of administration. Exemplary mucosal IgA is secretory IgA, e.g., detected in a mucosal lavage fluid selected from bronchoalveolar lavage fluid, intestinal lavage fluid, bowel lavage fluid, and vaginal lavage fluid.

[0020] A method for enhancing mucosal immunity to repeated presentation of an antigen in a subject comprises (i) administering an effective amount of a priming antigen via a systemic or mucosal route of administration and (ii) subsequently intranasally administering to the subject an effective amount of a boosting composition containing (i) the antigen or (ii) poly(amine-co-ester) or poly(amine-co-amide), (PACE), PACE-PEG block copolymers and / or PACE polymer blends, which may contain additional monomers that render the nanoparticles more acid-sensitive, and the antigen or a polynucleotide encoding the same. In one embodiment, the method comprises (i) administering via intramuscular (IM) injection an effective amount of a priming antigen containing lipid nanoparticles (LNPs) and a polynucleotide encoding the antigen, and (ii) subsequently intranasally administering to the subject an effective amount of the antigen or poly(amine-co-ester) or poly(amine-co-amide), (PACE), PACE-PEG block copolymers and / or PACE polymer blends, which may contain additional monomers that render the nanoparticles more acid-sensitive, and the antigen or a polynucleotide encoding the antigen. In another embodiment, the method comprises the steps of (i) intranasally (IN) administering to the subject an effective amount of a priming antigen comprising a poly(amine-co-ester) or poly(amine-co-amide), (PACE), PACE-PEG block copolymer and / or PACE polymer blend, which may include additional monomers that render the nanoparticles more acid-sensitive, and a polynucleotide encoding the antigen, and (ii) thereafter intranasally (IN) administering to the subject an effective amount of a boosting composition comprising a poly(amine-co-ester) or poly(amine-co-amide), (PACE), PACE-PEG block copolymer and / or PACE polymer blend, which may include additional monomers that render the nanoparticles more acid-sensitive, and a polynucleotide encoding the same antigen.

[0021] A method for providing protective immunity against one or more respiratory pathogens in a subject comprises (i) administering an effective amount of a priming immunogenic composition via a systemic route of administration, and then ii) administering to the subject via a systemic or, preferably, mucosal route, an effective amount of a boosting immunogenic composition containing an antigen or poly(amine-co-ester) or poly(amine-co-amide), (PACE), PACE-PEG block copolymers and / or PACE polymer blends, which may include additional monomers that render the nanoparticles more acid-sensitive, and a polynucleotide encoding the same antigen. Exemplary systemic routes of administration include intramuscular and intradermal administration. In one embodiment, the method comprises (i) administering, via intramuscular (IM) injection, an effective amount of a priming antigen containing lipid nanoparticles (LNPs) and a polynucleotide encoding the antigen, and (ii) subsequently administering, via intramuscular (IM) injection, an effective amount of a boosting composition containing poly(amine-co-ester) or poly(amine-co-amide) (PACE), PACE-PEG block copolymers and / or PACE polymer blends, which may include additional monomers that render the nanoparticles more acid-sensitive, and a polynucleotide encoding the same antigen. In some embodiments, the priming antigen comprises messenger RNA (mRNA) encoding the antigen bound or encapsulated within the lipid nanoparticles. Exemplary mucosal administration routes include the nasal or pulmonary systems, or administered to mucosal surfaces (vaginal, rectal, buccal, sublingual). In some embodiments, compositions for mucosal or intranasal administration are via aqueous suspension, respiratory droplets, or aerosol. In some embodiments, the PACE polymer and the polynucleotide encoding the antigen are in the form of particles, and optionally the PACE polymer is a polyethylene glycol (PEG)-conjugated PACE polymer.

[0022] In some embodiments, the PACE polymer has Formula I:

[0023] [ka] [In the formula, n is an integer of 1 to 30; m, o, and p are independently integers of 1 to 20; x, y, and q are independently integers of 1 to 1,000; R is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R1 and R2 are absent or are chemical entities containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. The PACE polymer with a high lactone content is used to form the solid nanoparticles.

[0024] Pharmaceutical compositions and kits for use in methods for enhancing mucosal immunity to an antigen are also provided. [Brief explanation of the drawings]

[0025] [Figure 1A]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1B]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1C]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1D]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1E]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1F]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1G]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1H]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1I]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1J]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1K]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1L]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1M]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 1N]Figures 1A-1N show that IN boosting with stabilized SARS-CoV-2 spike induces mucosal humoral memory. Figure 1A shows the experimental schema. Mice were immunized intramuscularly with 1 μg of mRNA-LNP (Pfizer / BioNTech BNT162b2) encoding the full-length SARS-CoV-2 (SCV2) spike protein, followed by IN immunization with 1 μg of prefusion-stabilized (Hexapro), trimeric, recombinant SCV2 spike protein 14 days after mRNA-LNP immunization. 14 days after the IN boost, serum, BALF, and nasal washes were collected to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis. Figures 1B–1G show measurements of SCV2 spike S1 subunit-specific (Figure 1B) nasal wash IgA, (Figure 1C) nasal wash IgG, (Figure 1D) BALF IgA, (Figure 1E) BALF IgG, (Figure 1F) serum IgA, and (Figure 1G) serum IgG in naive mice, mice immunized with mRNA-LNP IM (IM prime), mice immunized with spike protein IN (IN spike), or mice IM primed and IN boosted with spike (P&S). Figures 1H–1K show measurements of neutralizing titers against SCV2 spike-pseudotyped vesicular stomatitis virus (VSV) in BALF (Figure 1H) and serum (Figure 1K). Figures 1J–1N show that using CD45 IV labeling, various extravascular (IV-labeled antibody-negative) B cell subsets were measured in lung tissue obtained from IM-primed or P&S mice, including RBD tetramer-binding B cells, IgA+ BRM cells, IgG+ BRM cells, IgA+ ASCs, and IgG+ ASCs. Mean ± SEM. Statistical significance was calculated by one-way analysis of variance (ANOVA) [Figures 1B–1G] or Student's t-test [Figures 1H–1N]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2A]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2B]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2C]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2D]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2E]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2F]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2G]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2H]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2I]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2J]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2K]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2L]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2M]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2N]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 2O]Figures 2A-2O show that IN boosting with a stabilized SARS-CoV-2 spike induces mucosal T cell memory. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and boosted intranasally with 1 μg of SCV2 spike 14 days later. Lung tissue, BALF, and nasal turbinates were collected for extravascular T cell analysis. Lung tissue was collected 14 days post-boost, while BALF and nasal turbinates were obtained 7 days post-boost. Figures 2A-2I show extravascular CD8 T cell responses. Quantification of SCV2 spike-specific tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue [Figures 2A-2C], BALF [Figures 2D-2F], or nasal turbinates [Figures 2G-2I] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Figures 2J-2O show extravascular CD4 T cell responses. Quantification of activated polyclonal CD4 T cells, CD69+CD103- CD4 T cells, or CD69+CD103+ CD4 T cells in lung tissue [Figures 2J-2L] or BALF [Figures 2M-2O] from untreated, IM-primed, IN-spiked, or P&S mice is shown. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 2B-2O) followed by Tukey's correction; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two or three independent experiments. [Figure 3A]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3B]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3C]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3D]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3E]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3F]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3G]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3H]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3I]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3J]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3K]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 3L]Figures 3A-3L show that an IN SARS-CoV-2 spike-boost protects against COVID-19-like disease. Figure 3A shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 1 μg of spike 14 days after the IM priming. Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). The first cohort was used to assess weight loss and survival up to 14 days post-infection. The second cohort was used to collect lung and nasal turbinate tissues 2 days post-infection for virus titer determination. The third cohort was used to collect lung tissues 5 days post-infection for histological evaluation. Figures 3B-3D show weight loss and survival in untreated, IM-primed, or P&S mice from 1 to 14 days post-infection. Figures 3E and 3F show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days after infection by plaque assay. Figure 3G shows the pathology scores of lung sections 5 days after infection by H&E staining. Figure 3H shows representative H&E staining from uninfected, IM primed, or P&S mice. Scale bar, 250 mm. Sections are representative of multiple sections from at least five mice per group. Figure 3I shows the experimental schema. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and boosted intranasally with 10 μg of PACE-encapsulated mRNA 14 days after IM priming (IN PACE-Spike). Six weeks after the boost, mice were challenged with 6 × 10 PFU SCV2 (2019n-CoV / USA_WA1 / 2020). Weight loss and survival were assessed up to 14 days post-infection. Figures 3J–3L show weight loss and survival in untreated, IM primed, or primed and PACE-spiked K18-hACE2 mice 1–14 days postinfection. Mean ± SEM. Statistical significance was calculated by log-rank Mantel-Cox test (Figures 3D and 3L), one-way ANOVA (Figures 3E and 3F), or Student's t-test (Figure 3G); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.Each data point represents and is pooled from two independent experiments. [Figure 4A]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4B]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4C]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4D]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4E]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4F]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4G]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4H]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4I]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4J]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4K]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4L]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4M]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4N]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4O]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4P]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4Q]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4R]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4S]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4T]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 4U]Figures 4A-4U show that IN spike-boosting induces enhanced mucosal immunity and has a similar systemic humoral response to IM mRNA-LNP boosting. Figure 4A, experimental schema. K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and subsequently boosted 14 days later with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike-in. 45 days after priming, lung tissue was collected for T cell analysis by flow cytometry, and BALF and serum were collected for antibody measurements. K18-hACE2 mice were primed intramuscularly with 0.05 μg of mRNA-LNP and subsequently boosted 14 days later with 0.05 μg of mRNA-LNP intramuscularly or 1 μg of SCV2 spike-in. 118 days after priming, they were challenged with 6 × 104 PFU of SCV2. Figures 4B-4D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from untreated, mRNA-LNP prime-boost, or P&S mice. Figures 4E and 4F show quantification of total tetramer+ CD4+ T cells or CD69+CD103- tetramer+ CD4+ T cells in lung tissue. Figures 4G-4K show lung lymphocytes isolated by Percoll gradient and restimulated with a spike peptide megapool derived from SCV2. Intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 4L-4O show measurements of SCV2 spike S1 subunit-specific (Figure 4L) BALF IgA, (Figure 4M) BALF IgG, (Figure 4N) serum IgA, and (Figure 4O) serum IgG in naive, mRNA-LNP prime-boost, or P&S mice. Figure 4P shows measurements of neutralizing titers against SCV2 spike-pseudotyped VSV. Figures 4Q-4S show weight loss, survival, and disease-free survival (<5% maximum weight loss) in mRNA-LNP prime-boost or P&S mice 1-14 days postinfection. Figures 4T and 4U show measurements of infectious virus titers in lung and nasal turbinate tissues 2 days postinfection by plaque assay.To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4 and 6. Mean ± SEM. Statistical significance was calculated by one-way ANOVA [Figures 4B-4O] followed by Tukey's correction or Student's t-test [Figures 4P, 4T, and 4U], and log-rank Mantel-Cox test [Figures 4R and 4S]; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 5A]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5B]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5C]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5D]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5E]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5F]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5G]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5H]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5I]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5J]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5K]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5L]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5M]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5N]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 5O]Figures 5A-5O show that IN spike-boosting leads to reduced virus spread in a hamster model. Figure 5A shows the experimental schema. Syrian hamsters were primed intramuscularly with 0.5 μg of mRNA-LNP and subsequently boosted intramuscularly with 0.5 μg of mRNA-LNP or intranasally with 5 μg of SCV2 spike 21 days later (Figures 5B and 5C). Serum IgG and IgA were assessed by ELISA 60 days after priming. At 93 days after priming, naive, mRNA-LNP prime-boost, and P&S hamsters were infected with 6 × 10 PFU SCV2. Figure 5D shows weight loss as a percentage of starting weight. Figure 5D shows histopathological analysis of lung samples 7 days after infection. Figures 5F-5H show virus titers from oropharyngeal swabs, shown as mean (symbols) and standard deviation (shaded area), with P values ​​relative to controls at the same time points. Figure 5I shows AUC analysis of virus titers over 6 days post-infection. (Figure 5J) Transmission experimental schema. Syrian hamsters vaccinated as described above were co-housed for 4 hours with naive donor hamsters infected with 6 x 103 PFU SCV2 24 hours prior. (Figure 5K) Histopathological analysis of lung samples 7 days post-challenge. (Figures 5L-5N) Viral titers from oropharyngeal swabs are shown as mean (symbols) and standard deviation (shaded), with P values ​​relative to controls at the same time points. (Figure 5O) AUC analysis of virus titers over 6 days post-infection. Mean ± SEM. Statistical significance was calculated by one-way ANOVA (Figures 5B, 5C, 5E, 5I, 5K, and 5O) followed by Tukey's correction, mixed-effects analysis (Figures 5F-5H) followed by Tukey's multiple comparison test, or two-way ANOVA (Figures 5L-5N) followed by Dunnett's multiple comparison test; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents one independent experiment. [Figure 6A]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6B]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6C]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6D]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6E]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6F]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6G]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6H]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6I]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6J]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6K]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6L]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6M]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6N]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6O]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6P]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6Q]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6R]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6S]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6T]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6U]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6V]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6W]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 6X]Figures 6A-6X show that heterologous IN boosting with SARS-CoV-1 spike enhances pre-existing SCV2-specific immunity and broadens responsiveness to SCV1. (Figure 6A) Experimental schema. K18-hACE2 mice were primed intramuscularly with 1 μg of mRNA-LNP and subsequently boosted 14 days after prime with either 1 μg of mRNA-LNP or 5 μg of prefusion-stabilized, trimer-, recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). Figures 6B-6D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue from naive, mRNA-LNP prime-boost, or P&Sx mice. (Figures 6E-6N) Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from [(Figures 6E)-(Figure 6I)] SCV1 or [(Figures 6J)-(Figure 6N)] SCV2, and intracellular cytokine staining was performed to assess antigen-specific production of TNF-α, IL-2, IFN-γ, IL-17, and IL-4 by extravascular IV-CD45-CD44+ CD4+ T cells. Figures 6O-6S show measurements of SCV1 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. (Figure 6S) Measurement of neutralizing titers against SCV1 spike pseudotyped VSV. Figures 6T-6W show measurements of SCV2 spike S1 subunit-specific BALF IgA and IgG and serum IgA and IgG. Figure 6X shows measurements of neutralizing titers against SCV2 spike pseudotyped VSV. To reduce the overall number of experimental animals used, control data points from untreated and mRNA prime-boost are shared across Figures 4A-4U and 6A-6X. Mean ± SEM. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction, except for Student's t-tests (Figures 6S and 6X); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 7A]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7B]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7C]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7D]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7E]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7F]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7G]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7H]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 7I]Figures 7A-7I show that the IN spike-boost mediated by mucosal immunization is not affected by host genotype, boosting interval, or intranasal volume. (Figure 7A) Experimental schema: K18-hACE2 or C57BL / 6J (B6J) mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 2 or 4 weeks after IM prime in an inoculum volume of 25 or 50 μl. 14 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 7B-7D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue. Figures 7E–7H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 7E), BALF IgG (Figure 7F), serum IgA (Figure 7G), and serum IgG (Figure 7H). Measurement of neutralization titers against SCV2 spike-pseudotyped VSV (Figure 7I) is mean ± s.e.m. Statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 7B–7H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are pooled and represent data from two independent experiments. [Figure 8A]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8B]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8C]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8D]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8E]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8F]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8G]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8H]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8I]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8J]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 8K]Figures 8A-8K show that delayed boosting with an IN spike induces durable mucosal immunity. Figure 8A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and IN boosted with 1 μg of SCV2 spike 12 weeks after the IM prime. At 7 and 56 days after the boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 8B-8D show quantification of total tetramer+ CD8+ T cells (Figure 8B), CD69+CD103- tetramer+ CD8+ T cells (Figure 8C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 8D) in lung tissue from IM-primed or P&S mice at 7 and 56 days after the boost. (Figures 8E-8G) Quantification of total activated polyclonal CD4+ T cells (Figure 8E), CD69+CD103- CD4+ T cells (Figure 8F), or CD69+CD103+ CD4+ T cells (Figure 8G) in lung tissue obtained from IM-primed or P&S mice 7 and 56 days after boost. Figures 8H-8K show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 8H), BALF IgG (Figure 8I), serum IgA (Figure 8J), and serum IgG (Figure 8K) in IM-primed or P&S mice 7 and 56 days after boost. Mean ± s.e.m.; statistical significance was calculated by two-way ANOVA followed by Tukey's correction (H-K); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Each data point represents and is pooled from two independent experiments. [Figure 9A]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9B]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9C]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9D]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9E]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9F]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9G]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 9H]Figures 9A-9H show that IN delivery of SCV2 spike mRNA encapsulated in poly(amine-co-ester) (PACE) terpolymer mediates mucosal boosting. Figure 9A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP, followed by IN boosting with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE-encapsulated mRNA (IN PACE-spiked) 14 days after the IM prime. BALF and blood were collected for antibody measurements 14 days after the IN boost. Lung tissue was collected for CD8 T cell analysis. Figures 9B-9D show quantification of total tetramer+ CD8+ T cells (Figure 9B), CD69+CD103- tetramer+ CD8+ T cells (Figure 9C), or CD69+CD103+ tetramer+ CD8+ T cells (Figure 9D) in lung tissue obtained from naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Figures 9E-9H show measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (Figure 9E), BALF IgG (Figure 9F), serum IgA (Figure 9G), and serum IgG (Figure 9H) in naive, IM primed, IN PACE-spiked, IM primed + IN naked mRNA, or primed and PACE-spiked mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 9B-9D) or two-way ANOVA followed by Tukey's correction (Figures 9E-9H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Data are pooled from two independent experiments. [Figure 10A]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10B]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10C]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10D]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10E]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10F]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10G]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 10H]Figures 10A-10H show that protection induced by IN spike-boost against COVID-19 correlates with enhanced mucosal immunity. Figure 10A shows the experimental schema: K18-hACE2 mice were IM primed with 0.05 μg of mRNA-LNP and IN boosted with 1 μg of spike-IN 14 days after IM prime. Six weeks after the boost, lung tissue was collected for CD8+ T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements. Figures 10B-10D show quantification of total tetramer+ CD8+ T cells, CD69+CD103- tetramer+ CD8+ T cells, or CD69+CD103+ tetramer+ CD8+ T cells in lung tissue obtained from naive, IM primed, or P&S mice. Figures 10E-10H show measurements of SCV2 spike S1 subunit-specific BALF IgA (Figure 10E), BALF IgG (Figure 10F), serum IgA (Figure 10G), and serum IgG (Figure 10H) in naive, IM-primed, or P&S mice. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 10B-10D) or two-way ANOVA followed by Tukey's correction (Figures 10E-10H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented by one independent experiment. [Figure 11A]Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11B] Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11C]Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11D] Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11E]Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11F] Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 11G]Figures 11A-11G show MHC-peptide tetramer-based identification of SARS-CoV-2 spike-specific CD8 and CD4 T cells. Figure 11A shows the sequences of the CD4 epitope (VTWFHAIHVSGTNGT (SEQ ID NO: 5), spike 62-76) and CD8 epitope (VNFNFNGL (SEQ ID NO: 6), spike 539-546) from the sarbecovirus spike protein. MHC class II sequences are shown as SEQ ID NOs: 8-17, and MHC class I sequences are shown as SEQ ID NOs: 18-22. Figures 11B-11D show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11C) and CD4 T cells (Figure 11D) in the lung 42 days after the IM prime and 28 days after the IN spike boost. Figures 11E-11G show the identification and phenotypic analysis of spike-specific CD8 T cells (Figure 11F) and CD4 T cells (Figure 11G) in the lungs 14 days after SARS-CoV-2 infection. Contour plots (Figures 11C and 11F) are representative of two independent experiments. [Figure 12A]Figures 12A-12E show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figure 12A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and 14 days after IM prime, IN boosted with 1 μg of spike IN or 1 μg of mRNA-LNP. Forty-five days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with a spike peptide megapool from SARS-CoV-2, and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD4+CD44+ T cells in naive, mRNA-LNP prime-boost, or P&S mice. Figures 12B-12E show assessment of the simultaneous production of TNF-α and IL-2 (Figure 12B), TNF-α and IFN-γ (Figure 12C), IL-2 and IFN-γ (Figure 12D), or TNF-α, IL-2, and IFN-γ (Figure 12E). Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 12B-12E); *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 12B]Figures 12A-12E show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figure 12A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and 14 days after IM prime, IN boosted with 1 μg of spike IN or 1 μg of mRNA-LNP. Forty-five days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with a spike peptide megapool from SARS-CoV-2, and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD4+CD44+ T cells in naive, mRNA-LNP prime-boost, or P&S mice. Figures 12B-12E show assessment of the simultaneous production of TNF-α and IL-2 (Figure 12B), TNF-α and IFN-γ (Figure 12C), IL-2 and IFN-γ (Figure 12D), or TNF-α, IL-2, and IFN-γ (Figure 12E). Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 12B-12E); *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 12C]Figures 12A-12E show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figure 12A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and 14 days after IM prime, IN boosted with 1 μg of spike IN or 1 μg of mRNA-LNP. Forty-five days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with a spike peptide megapool from SARS-CoV-2, and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD4+CD44+ T cells in naive, mRNA-LNP prime-boost, or P&S mice. Figures 12B-12E show assessment of the simultaneous production of TNF-α and IL-2 (Figure 12B), TNF-α and IFN-γ (Figure 12C), IL-2 and IFN-γ (Figure 12D), or TNF-α, IL-2, and IFN-γ (Figure 12E). Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 12B-12E); *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 12D]Figures 12A-12E show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figure 12A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and 14 days after IM prime, IN boosted with 1 μg of spike IN or 1 μg of mRNA-LNP. Forty-five days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with a spike peptide megapool from SARS-CoV-2, and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD4+CD44+ T cells in naive, mRNA-LNP prime-boost, or P&S mice. Figures 12B-12E show assessment of the simultaneous production of TNF-α and IL-2 (Figure 12B), TNF-α and IFN-γ (Figure 12C), IL-2 and IFN-γ (Figure 12D), or TNF-α, IL-2, and IFN-γ (Figure 12E). Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 12B-12E); *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 12E]Figures 12A-12E show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figure 12A shows the experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and 14 days after IM prime, IN boosted with 1 μg of spike IN or 1 μg of mRNA-LNP. Forty-five days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with a spike peptide megapool from SARS-CoV-2, and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD4+CD44+ T cells in naive, mRNA-LNP prime-boost, or P&S mice. Figures 12B-12E show assessment of the simultaneous production of TNF-α and IL-2 (Figure 12B), TNF-α and IFN-γ (Figure 12C), IL-2 and IFN-γ (Figure 12D), or TNF-α, IL-2, and IFN-γ (Figure 12E). Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 12B-12E); *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13A]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13B]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13C]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13D]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13E]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13F]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13G]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 13H]Figures 13A-13H show that IN spike-boost mediates the expansion of polyfunctional spike-specific CD4+ T cells in lung parenchyma. Figures 13A-13H, experimental schema: K18-hACE2 mice were IM primed with 1 μg of mRNA-LNP and, 14 days after IM prime, IN boosted with 5 μg of SARS-CoV-1 spike or IM boosted with 1 μg of mRNA-LNP. 45 days after priming, lung tissue was harvested for assessment of antigen-specific CD4+ T cell cytokine production by flow cytometry. Percoll gradient-purified lung lymphocytes were restimulated with spike peptide megapools from SARS-CoV-1 (Figures 13A-13D) or SARS-CoV-2 (Figures 13E-13H), and intracellular cytokine staining was performed to assess cytokine production by extravascular IV-CD45-CD44+ CD4+ T cells in naive, mRNA prime-boost, or P&Sx mice. Figures 13A-13D show assessment of simultaneous production of TNF-α and IL-2 (Figure 13A), TNF-α and IFN-γ (Figure 13B), IL-2 and IFN-γ (Figure 13C), or TNF-α, IL-2, and IFN-γ (Figure 13D) by extravascular SARS-CoV-1 spike-specific CD4+ T cells. (Figures 13E-13H) Assessment of simultaneous production of TNF-α and IL-2 (Figure 13E), TNF-α and IFN-γ (Figure 13F), IL-2 and IFN-γ (Figure 13G), or TNF-α, IL-2, and IFN-γ (Figure 13H) by extravascular SARS-CoV-2 spike-specific CD4+ T cells. Mean ± sem; statistical significance was calculated by one-way ANOVA followed by Tukey's correction (Figures 13A-13H); *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Individual data points are represented and pooled from two independent experiments. [Figure 14]Figure 14 shows the integrity of mRNA extracted from Comirnaty mRNA-LNP. The length and integrity of the extracted mRNA were analyzed using agarose gel electrophoresis. The extracted mRNA was mixed with SYBR Safe dye, then loaded onto a 1% agarose gel, run in TAE buffer, and imaged using a gel imaging system. [Figure 15A] Figures 15A-15C show gating strategies for analyzing extravascular adaptive immune responses in the airways. Figure 15A shows the gating strategy for identifying extravascular spike-specific CD8 and CD4 T cells. Figure 15B shows the gating strategy for identifying extravascular antigen-specific and polyclonal B cells. Figure 15C shows the gating strategy for identifying cytokine-producing extravascular spike-specific CD4 T cells. [Figure 15B] Figures 15A-15C show gating strategies for analyzing extravascular adaptive immune responses in the airways. Figure 15A shows the gating strategy for identifying extravascular spike-specific CD8 and CD4 T cells. Figure 15B shows the gating strategy for identifying extravascular antigen-specific and polyclonal B cells. Figure 15C shows the gating strategy for identifying cytokine-producing extravascular spike-specific CD4 T cells. [Figure 15C] Figures 15A-15C show gating strategies for analyzing extravascular adaptive immune responses in the airways. Figure 15A shows the gating strategy for identifying extravascular spike-specific CD8 and CD4 T cells. Figure 15B shows the gating strategy for identifying extravascular antigen-specific and polyclonal B cells. Figure 15C shows the gating strategy for identifying cytokine-producing extravascular spike-specific CD4 T cells. [Figure 16] Figure 16 is a bar graph of PEG-conjugated PACE-mediated delivery and expression of mRNA cargo to the lung, showing RLU / mg protein (0-10,000) across % PACE-PEG content (0-50%) for the control and samples containing 0%, 0.25%, 1%, 5%, 10%, or 50% PACE-PEG, respectively. [Figure 17] Figure 17 is a schematic diagram illustrating the methodology of using mRNA-PACE as a mucosal booster to establish tissue-resident immunity against SARS-CoV-2. The bottom panel is a schematic diagram of the method applied to the K18-hACE2 mouse model, including the priming step on day -14 (injecting 1 microgram of RNA mRNA-LNP (lipid nanoparticle) via the intramuscular (IM) route); the boosting step on day 0 (injecting 1 microgram of RNA mRNA-PACE particles via the intramuscular (IM) or intranasal (IN) route; 2 weeks after priming); and the immunophenotyping analysis on day 14 (consisting of IV antibody labeling followed by lung, bronchoalveolar lavage fluid (BALF), and serum collection; 2 weeks after boosting). The upper right panel is a schematic diagram of the process of IV antibody labeling and flow cytometry analysis. [Figure 18A] 18A-18C are bar graphs of lung-resident (IV-) spike-specific CD8+ T cell immunity induced by IM mRNA-LNP priming and IN mRNA-PACE boosting, respectively, for total spike-specific (IV-tetramer+CD8+ T cells) (FIG. 18A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (FIG. 18B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (FIG. 18C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, as follows: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ); and absolute cell counts of each of the IM LNP>IN PACE (lower triangle) cell samples (#100 ~10 5 ) is shown. [Figure 18B] 18A-18C are bar graphs of lung-resident (IV-) spike-specific CD8+ T cell immunity induced by IM mRNA-LNP priming and IN mRNA-PACE boosting, respectively, for total spike-specific (IV-tetramer+CD8+ T cells) (FIG. 18A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (FIG. 18B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (FIG. 18C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, as follows: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ); and absolute cell counts of each of the IM LNP>IN PACE (lower triangle) cell samples (#10 0 ~10 5 ) is shown. [Figure 18C]18A-18C are bar graphs of lung-resident (IV-) spike-specific CD8+ T cell immunity induced by IM mRNA-LNP priming and IN mRNA-PACE boosting, respectively, for total spike-specific (IV-tetramer+CD8+ T cells) (FIG. 18A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (FIG. 18B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (FIG. 18C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, as follows: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ); and absolute cell counts of each of the IM LNP>IN PACE (lower triangle) cell samples (#10 0 ~10 5 ) is shown. [Figure 19A] 19A-19C are bar graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting did not affect circulating (IV+) CD8+ T cell responses. The graphs show the relationship between total spike-specific (IV+ tetramer+ CD8+ T cells) (FIG. 19A); antigen-experienced activated (IV+ tetramer+ CD69+ CD8+ T cells) (FIG. 19B); and tissue-resident memory (IV+ tetramer+ CD69+ CD103+ CD8+ T cells) (FIG. 19C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, in the following groups: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ) and IM LNP>IN PACE (lower triangle) cell samples, absolute cell counts (#10 0 ~10 5 ) is shown. [Figure 19B] 19A-19C are bar graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting did not affect circulating (IV+) CD8+ T cell responses. The graphs show the relationship between total spike-specific (IV+ tetramer+ CD8+ T cells) (FIG. 19A); antigen-experienced activated (IV+ tetramer+ CD69+ CD8+ T cells) (FIG. 19B); and tissue-resident memory (IV+ tetramer+ CD69+ CD103+ CD8+ T cells) (FIG. 19C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, in the following groups: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ) and IM LNP>IN PACE (lower triangle) cell samples, absolute cell counts (#10 0 ~10 5 ) is shown. [Figure 19C]19A-19C are bar graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting did not affect circulating (IV+) CD8+ T cell responses. The graphs show the relationship between total spike-specific (IV+ tetramer+ CD8+ T cells) (FIG. 19A); antigen-experienced activated (IV+ tetramer+ CD69+ CD8+ T cells) (FIG. 19B); and tissue-resident memory (IV+ tetramer+ CD69+ CD103+ CD8+ T cells) (FIG. 19C) cells acquired with / without (+ / -) IM mRNA-LNP priming; IN naked mRNA boosting; IM mRNA-PACE boosting; and IN mRNA-PACE boosting, respectively, in the following groups: naive (circles); IM LNP (upper triangles); IM LNP > IN naked mRNA ( [ka] );IN PACE(square);IM LNP>IM PACE( [ka] ) and IM LNP>IN PACE (lower triangle) cell samples, absolute cell counts (#10 0 ~10 5 ) is shown. [Figure 20A] 20A-20D are graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the results of naive (circles); IM LNP (upper triangles); IN PACE (squares); and IM LNP>IM PACE ( ), respectively, obtained from BALF at a dilution of 1:36 ( FIG. 20A ) or 1:216 ( FIG. 20B ) and serum at a dilution of 1:1250 ( FIG. 20C ) or 1:62500 ( FIG. 20D ). [ka] The optical density (OD) (1-4) of anti-S1 IgA in each of the IM LNP>IN PACE (lower triangle) cell samples is shown. [Figure 20B] 20A-20D are graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the results of naive (circles); IM LNP (upper triangles); IN PACE (squares); and IM LNP>IM PACE ( ), respectively, obtained from BALF at a dilution of 1:36 ( FIG. 20A ) or 1:216 ( FIG. 20B ) and serum at a dilution of 1:1250 ( FIG. 20C ) or 1:62500 ( FIG. 20D ). [ka] The optical density (OD) (1-4) of anti-S1 IgA in each of the IM LNP>IN PACE (lower triangle) cell samples is shown. [Figure 20C] 20A-20D are graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the results of naive (circles); IM LNP (upper triangles); IN PACE (squares); and IM LNP>IM PACE ( ), respectively, obtained from BALF at a dilution of 1:36 ( FIG. 20A ) or 1:216 ( FIG. 20B ) and serum at a dilution of 1:1250 ( FIG. 20C ) or 1:62500 ( FIG. 20D ). [ka] The optical density (OD) (1-4) of anti-S1 IgA in each of the IM LNP>IN PACE (lower triangle) cell samples is shown. [Figure 20D] 20A-20D are graphs showing that IM mRNA-LNP priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the results of naive (circles); IM LNP (upper triangles); IN PACE (squares); and IM LNP>IM PACE ( ), respectively, obtained from BALF at a dilution of 1:36 ( FIG. 20A ) or 1:216 ( FIG. 20B ) and serum at a dilution of 1:1250 ( FIG. 20C ) or 1:62500 ( FIG. 20D ). [ka] The optical density (OD) (1-4) of anti-S1 IgA in each of the IM LNP>IN PACE (lower triangle) cell samples is shown. [Figure 21] Figure 21 is a schematic diagram of the method applied in the K18-hACE2 mouse model, including a priming step on day 0 (injection of 0.05 micrograms of RNA with mRNA-LNPs (lipid nanoparticles) via the intramuscular (IM) route); a boosting step on day 14 (injection of 10 micrograms of RNA with mRNA-PACE particles via the intramuscular (IM) or intranasal (IN) route; 2 weeks after priming); pre-infection bleeding on day 49; IN SARS-CoV-2 challenge on day 56 and 2 weeks of monitoring of weight loss and survival until day 70. [Figure 22A] Figures 22A-22C show the results of untreated (circles); IM primed (upper triangles); IM primed > IM PACE ( [ka] 22A and 22B are line graphs showing OD anti-S1 IgG as a function of log dilution from 2 to 5 for IM primed > IN PACE (lower triangle) and IM primed > IN PACE (lower triangle) samples, respectively (Figure 22A), % body weight on day 0 over a 14-day period from day 0 to day 14 post-infection (Figure 22B), and % survival over a 14-day period from day 0 to day 14 post-infection (Figure 22C). [Figure 22B] Figures 22A-22C show the results of untreated (circles); IM primed (upper triangles); IM primed > IM PACE ( [ka] 22A and 22B are line graphs showing OD anti-S1 IgG as a function of log dilution from 2 to 5 for IM primed > IN PACE (lower triangle) and IM primed > IN PACE (lower triangle) samples, respectively (Figure 22A), % body weight on day 0 over a 14-day period from day 0 to day 14 post-infection (Figure 22B), and % survival over a 14-day period from day 0 to day 14 post-infection (Figure 22C). [Figure 22C] Figures 22A-22C show the results of untreated (circles); IM primed (upper triangles); IM primed > IM PACE ( [ka] 22A and 22B are line graphs showing OD anti-S1 IgG as a function of log dilution from 2 to 5 for IM primed > IN PACE (lower triangle) and IM primed > IN PACE (lower triangle) samples, respectively (Figure 22A), % body weight on day 0 over a 14-day period from day 0 to day 14 post-infection (Figure 22B), and % survival over a 14-day period from day 0 to day 14 post-infection (Figure 22C). [Figure 23] Figure 23 is a schematic diagram of the method applied to the K18-hACE2 mouse model, including a priming step on day 0 (administration of 10 micrograms of RNA mRNA-PACE particles via the intranasal (IN) route); a boosting step on day 28 (administration of 10 micrograms of RNA mRNA-PACE particles via the intranasal (IN) route; 4 weeks after priming) and immunophenotyping on day 42 (consisting of IV antibody labeling followed by lung, bronchoalveolar lavage fluid (BALF) and serum collection; 2 weeks after boosting). [Figure 24A]Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 24B] Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 24C]Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 24D] Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 24E]Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 24F] Figures 24A-24F are bar graphs of tissue-resident (IV-) spike-specific CD8+ T cell immunity induced by IN mRNA-PACE priming and IN mRNA-PACE boosting, respectively, showing the proportion of total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24A); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24B); and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24C) cells in each of naive (circles) and IN PACE>IN Absolute cell counts in the lungs of the PACE (upper triangle) group and absolute cell counts in the mediastinal lymph nodes of the naive (circles) and IN PACE > IN PACE (upper triangle) groups for total spike-specific (IV-tetramer+CD8+ T cells) (Figure 24D); antigen-experienced activated (IV-tetramer+CD69+CD8+ T cells) (Figure 24E), and tissue-resident memory (TRM) (IV-tetramer+CD69+CD103+CD8+ T cells) (Figure 24F) cells, respectively, are shown. [Figure 25A]Figures 25A-25D are bar graphs of humoral immune responses induced by IN mRNA-PACE priming and IN mRNA-PACE boosting in draining lymph nodes, showing absolute cell counts in the naive (circles) and IN PACE>IN PACE (upper triangles) groups for CXCR5+PD1+ TFH cells (Figure 25A); GL7+ germinal center (GC) B cells (Figure 25B); CD138+ antibody-secreting cells (ASCs) (Figure 25C), and tetramer+ B cells (Figure 25D), respectively. [Figure 25B] Figures 25A-25D are bar graphs of humoral immune responses induced by IN mRNA-PACE priming and IN mRNA-PACE boosting in draining lymph nodes, showing absolute cell counts in the naive (circles) and IN PACE>IN PACE (upper triangles) groups for CXCR5+PD1+ TFH cells (Figure 25A); GL7+ germinal center (GC) B cells (Figure 25B); CD138+ antibody-secreting cells (ASCs) (Figure 25C), and tetramer+ B cells (Figure 25D), respectively. [Figure 25C] Figures 25A-25D are bar graphs of humoral immune responses induced by IN mRNA-PACE priming and IN mRNA-PACE boosting in draining lymph nodes, showing absolute cell counts in the naive (circles) and IN PACE>IN PACE (upper triangles) groups for CXCR5+PD1+ TFH cells (Figure 25A); GL7+ germinal center (GC) B cells (Figure 25B); CD138+ antibody-secreting cells (ASCs) (Figure 25C), and tetramer+ B cells (Figure 25D), respectively. [Figure 25D] Figures 25A-25D are bar graphs of humoral immune responses induced by IN mRNA-PACE priming and IN mRNA-PACE boosting in draining lymph nodes, showing absolute cell counts in the naive (circles) and IN PACE>IN PACE (upper triangles) groups for CXCR5+PD1+ TFH cells (Figure 25A); GL7+ germinal center (GC) B cells (Figure 25B); CD138+ antibody-secreting cells (ASCs) (Figure 25C), and tetramer+ B cells (Figure 25D), respectively. [Figure 26A]Figures 26A-26D are bar graphs showing that IN mRNA-PACE priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the optical densities (OD) (1-3) of anti-S1 IgG from untreated (circles) and IN PACE > IN PACE (upper triangles) samples obtained from BALF diluted 1:36 or 1:216 (Figure 26A) and serum diluted 1:1250 or 1:62500 (Figure 26B), respectively; and the optical densities (OD) (1-3) of anti-S1 IgA from BALF diluted 1:6 or 1:36 (Figure 26C) and serum diluted 1:250 or 1:1250 (Figure 26D), respectively, from untreated (circles) and IN PACE > IN PACE (upper triangles) samples obtained from BALF diluted 1:6 or 1:36 (Figure 26C) and serum diluted 1:250 or 1:1250 (Figure 26D). [Figure 26B] Figures 26A-26D are bar graphs showing that IN mRNA-PACE priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the optical densities (OD) (1-3) of anti-S1 IgG from untreated (circles) and IN PACE > IN PACE (upper triangles) samples obtained from BALF diluted 1:36 or 1:216 (Figure 26A) and serum diluted 1:1250 or 1:62500 (Figure 26B), respectively; and the optical densities (OD) (1-3) of anti-S1 IgA from BALF diluted 1:6 or 1:36 (Figure 26C) and serum diluted 1:250 or 1:1250 (Figure 26D), respectively, from untreated (circles) and IN PACE > IN PACE (upper triangles) samples obtained from BALF diluted 1:6 or 1:36 (Figure 26C) and serum diluted 1:250 or 1:1250 (Figure 26D). [Figure 26C]Figures 26A-26D are bar graphs showing that IN mRNA-PACE priming followed by IN mRNA-PACE boosting induced robust mucosal and systemic spike-specific IgA and IgG. The graphs show the optical densities (OD) (1-3) of anti-S1 IgG from untreated (circles) and IN PACE > IN PACE (upper triangles) samples obtained from BALF diluted 1:36 or 1:216 (Figure 26A) and serum diluted 1:1250 or 1:62500 (Figure 26B), respectively; and the optical densities (OD) (1-3) of anti-S1 IgA from BALF diluted 1:6 or 1:36 (Figure 26C) and serum diluted 1:250 or 1:1250 (Figure 26D), respectively, from untreated (circles) and ...

Claims

1. 1. A method for enhancing mucosal immunity to an antigen in a subject, comprising: administering to a mucosal tissue in a subject an effective amount of a vaccine composition comprising (a) an antigen or (b) a polynucleotide encoding the antigen in a carrier. Including, The method wherein the subject has been previously exposed to the antigen.

2. 1. A method for enhancing mucosal immunity to repeated presentation of an antigen in a subject, comprising: (i) administering an effective amount of a priming antigen via a systemic or mucosal route of administration; and (ii) then administering to the subject by a mucosal route an effective amount of a boosting composition comprising (a) the antigen or (b) a polynucleotide encoding the antigen in a carrier. A method comprising:

3. 3. The method of claim 1 or 2, wherein the carrier comprises a poly(amine-co-ester) (PACE) polymer, a poly(amine-co-ester) polymer or a poly(amine-co-amide) polymer, or is a carrier material selected from the group consisting of liposomes, liposome-like nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, absorbed lipid-polymer hybrid nanoparticles, encapsulated lipid-polymer hybrid nanoparticles, cationic nanoemulsions, exosomes, natural lipoproteins and synthetic lipoproteins.

4. The prior exposure to the antigen comprises administering an effective amount of a priming antigen via a systemic or mucosal route of administration; The method of claim 1 , wherein the priming antigen shares one or more antigenic sites with the antigen encoded by the polynucleotide.

5. 5. The method of any one of claims 2 to 4, wherein the systemic administration route is intramuscular or intradermal administration, and / or the mucosal administration route is via one or more mucosal tissues selected from the group consisting of lung, nasal cavity, oral cavity, digestive system, vaginal, and rectal mucosa, optionally the mucosal administration route is intranasal or pulmonary administration, and optionally the intranasal administration is via respiratory droplets and aerosols.

6. An enhanced number of CD8 cells compared to the number of cells prior to administration to the mucosal tissue. ＋ Tissue resident memory (T ＲＭ ) cells, CD4 ＋ Tissue resident memory (T ＲＭ The method of any one of claims 1 to 5, wherein cells selected from the group consisting of immune-mediated immune (IM) cells and memory B cells are induced to an antigen in a mucosal tissue in a subject.

7. The method of any one of claims 1 to 6, which induces a higher titer of mucosal IgA compared to the IgA titer prior to the step of administering to the mucosal tissue.

8. The method of any one of claims 2 to 7, wherein the priming antigen comprises messenger RNA (mRNA) encoding the antigen bound or encapsulated within a lipid nanoparticle.

9. The method according to any one of claims 1 to 8, wherein the mucosal tissue is one or more selected from the group consisting of lung, nasal cavity, oral cavity, digestive system, vaginal, and rectal mucosa.

10. The method according to any one of claims 1 to 8, wherein the mucosal tissue is lung and nasal mucosa.

11. 10. The method of any one of claims 1 to 9, wherein the antigen is derived from or generates a protective immune response against one or more respiratory viruses selected from the group consisting of orthomyxovirus, paramyxovirus, coronavirus, adenovirus, herpesvirus and human bocavirus.

12. 12. The method of any one of claims 1 to 11, wherein the antigen is derived from or generates a protective immune response against one or more respiratory viruses selected from the group consisting of influenza virus, parainfluenza virus, measles virus, mumps virus and respiratory syncytial virus, human metapneumovirus, severe acute respiratory syndrome (SARS) virus, herpes simplex virus (HSV), cytomegalovirus (CMV), Epstein-Barr virus (EBV) and varicella zoster virus (VZV).

13. The method of any one of claims 1 to 12, wherein the antigen is derived from or generates a protective immune response against an influenza virus.

14. 13. The method of any one of claims 1 to 12, wherein the antigen is derived from or generates a protective immune response against the SARS-CoV-2 virus, optionally wherein the antigen is derived from a coronavirus spike protein and the coronavirus is a variant of SARS-CoV-2.

15. The method according to any one of claims 1 to 14, wherein the polynucleotide encoding the antigen is a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule.

16. The method according to any one of claims 1 to 15, wherein the polynucleotide encoding the antigen is messenger RNA (mRNA).

17. 17. The method of any one of claims 1 to 16, wherein the mRNA comprises an open reading frame encoding a spike protein sequence, the spike protein sequence comprising all or a portion of a coronavirus spike protein, and the coronavirus is a variant of SARS-CoV-2.

18. 18. The method of claim 17, wherein the variant is selected from SARS-CoV-2 B.1.1.7 (alpha variant), SARS-CoV-2 B.1.351 (beta variant), SARS-CoV-2 P.1 (gamma variant), SARS-CoV-2 B.1.

617.

19. 18. The method of claim 17, wherein the variant is selected from SARS-CoV-2 B.1.617.1 (kappa variant), SARS-CoV-2 B.1.617.2 (delta variant), SARS-CoV-2 B.1.617.3 and SARS-CoV-2 B.1.1.529 (omicron variant).

20. The vaccine composition induces CD8 expression in mucosal tissue against an antigen. ＋ Tissue resident memory (T ＲＭ ) cells, CD4 ＋ Tissue resident memory (T ＲＭ 20. The method of any one of claims 1 to 19, wherein the antibody is administered in an amount effective to induce one or more cells selected from the group consisting of: IgG1-specific B cells and memory B cells.

21. The vaccine composition produced increased numbers of CD8 ＋ Tissue resident memory (T ＲＭ ) cells, CD4 ＋ Tissue resident memory (T ＲＭ The method according to any one of claims 1 to 20, wherein cells selected from the group consisting of immune-mediated immune (IM) cells and memory B cells are induced in a mucosal tissue against an antigen.

22. The method of any one of claims 1 to 21, wherein the vaccine composition is administered in an amount effective to induce mucosal immunoglobulin A (IgA) against the antigen.

23. 22. The method of any one of claims 1 to 21, wherein the vaccine composition induces increased amounts of mucosal IgA against the antigen compared to a vaccine composition delivered in the absence of the poly(amine-co-ester) (PACE) polymer via the same route of administration.

24. 24. The method of claim 23, wherein the IgA is secretory IgA in a mucosal lavage fluid selected from bronchoalveolar lavage fluid, intestinal lavage fluid, intestinal lavage fluid and vaginal lavage fluid.

25. 25. The method of any one of claims 3 to 24, wherein the PACE polymer and the polynucleotide encoding the antigen are in the form of a particle selected from the group consisting of microparticles and nanoparticles, and optionally the PACE polymer is a polyethylene glycol (PEG) conjugated PACE polymer.

26. The PACE polymer has the formula I: 【Chemistry 1】 [wherein n is an integer from 1 to 30; m, o and p are independently integers from 1 to 20; x, y and q are independently integers from 1 to 1000; Rx is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z′ are independently O or NR′, R′ is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R １ and R ２ is absent or is a chemical entity that contains a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. The method of any one of claims 3 to 25, comprising a polymer of the formula:

27. R1 and / or R2 are 【Chemistry 2】 27. The method of claim 26, wherein:

28. R １ and / or R ２ but, 【Chemistry 3】 28. The method of claim 27, wherein the particles (e.g., microparticles and / or nanoparticles) formed from the polymer exhibit improved loading, improved cellular transfection, improved intracellular endosomal release, or a combination thereof, of mRNA compared to a corresponding particle selected from the group consisting of microparticles and nanoparticles consisting of:

29. The polymer has formula II: 【Chemistry 4】 [In the formula, J １ and J. ２ is independently a linking moiety or absent, R ３ and R ４ is a substituted alkyl containing a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof.

28. The method of claim 27 having the structure:

30. The polymer has formula III: 【Chemistry 5】 28. The method of claim 27 having the structure:

31. (a) Z is the same as Z′; (b) n is 4, 10, 13, or 14; (c) m is 5, 6, or 7; or (d) R ｘ 28. The method of claim 27, wherein is substituted or unsubstituted alkyl.

32. 28. The method of claim 27, wherein the weight average molecular weight, as measured by gel permeation chromatography using narrow polydispersity polystyrene standards, is between about 2,000 and 20,000 daltons, preferably between about 2,000 and about 10,000 daltons, and most preferably between about 2000 and about 7,000 daltons.

33. R ３ But, R ４ The method of claim 29, wherein

34. R ３ and / or R ４ Independent 【Chemistry 6】 【change】 30. The method of claim 29, selected from the group consisting of:

35. (a) J １ is —O— or —NH; J ２ is —C(O)NH— or —C(O)O— or a combination thereof, and (b) R ３ , R ４ or both contain a primary amine group, and optionally one or more secondary or tertiary amine groups; and (c) R ３ , R ４ or both contain a hydroxyl group and, optionally, one or more amine groups; and (d) R ３ , R ４ or both contain hydroxyl groups and no amine groups, or (e) R ３ and R ４ 30. The method of claim 29, wherein at least one of the groups does not contain a hydroxyl group.

36. R ３ , R ４ or both are -unsubstituted C １ ~C １０ Alkylene-Aq-unsubstituted C １ ~C １０ Alkylene-Bq, -unsubstituted C １ ~C １０ Alkylene-Aq-substituted C １ ~C １０ Alkylene-Bq, -substituted C １ ~C １０ Alkylene-Aq-unsubstituted C １ ~C １０ Alkylene-Bq or -substituted C １ ~C １０ Alkylene-Aq-substituted C １ ~C １０ alkylene-Bq, Aq is absent or -NR ５ -, Bq is hydroxyl, primary amine, secondary amine or tertiary amine, R ５ 30. The method of claim 29, wherein is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl.

37. The carrier has the following structure: 【Chemistry 7】 [In the formula, n is an integer from 1 to 30, m, o and p are independently integers from 1 to 20; x, y and q are independently integers from 1 to 1000; R ｘ is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted alkoxy; Z and Z' are independently O or NR', where R' is hydrogen, substituted or unsubstituted alkyl, or substituted or unsubstituted aryl; R １ and R ２ is a chemical entity that contains a hydroxyl group, a primary amine group, a secondary amine group, a tertiary amine group, or a combination thereof. The method of claim 26, comprising a poly(amine-co-ester) or poly(amine-co-amide) having the formula:

38. 38. The method of claim 37, wherein (a) Z is the same as Z'; (b) Z is O and Z' is NR', or Z is NR' and Z' is O; (c) Z' is O and n is an integer from 1 to 24, e.g., 4, 10, 13 or 14, optionally Z is also O; or (d) Z' is O and n is an integer from 1 to 24, e.g., 4, 10, 13 or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7 or 8, and o and p are the same integers from 1 to 6, such as 2, 3 or 4, optionally Z is also O.

39. 39. The method of claim 38, wherein Z is O, Z' is O, Z is NR', and Z' is NR'.

39. 39. The method of claim 38, wherein m is an integer from 1 to 10, for example 4, 5, 6, 7 or 8, and optionally Z is also O.

40. 38. The method of claim 37, wherein Z' is O, n is an integer from 1 to 24, e.g., 4, 10, 13 or 14, m is an integer from 1 to 10, e.g., 4, 5, 6, 7 or 8, R is alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, or homologs and isomers thereof, e.g., n-pentyl, n-hexyl, n-heptyl and n-octyl, or aryl, e.g., phenyl, naphthalyl, anthracenyl, phenanthryl, chrysenyl, pyrenyl, tolyl or xylyl, and optionally Z is also O.

41. 38. The method of claim 37, wherein n is 14, optionally pentadecalactone (PDL), m is 7, optionally sebacic acid, and o and p are 2, optionally N-methyldiethanolamine (MDEA).

42. The polymer has formula II: 【Chemistry 8】 wherein Rx, Z, Z′, m, n, o, p, q, x, and y are as described above for formula I; J １ and J. ２ is independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R in Formula II ３ and R ４ are independently substituted alkyls containing hydroxyl groups, primary amine groups, secondary amine groups, tertiary amine groups, or combinations thereof.

27. The method of claim 26 having the structure:

43. (a) J １ is -O- or -NH-, J ２ is —C(O)—, —C(O)NH—, or —C(O)O—, or (b) R ３ But, R ４ and optionally R ３ and / or R ４ The method of claim 42, wherein is linear.

44. R ３ , R ４ or both contain a hydroxyl group, optionally R ３ , R ４ 44. The method of claim 43, wherein either or both contain a hydroxyl group and one or more amine groups, optionally secondary or tertiary amine groups.

45. (a) R ３ , R ４ or both contain hydroxyl groups and no amine groups, or (b) R ３ and R ４ The method of claim 42, wherein at least one of the groups does not contain a hydroxyl group.

46. The polymer has formula III: 【Chemistry 9】 wherein Rx, Z, Z', m, n, o, p, q, x and y are as defined above for formula I; J １ and J. ２ is independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—; R in Formula II ３ and R ４ are independently substituted alkyls containing hydroxyl groups, primary amine groups, secondary amine groups, tertiary amine groups, or combinations thereof.

27. The method of claim 26 having the structure:

47. (a) J １ is -O- or -NH-, J ２ is —C(O)—, —C(O)NH—, or —C(O)O—; (b) R ３ R ４ and optionally R ３ and / or R ４ is linear; (c) R ３ , R ４ or both contain a hydroxyl group, optionally R ３ , R ４ or both contain a hydroxyl group and one or more amine groups, optionally secondary or tertiary amine groups; (d) R ３ , R ４ or both contain hydroxyl groups and no amine groups, or (d) R ３ and R ４ 47. The method of claim 46, wherein at least one of the following does not contain a hydroxyl group.

48. The method of any of claims 37 to 47, wherein the weight average molecular weight of the polymer is greater than 20,000 Daltons, greater than 15,000 Daltons, greater than 10,000 Daltons, greater than 5,000 Daltons, greater than 2,000 Daltons.

49. 49. The method of claim 48, wherein the weight average molecular weight of the polymer is between about 2,000 Daltons and about 20,000 Daltons, more preferably between about 5,000 Daltons and about 10,000 Daltons.

50. 50. The method of any of claims 37 to 49, prepared from one or more lactones, one or more amine-diols (Z and Z' = O) or triamines (Z and Z' = NR'), and one or more diacids or diesters, where the values ​​of n, o, p, and / or m may be the same or different when two or more different lactone, diacid or diester and / or triamine or amine-diol monomers are used.

51. The method of any of claims 37 to 50, wherein the nanoparticles are formed into particles selected from the group consisting of microparticles and nanoparticles.

52. 52. The method of claim 51 , wherein the nucleic acid is formed into a particle selected from the group consisting of a microparticle and a nanoparticle, the particle having one or more nucleic acids encapsulated therein.

53. The particles selected from the group consisting of microparticles and nanoparticles are R １ and / or R ２ but 【Chemistry 10】 53. The method of claim 52, wherein the polymer is formed from a polymer that does not consist of or does not contain

54. 54. The method of claim 53, wherein the poly(amine-co-ester) or poly(amine-co-amide) is formed into particles selected from the group consisting of microparticles and nanoparticles and is in a mixture containing a poly(amine-co-ester) or poly(amine-co-amide) conjugated to polyethylene glycol (PEG), i.e., a PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide).

55. The PEG-conjugated poly(amine-co-ester) or poly(amine-co-amide) has the formula XI: 【Chemistry 11】 [In the formula, m′ and m″ are independently 0 or 1, provided that m′+m″ is 1 or 2; J in formula XI １ and J. ２ is independently absent or a linking moiety, e.g., —C(O)—, —C(O)NH—, —C(O)O—, —O—, and —NH—. The structure is In some forms of formula XI, J １ is —O— or —NH—, In some forms of formula XI, J ２ The method of claim 54, wherein is -C(O)-, -C(O)NH-, or -C(O)O-.

56. A pharmaceutical composition for use in any one of claims 1 to 55.

57. 1. A method of immunizing a subject against infection by a virus, comprising:

1. A method comprising: parenterally administering to a subject a first composition comprising a first antigen in an amount sufficient to induce a systemic T and / or B cell response in the subject against a virus; and administering to a subject a second composition comprising a viral protein by a route of administration including intranasal, inhalational, intratracheal, intrapulmonary and intrabronchial, in an amount sufficient to result in the establishment of lung resident T and / or B cells specific for the virus in the subject, wherein the virus comprises at least one selected from the group consisting of coronavirus, influenza virus and herpes simplex virus.

58. 1. A method of treating, ameliorating, and / or preventing infection by a virus in a subject, comprising:

1. A method comprising parenterally administering to a subject a first composition comprising a first antigen in an amount sufficient to induce a systemic T and / or B cell response in the subject against a coronavirus, and administering to a subject a second composition comprising a viral protein by a route of administration including intranasal, inhalational, intratracheal, intrapulmonary and intrabronchial, in an amount sufficient to result in the establishment of lung resident T and / or B cells specific for the virus in the subject, wherein the viral infection is caused by at least one virus selected from the group consisting of coronavirus, influenza virus and herpes simplex virus.

59. 59. The method of any one of claims 57 or 58, wherein the virus comprises a coronavirus, and the coronavirus is at least one virus selected from the group consisting of an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and a deltacoronavirus.

60. 60. The method of any one of claims 57 to 59, wherein the virus comprises a coronavirus, and the coronavirus is at least one virus selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2.

61. 61. The method of any one of claims 57 to 60, wherein the virus comprises a coronavirus, and the coronavirus is SARS-CoV-2.

62. 62. The method of any one of claims 57-61, wherein the virus comprises a coronavirus, the coronavirus is SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

63. 63. The method of any one of claims 57-62, wherein the virus comprises a coronavirus, the coronavirus being SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.

1.

64. The virus comprises a coronavirus, the coronavirus being the B.1 lineage SARS-CoV-2, the B.1 lineage being B.1, B.1.1, B.1.1.7, B.1.1.7 with E484K, B.1.2, B.1.5 to B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3 to B.1.66, B.1.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.526.3, B.1.526.4, B.1.526.5, B.1.526.6, B.1.526.7, B.1.526.8, B.1.526.9 ...3 64. The method of any one of claims 57 to 63, comprising at least one of B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620 and B.1.

621.

65. The method of any one of claims 57 to 64, wherein the first composition comprises a vaccine against an infection.

66. 66. The method of any one of claims 57 to 65, wherein the first composition comprises at least one selected from the group consisting of an mRNA-lipid nanoparticle (LNP) based vaccine against infection, a viral vector vaccine against infection, an inactivated viral vaccine against infection, a viral subunit / peptide vaccine against infection, a virus-like particle (VLP) based vaccine against infection, and a DNA vaccine against infection.

67. 67. The method of any one of claims 57 to 66, wherein the virus comprises a coronavirus and the second composition comprises a coronavirus spike protein.

68. 68. The method of any one of claims 57-67, wherein the virus comprises a first coronavirus and the second composition comprises a spike protein from a second coronavirus.

69. 69. The method of claim 68, wherein (a) the first coronavirus and the second coronavirus are the same, or (b) the first coronavirus and the second coronavirus are different.

70. 70. The method of claim 69, wherein the first coronavirus is SARS-CoV and the second coronavirus is SARS-CoV-2, or the first coronavirus is SARS-CoV-2 and the second coronavirus is SARS-CoV.

71. The method of any one of claims 57 to 70, wherein the second composition does not comprise an adjuvant.

72. 72. The method of any of claims 57-71, wherein the second composition comprises a pharma- ceutically acceptable carrier suitable for intranasal, inhalational, intratracheal, intrapulmonary and / or intrabronchial administration of the second composition.

73. 73. The method of any one of claims 57 to 72, which results in the expansion of an antigen-specific tissue-resident memory T and / or B cell response against the virus in the lung parenchyma and airways of a subject.

74. 74. The method of any one of claims 57 to 73, wherein subjects immunized with the method develop higher levels of airway and systemic IgA and IgG antibodies against the virus compared to control subjects immunized without intranasal, inhaled, intratracheal, intrapulmonary, and / or intrabronchial administration of the second composition.

75. 75. The method of any one of claims 57 to 74, wherein the method reduces viral titers in the upper and lower respiratory tract and minimizes and / or prevents the occurrence of a lethal coronavirus virus infection in a subject.

76. The method of any one of claims 57 to 75, wherein the subject is a mammal.

77. The method of any one of claims 57 to 76, wherein the subject is a human.

78. 1. A composition for boosting existing immunity to infection by a virus in a subject, comprising: a carrier material comprising a poly(amine-co-ester) (PACE) polymer or a carrier material selected from the group consisting of liposomes, liposome-like nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, absorbed lipid-polymer hybrid nanoparticles, encapsulated lipid-polymer hybrid nanoparticles, cationic nanoemulsions, exosomes, natural lipoproteins and synthetic lipoproteins, a viral protein or a polynucleotide encoding a viral protein in / on a carrier material; A pharma- ceutically acceptable carrier suitable for intranasal, inhalational, intratracheal, intrapulmonary and / or intrabronchial administration of a coronavirus protein, the virus comprising at least one selected from the group consisting of coronavirus, influenza virus and herpes simplex virus. A composition comprising:

79. 79. The composition of claim 78, wherein the virus comprises a coronavirus, and the coronavirus is at least one virus selected from the group consisting of an alphacoronavirus, a betacoronavirus, a gammacoronavirus, and a deltacoronavirus.

80. 80. The composition of any one of claims 78 to 79, wherein the virus comprises a coronavirus, the coronavirus being at least one virus selected from the group consisting of MERS CoV, SARS-CoV and SARS-CoV-2.

81. 81. The composition of any one of claims 78 to 80, wherein the virus comprises a coronavirus, and the coronavirus is SARS-CoV-2.

82. 81. The composition of any one of claims 78-80, wherein the virus comprises a coronavirus, the coronavirus being SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

83. 83. The composition of any one of claims 78-82, wherein the virus comprises a coronavirus, the coronavirus being SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.

1.

84. The virus comprises a coronavirus, the coronavirus being the B.1 lineage SARS-CoV-2, the B.1 lineage being B.1, B.1.1, B.1.1.7, B.1.1.7 with E484K, B.1.2, B.1.5 to B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3 to B.1.66, B.1.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.526.3, B.1.526.4, B.1.526.5, B.1.526.6, B.1.526.7, B.1.526.8, B.1.526.9 ...3 The composition of any one of claims 78 to 83, comprising at least one of B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620 and B.1.

621.

85. 85. The composition of any one of claims 78 to 84, wherein the virus comprises a coronavirus and the coronavirus protein comprises a coronavirus spike protein.

86. 86. The composition of any one of claims 78 to 85, wherein the virus comprises a first coronavirus and the viral protein is a spike protein from a second coronavirus.

87. 87. The composition of claim 86, wherein (a) the first coronavirus and the second coronavirus are the same virus, or (b) the first coronavirus and the second coronavirus are different.

88. 87. The composition of claim 86, wherein the first coronavirus is SARS-CoV and the second coronavirus is SARS-CoV-2, or the first coronavirus is SARS-CoV-2 and the second coronavirus is SARS-CoV.

89. A composition according to any one of claims 78 to 88, which does not contain an adjuvant.

90. The composition of any one of claims 78 to 89, wherein the subject is a mammal.

91. The composition of any one of claims 78 to 90, wherein the subject is a human.

92. A composition according to claim 78 comprising a polymer or particle according to any one of claims 26 to 55.

93. 1. A kit for immunizing a subject against infection with a virus, comprising:

1. A kit comprising: a first composition comprising a first antigen in an amount sufficient to induce a systemic T and / or B cell response in a subject against a virus, the first composition formulated for parenteral administration to the subject; and a second composition comprising a viral protein in an amount sufficient to result in establishment of lung resident T and / or B cells against the virus in the subject, the second composition formulated for intranasal, inhalational, intratracheal, intrapulmonary, and / or intrabronchial administration, wherein the virus comprises at least one selected from the group consisting of a coronavirus, influenza virus, and herpes simplex virus.

94. 94. The kit of claim 93, wherein the virus comprises a coronavirus, and the coronavirus is at least one virus selected from the group consisting of MERS-CoV, SARS-CoV, and SARS-CoV-2.

95. The kit of any one of claims 93 to 94, wherein the virus comprises a coronavirus, and the coronavirus is SARS-CoV-2.

96. 96. The kit of any one of claims 93-95, wherein the virus comprises a coronavirus, the coronavirus being SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma), B.1.617.2 (delta), B.1.429 / B.1.427 (epsilon), B.1.617.1 (kappa), B.1.525 (eta), B.1.526 (iota), P.3 (theta), P.2 (zeta), and B.1.1.529 (omicron).

97. 97. The kit of any one of claims 93-96, wherein the virus comprises a coronavirus, the coronavirus being SARS-CoV-2, and the SARS-CoV-2 comprises at least one variant selected from the group consisting of A.1-A.6, B.3-B.7, B.9, B.10, B.13-B.16, B.2, B.1 lineage, P.1, P.2, P.3, and R.

1.

98. The virus comprises a coronavirus, the coronavirus being the B.1 lineage SARS-CoV-2, the B.1 lineage being B.1, B.1.1, B.1.1.7, B.1.1.7 with E484K, B.1.2, B.1.5 to B.1.72, B.1.9, B.1.13, B.1.22, B.1.26, B.1.37, B.1.3 to B.1.66, B.1.177, B.1.243, B.1.313, B.1.351, B.1.427, B.1.429, B.1.525, B.1.526, B.1.526.1, B.1.526.2, B.1.526.3, B.1.526.4, B.1.526.5, B.1.526.6, B.1.526.7, B.1.526.8, B.1.526.9, B.1.526.1, B.1.526.2 ...3, B.1.526.4, B.1.526.5, B.1.526.6, B.1.526.7, B.1.526.8, B.1.526.9, B.1.526.1, B.1.526.2 98. The kit of any one of claims 93 to 97, comprising at least one of B.1.617, B.1.617.1, B.1.617.2, B.1.617.3, B.1.619, B.1.620 and B.1.

621.

99. 99. The kit of any one of claims 93 to 98, wherein the virus comprises a coronavirus and the first composition comprises a vaccine against coronavirus infection.

100. The kit of any one of claims 93 to 99, wherein the first composition comprises at least one selected from the group consisting of an mRNA-lipid nanoparticle (LNP) based vaccine against infection, a viral vector vaccine against infection, an inactivated viral vaccine against infection, a viral subunit / peptide vaccine against infection, a virus-like particle (VLP) based vaccine against infection, and a DNA vaccine against infection.

101. 101. The kit of any one of claims 93 to 100, wherein the virus comprises a coronavirus and the second composition comprises a coronavirus spike protein.

102. 102. The kit of any one of claims 93 to 101, wherein the virus comprises a first coronavirus and the viral protein is a spike protein from a second coronavirus.

103. 103. The kit of claim 102, wherein (a) the first coronavirus and the second coronavirus are the same virus, or (b) the first coronavirus and the second coronavirus are different.

104. 103. The kit of claim 102, wherein the first coronavirus is SARS-CoV and the second coronavirus is SARS-CoV-2, or the first coronavirus is SARS-CoV-2 and the second coronavirus is SARS-CoV.

105. The kit of any one of claims 93 to 104, wherein the second composition does not comprise an adjuvant.

106. 106. The kit of any one of claims 93 to 105, wherein the second composition comprises a pharma- ceutically acceptable carrier suitable for intranasal, inhalational, intratracheal, intrapulmonary and / or intrabronchial administration of the second composition.

107. The kit of any one of claims 93 to 106, further comprising instructions for use.

108. The kit of any one of claims 93 to 107, wherein the subject is a mammal, optionally a human.

109. A kit according to any one of claims 38 to 54, comprising a composition according to any one of claims 56 or 78 to 92.

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