Methods for enhancing immunity

Abstract

The invention relates to a method for enhancing immunity.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 336,499, filed on April 29, 2022, entitled “Methods for Enhancing Immunity,” and U.S. Provisional Patent Application No. 63 / 648,451, filed on June 2, 2022, entitled “Methods for Enhancing Immunity,” each of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to a method for enhancing immunity. Sequence Listing

[0003] This application contains a sequence listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The sequence listing of this application is marked as "130481-5002-WO.XML", which was created on April 25, 2023 and is 8,500 bytes in size. Background Art

[0004] mRNA-based vaccines against SARS-CoV-2 have demonstrated great potential for safe and effective use of mRNA therapeutics in the general population. However, recent studies have shown that vaccine effectiveness in asymptomatic infection as well as symptomatic and severe infection decreases starting approximately 4 months after the second dose of an mRNA-lipid nanoparticle (LNP)-based regimen. In addition, continued viral evolution and enhanced immune evasion, especially β (B.1.351), δ (B.1.617.2), and now ο (B.1.529) variants of concern (VOC), have also led to a decrease in vaccine effectiveness against COVID-19. Not only have current vaccines become less effective in preventing SARS-CoV-2 infection, but they have also become less capable of preventing viral transmission.

[0005] Therefore, there is still a need to enhance immunity against COVID-19. The present invention meets this need. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1AAn experiment is shown in which K18-hACE2 mice were immunized intramuscularly (IM) with 1 μg of mRNA-lipid nanoparticles (LNP) encoding full-length SARS-CoV-2 (SCV2) spike protein, and then intranasally (IN) with 1 μg of prefusion stabilized trimeric recombinant SARS-CoV-2 (SCV2) spike protein 14 days after mRNA-LNP immunization. Serum, bronchoalveolar lavage fluid (BALF), and nasal wash were collected 14 days after the IN boost to assess binding and neutralizing antibody responses. Lung tissue was collected for extravascular B cell analysis.

[0007] Figure 1B Shows naivety Measurement results of SCV2 Spike S1 subunit-specific nasal wash IgA (B), nasal wash IgG (C), BALF IgA (D), BALF IgG (E), serum IgA (F), and serum Ig (G) in mice, mice immunized IM with mRNA-LNP (IM prime mice), mice immunized IN with Spike protein (IN spike mice), or mice primed IM and boosted IN with Spike protein (prime and spike mice).

[0008] Figure 1C Shown are the results of measurement of neutralization titers against SCV2 spike pseudotyped vesicular stomatitis virus (VSV) in BALF (H, I) and serum (J, K).

[0009] Figure 1D The results show the measurement of multiple extravascular (negative for intravenous marker antibodies) B cell subsets in the lung tissue of IM-primed mice or primed and spiked mice, including RBD tetramer-binding B cells, IgA + Resident memory B cells (B RM ), IgG + B RM , IgA + Antibody Secreting Cells (ASC) and IgG + ASC.

[0010] Figure 2A An experiment is shown in which K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and boosted IN 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 after the boost, and BALF and nasal turbinates were collected 7 days after the boost.

[0011] Figure 2BExtravascular CD8 T cell responses are shown: SCV2 spike-specific tetramers in lung tissue (BD), BALF (EG) + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification results of CD8 T cells.

[0012] Figure 2C Extravascular CD8 T cell responses revealed: SCV2 spike-specific tetramers in the nasal turbinates + CD8T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification of CD8 T cells (HJ), extravascular CD4 T cell responses (KP): activated polyclonal CD4 T cells in lung tissue (KM) or BALF (NP) from naive mice, IM primed mice, IN spiked mice, or primed and spiked mice, CD69 + CD103 - CD4 T cells or CD69 + CD103 + Quantification results of CD4 T cells.

[0013] Figure 3A An experiment is shown in which K18-hACE2 mice were primed IM with 1 μg of mRNA-LNPs and then boosted IN with 1 μg of naked mRNA (IN naked mRNA) or 1 μg of PACE (IN PACE-Spike) encapsulated mRNA 14 days after IM prime. Fourteen (14) days after IN boost, BALF and blood were collected for antibody measurement. Lung tissue was collected for CD8 T cell analysis.

[0014] Figure 3B Total tetramers in lung tissue of naive mice, IM primed mice, IN PACE-Spike mice, IM primed + IN naked mRNA mice, or primed and PACE-Spike mice are shown. + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 +Tetramer + Quantification results of CD8 T cells.

[0015] Figure 3C Naive mice, IM primed mice, IN PACE-spike, IM primed mice + Measurement results of SARS-CoV-2 Spike S1 subunit-specific BALF IgA (E), BALF IgG (F), serum IgA (G), and serum IgG (H) in IN nude mRNA mice or primed and PACE-Spike mice.

[0016] Figure 4A An experiment is shown in which K18-hACE2 mice were primed IM with 0.05 μg of mRNA LNP and boosted IN with 1 μg of spike IN 14 days after IM priming. Six weeks after boosting, mice were treated with 6×10 4 PFU SARS-CoV-2 (2019n-CoV / USA_WA1 / 2020) challenged mice. The first group was used to evaluate weight loss and survival up to 14 DPI (days post infection). The second group was used to collect lung and nasal turbinate tissues at 2 DPI for viral titer measurement. The third group was used to collect lung tissues at 5 DPI for histological evaluation.

[0017] Figure 4B Weight loss and survival of naive mice, IM primed mice, or primed and spiked mice from 1 DPI to 14 DPI are shown. (EF) Infectious virus titers in lung and nasal turbinates at 2 DPI were measured by plaque assay. (G) Lung sections at 5 DPI were pathologically scored by hematoxylin and eosin (H&E) staining.

[0018] Figure 4C Representative H&E staining results of uninfected mice, IM-primed mice, or primed and spiked mice are shown.

[0019] Figure 4D An experiment is shown in which K18-hACE2 mice were primed IM with 0.05 μg of mRNA-LNP and boosted IN with 10 μg of PACE-encapsulated mRNA (IN PACE-Spike) 14 days after IM priming. Six weeks after boosting, mice were treated with 6×10 4 Mice were challenged with PFU SARS-CoV-2 (2019n-CoV / USA_WA1 / 2020).

[0020] Figure 4EShown are weight loss and survival rates of naive K18-hACE2 mice, IM-primed K18-hACE2 mice, or primed and PACE-Spike K18-hACE2 mice from 1 DPI to 14 DPI.

[0021] Figure 5A An experiment is shown in which K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and then boosted 14 days after IM prime with 1 μg of mRNALNP IM or 5 μg of prefusion stabilized trimeric recombinant SARS-CoV-1 (SCV1) spike IN (IN SpikeX). At 31 days after boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurement.

[0022] Figure 5B Total tetramers in lung tissue of naive mice, mRNA-LNP prime / boost mice, or prime and spike-X mice are shown. + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification results of CD8 T cells.

[0023] Figure 5C The results of measurement of BALF IgA (E), BALF IgG (F), serum IgA (G), and serum IgG (H) specific for SCV1 spike S1 subunit in naive mice, mRNA-LNP prime / boost mice, or prime and spike X mice are shown. (IL) The results of measurement of BALF IgA (I), BALF IgG (J), serum IgA (K), and serum IgG (L) specific for SCV2 spike S1 subunit in naive mice, mRNA-LNP prime / boost mice, or prime and spike X mice. (M,N) The results of measurement of neutralization titers against SCV1 spike pseudotyped VSV. (O,P) The results of measurement of neutralization titers against SCV2 spike pseudotyped VSV.

[0024] Fig. 6A An experiment is shown in which K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and boosted IN with 1 μg of SCV2 spike 12 weeks after IM priming. Lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements 7 and 56 days after boosting.

[0025] Figure 6B(BD) Total tetramers in lung tissue of IM primed or primed and primed mice at 7 and 56 days after boost + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification of CD8 T cells. (EG) Total activated polyclonal CD4 T cells, CD69 T cells, and CD8 T cells in lung tissues of IM-primed mice or primed and primed mice at 7 and 56 days after boost. + CD103 - CD4 T cells or CD69 + CD103 + Quantification results of CD4 T cells.

[0026] Figure 6C Shown are the results of measurements of SCV2 spike S1 subunit-specific BALF IgA (H), BALF IgG (I), serum IgA (J), and serum IgG (K) in IM-primed or primed-and-primed mice at 7 and 56 days after boost.

[0027] Fig. 7A An experiment is shown in which K18-hACE2 mice were primed IM with 0.05 μg of mRNA-LNP and boosted IN with 1 μg of spike IN 14 days after IM prime. Six weeks after boost, lung tissue was collected for CD8 T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurements.

[0028] Figure 7B Total tetramers in lung tissue of naive mice, IM-primed mice, or primed and spiked mice are shown. + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification results of CD8 T cells.

[0029] Figure 7C Shown are the results of measurements of SCV2 spike S1 subunit-specific BALF IgA (E), BALF IgG (F), serum IgA (G), and serum IgG (H) in naive mice, IM-primed mice, or primed and primed mice.

[0030] Fig. 8AAn experiment is shown in which K18-hACE2 mice were primed IM with 1 μg of mRNA-LNP and then boosted 14 days after IM prime with 1 μg of mRNA-LNP IM or 1 μg of SCV2 spike IN (IN spike). At 45 days after boost, lung tissue was collected for T cell analysis by flow cytometry, and BALF and blood were collected for antibody measurement.

[0031] Figure 8B Total tetramers in lung tissue of naive mice, mRNA-LNP prime / boost mice, or prime and spike mice are shown + CD8 T cells, CD69 + CD103 - Tetramer + CD8 T cells or CD69 + CD103 + Tetramer + Quantification results of CD8 T cells.

[0032] Figure 8C Shown are the measurements of SARS-CoV-2 spike S1 subunit-specific BALF IgA (E), BALF IgG (F), serum IgA (G), and serum IgG (H) in naive mice, mRNA-LNP prime / boost mice, or primed and spiked mice. (I,J) Measurements of neutralization titers against SCV2 spike-pseudotyped VSV.

[0033] Fig. 9 Agarose gel electrophoresis is used to analyze the length and integrity of extracted mRNA. The extracted mRNA was mixed with SYBR Safe stain and then loaded onto a 1% agarose gel, run in TAE buffer, and imaged using a gel imaging system.

[0034] Figures 10A-10B It shows ( Fig. 10A ) Gating strategy for identifying extravascular antigen-specific CD8 T cells and polyclonal activated CD4 T cells. ( Fig. 10B ) Gating strategy for identifying extravascular antigen-specific and polyclonal B cell subsets. Summary of the invention

[0035] The present invention encompasses a method of enhancing an immune response to an antigen in a human in need thereof, the method comprising administering to a mucosal site of the subject an effective amount of a pharmaceutical composition comprising an antigen or a nucleic acid encoding the antigen, wherein the human has been previously vaccinated against a virus or has been infected with a virus. In some embodiments, the human has elevated antibodies, memory B cells, effector CD4 + and CD8 +In some embodiments, elevated antibodies, memory B cells, effector CD4 + and CD8 + The T cells are elicited by previous vaccination against the virus.

[0036] In some embodiments, elevated antibodies, memory B cells, effector CD4 + and CD8 + T cells are primed by a previous viral infection.

[0037] In some embodiments, the mucosal site is selected from the group consisting of rectal, vaginal, bladder, ocular, oral, sublingual, esophageal, nasal, gastrointestinal, pulmonary, and otic mucosal sites.

[0038] In some embodiments, the antigen comprises a protein or a polypeptide.

[0039] In some embodiments, the antigen is a multivalent antigen.

[0040] In some embodiments, the antigen comprises a nucleic acid encoding a protein or polypeptide.

[0041] In some embodiments, the nucleic acid is DNA or RNA.

[0042] In some embodiments, the nucleic acid is mRNA.

[0043] In some embodiments, the antigen is derived from a microbial pathogen.

[0044] In some embodiments, the microbial pathogen is a mycobacterium, a bacterium, a fungus, a virus, a parasite, or a prion.

[0045] In some embodiments, the virus is selected from the group consisting of rotavirus, norovirus, adenovirus, astrovirus, variants thereof, and any combination of these.

[0046] In some embodiments, the virus is selected from influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, variants thereof, and any combination of these.

[0047] In some embodiments, the virus is selected from the group consisting of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human papillomavirus (HPV), variants thereof, and any combination of these.

[0048] In some embodiments, the virus is selected from human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus, adenovirus, poliovirus, Japanese encephalitis virus, smallpox virus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, human papillomavirus (HPV), molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV-2, Henoch-Schönlein purpura (HSP) virus, RNA virus, DNA virus, variants thereof, and any combination of these.

[0049] In some embodiments, the RNA virus is selected from the group consisting of common cold virus, influenza virus, SARS virus, MERS virus, Covid-19 virus, dengue virus, hepatitis C virus, hepatitis E virus, West Nile virus, Ebola virus disease virus, rabies virus, polio virus, mumps virus, measles virus, variants thereof, and any combination thereof.

[0050] In some embodiments, the DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, roseola virus, human herpes virus-7, Kaposi's sarcoma-associated virus, variants thereof, and any combination of these.

[0051] In some embodiments, the pharmaceutical composition is administered by mucosal delivery.

[0052] In some embodiments, mucosal delivery is selected from rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, intranasal delivery, vaginal delivery, and oral delivery.

[0053] In some embodiments, the pharmaceutical composition is administered to a mucosal tissue of a human subject.

[0054] In some embodiments, the mucosal tissue is selected from the group consisting of anterior nares, sinuses, rectum, vagina, esophagus, urethra, sublingual, and buccal mucosal tissue.

[0055] In some embodiments, the pharmaceutical composition is administered orally, intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0056] In some embodiments, the pharmaceutical composition is administered by intranasal spray.

[0057] In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0058] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0059] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNPs).

[0060] In some embodiments, the antigen is encapsulated within lipid nanoparticles (LNPs).

[0061] In another aspect, the present invention relates to a method of enhancing an immune response to SARS-CoV-2 in a human in need thereof; the method comprising administering an effective amount of a pharmaceutical composition comprising at least one mRNA to a mucosal site of a subject, wherein the human has previously been vaccinated against SARS-CoV-2 or has been infected with SARS-CoV-2. In some embodiments, the mRNA encodes the spike protein of SARS-CoV-2 or a fragment thereof.

[0062] In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0063] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0064] In some embodiments, the pharmaceutical composition further comprises lipid nanoparticles (LNPs).

[0065] In some embodiments, the mRNA is encapsulated within lipid nanoparticles (LNPs).

[0066] In some embodiments, the lipid nanoparticle (LNP) comprises at least one cationic lipid.

[0067] In some embodiments, the at least one cationic lipid comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0068] In some embodiments, the lipid nanoparticle (LNP) further comprises at least one phospholipid.

[0069] In some embodiments, the at least one phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0070] In some embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 1000 nm.

[0071] In some embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 400 nm, about 50 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, or about 300 nm to about 600 nm.

[0072] In some embodiments, the immune response is a mucosal immune response.

[0073] In some embodiments, the mucosal immune response is antigen-specific IgA antibody production.

[0074] In some embodiments, the mucosal immune response is antigen-specific IgG antibody production.

[0075] In some embodiments, the human has elevated IgA antibodies.

[0076] In some embodiments, the human has elevated IgG antibodies. DETAILED DESCRIPTION definition

[0077] As used herein and in the appended claims, the articles "a" and "an" are used herein to refer to more than one of the grammatical objects of the article (ie, to at least one) unless the context clearly dictates otherwise. For example, "an element" means one element or more than one element.

[0078] The term "variant" refers to a polypeptide or nucleotide comprising a change (i.e., substitution, insertion and / or deletion) at more than one (e.g., several) positions. In some embodiments, the term "variant" refers to a SARS-CoV-2 virus variant.

[0079] The terms "spike," "boost," or "boosting agent" are used interchangeably.

[0080] As used herein, the term "immunogenic agent" encompasses any substance, composition of substances, or composition of organic material, such as a suspension of cells or cell components, which, when administered in an appropriate amount and mixed with an appropriate substance, can induce a significant immune response to a coronavirus in a human subject.

[0081] The term "immunologically equivalent" means that the polypeptide is functionally equivalent to a polypeptide having the amino acid sequence of any S protein with respect to the ability to elicit an immune response.

[0082] As used herein, the term "polypeptide" includes both short peptides of 2-10 amino acid residues in length, oligopeptides (11-100 amino acid residues) and longer peptides (generally understood as polypeptides, i.e., longer than 100 amino acid residues), and proteins (functional entities containing at least one peptide, oligopeptide or polypeptide, which may be chemically modified by glycosylation or conjugated to other chemical groups). The definition of polypeptide also includes polypeptides or proteins in natural form in SARS-CoV-2, recombinant proteins or peptides in any type of expression vector that transforms any type of host, and chemically synthesized peptides.

[0083] As used herein, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form, including DNA, RNA, and mixtures thereof. DNA can be in the form of an antisense molecule, plasmid DNA, cDNA, PCR product, or vector. RNA can be in the form of a small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA, Dicer substrate RNA, or viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to reference nucleic acids.

[0084] The present invention relates to a method for vaccinating a human subject with a virus, wherein the human subject has previously been systemically vaccinated with a vaccine against the virus or infected with the virus. The method comprises administering an effective amount of a pharmaceutical composition comprising an antigen to a mucosal site of the subject. This method is also referred to as "primary immunization and spike" or "primary immunization and boost". In some embodiments, the primary immunization and spike method utilizes an adjuvant-free intranasal spike boost, which utilizes the existing immunity generated by the initial systemic vaccination to induce mucosal immune memory in the respiratory tract. In addition, by using different spike proteins, the primary immunization and spike can induce cross-reactive immunity against sarbecovirus. In some embodiments, the primary immunization and spike can produce a multivalent response against sarbecovirus (e.g., mERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof).

[0085] In one aspect, the invention encompasses a method of enhancing an immune response to an antigen in a human in need thereof, the method comprising administering to a mucosal site of the subject an effective amount of a pharmaceutical composition comprising an antigen or a nucleic acid encoding the antigen, wherein the human has been previously vaccinated against a virus or has been infected with a virus. In some embodiments, the human has elevated antibodies, memory B cells, effector CD4 + and CD8 +In some embodiments, elevated antibodies, memory B cells, effector CD4 + and CD8 + The T cells are elicited by a previous vaccination against the virus. In some embodiments, the previous vaccination was performed by parenteral administration.

[0086] In some embodiments, elevated antibodies, memory B cells, effector CD4 + and CD8 + T cells are induced by a previous viral infection. In some embodiments, the elevated antibodies are immunoglobulin G (IgG), IgM and IgA.

[0087] In some embodiments, the mucosal site is selected from the group consisting of rectal, vaginal, bladder, ocular, oral, sublingual, esophageal, nasal, gastrointestinal, pulmonary, and otic mucosal sites.

[0088] In some embodiments, the antigen comprises a protein or a polypeptide.

[0089] In some embodiments, the antigen comprises at least one nucleic acid encoding a protein or polypeptide.

[0090] In some embodiments, the nucleic acid is DNA or RNA.

[0091] In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is mRNA modified with N1-methyl-pseudouridine. In some embodiments, the mRNA is mRNA modified with pseudouridine. In some embodiments, the antigen comprises two or more different mRNAs. The two or more mRNAs encode two or more different proteins to induce a multivalent reaction.

[0092] In some embodiments, the antigen is derived from a microbial pathogen.

[0093] In some embodiments, the microbial pathogen is a mycobacterium, a bacterium, a fungus, a virus, a parasite, or a prion.

[0094] In some embodiments, the virus is selected from the group consisting of rotavirus, norovirus, adenovirus, astrovirus, variants thereof, and any combination of these.

[0095] In some embodiments, the virus is selected from influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, variants thereof, and any combination of these.

[0096] In some embodiments, the virus is selected from the group consisting of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human papillomavirus (HPV), variants thereof, and any combination of these.

[0097] In some embodiments, the virus is selected from human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus, adenovirus, poliovirus, Japanese encephalitis virus, smallpox virus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, human papillomavirus (HPV), molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV-2, Henoch-Schönlein purpura (HSP) virus, RNA virus, DNA virus, variants thereof, and any combination of these.

[0098] In some embodiments, the RNA virus is selected from the group consisting of common cold virus, influenza virus, SARS virus, MERS virus, Covid-19 virus, dengue virus, hepatitis C virus, hepatitis E virus, West Nile virus, Ebola virus disease virus, rabies virus, polio virus, mumps virus, measles virus, variants thereof, and any combination thereof.

[0099] In some embodiments, the DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, roseola virus, human herpes virus-7, Kaposi's sarcoma-associated virus, variants thereof, and any combination of these.

[0100] In some embodiments, the pharmaceutical composition is administered by mucosal delivery.

[0101] In some embodiments, mucosal delivery is selected from rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, intranasal delivery, vaginal delivery, and oral delivery.

[0102] In some embodiments, the pharmaceutical composition is administered to a mucosal tissue of a human subject.

[0103] In some embodiments, the mucosal tissue is selected from the group consisting of anterior nares, sinuses, rectum, vagina, esophagus, urethra, sublingual, and buccal mucosal tissue.

[0104] In some embodiments, the pharmaceutical composition is administered orally, intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0105] In some embodiments, the pharmaceutical composition is administered by intranasal spray.

[0106] In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0107] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0108] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNP). In some embodiments, lipid nanoparticles (LNP) comprise poly(amine-to-ester) (PACE) polymers. In some embodiments, PACE polymers are described in U.S. Patent Nos. 10,682,422, 10,465,042, 9,272,043, 9,895,451, PCT / US2012 / 067447, and U.S. Patent Publication No. US20200399424, which are incorporated herein by reference in their entirety.

[0109] In some embodiments, the antigen is encapsulated within lipid nanoparticles (LNPs).

[0110] In some embodiments, the human was vaccinated against the virus or was infected with the virus about a week ago, two weeks ago, three weeks ago, one month ago, two months ago, three months ago, four months ago, five months ago, six months ago, seven months ago, eight months ago, nine months ago, ten months ago, eleven months ago, or twelve months ago.

[0111] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is selected from one or more RNAs of small RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), micro RNA (miRNA), Dicer substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA) and self-amplifying mRNA (SAM). In some embodiments, the nucleic acid is DNA. In some embodiments, once administered to a human subject, the nucleic acid will eventually be translated into a protein, wherein the protein will affect therapeutic function or vaccination.

[0112] In some embodiments, the nanoparticle comprises one or more compounds described in U.S. Patent Nos. 10,106,490, 10,723,692, 9,737,619, 9,738,593, and WO2015199952A1, which are incorporated herein by reference in their entireties.

[0113] In some embodiments, the nanoparticles comprise one or more compounds described in U.S. Pat. Nos. 10,682,422, 10,465,042, 9,272,043, 9,895,451, PCT / US2012 / 067447, and U.S. Patent Publication No. US20200399424, which are incorporated herein by reference in their entireties.

[0114] In some embodiments, the particles have an average particle size of about 100 nm to about 300 nm, preferably about 150 nm to about 275 nm. In some embodiments, the weight:weight ratio of polymer:polypeptide is about 25:1 to 250:1.

[0115] Currently approved SARS-CoV-2 mRNA-LNP-based and vector-based vaccines rely on intramuscular administration, which induces high levels of circulating antibodies, memory B cells, and circulating effector CD4 + and CD8 + However, parenteral vaccines do not induce high levels of potent antiviral immune memory, such as tissue-resident memory T cells (T RM ) and B cells (B RM ) and mucosal IgG and dimeric IgA. This is consistent with humans and mice (where CD8 + T RM Vaccines targeting the respiratory mucosa could address the shortcomings of parenteral vaccination, as recent preclinical evaluations of intranasally delivered adenoviral vectors encoding the SARS-CoV-2 spike showed impressive mucosal immunogenicity as well as protection and reduced viral shedding in mice, hamsters, and nonhuman primates. Preclinical mucosal influenza vaccine studies have also shown that mucosal immunity can be enhanced through CD8 + T RM or dimeric IgA to enhance protection against heterosubtypic attacks and improve the durability of immunity.

[0116] Although initial respiratory administration of the vaccine induces a potent mucosal immune response, a systemic prime followed by an intranasal boost will result in similar systemic immunity with the additional benefit of enhanced mucosal immunity.

[0117] Recent studies have shown that the effectiveness of FDA-approved mRNA vaccines against COVID-19 decreases in asymptomatic infection and in symptomatic and severe infection starting approximately 4 months after the second dose. In the context of immune waning with this parenteral vaccination regimen, the present disclosure describes a method for enhancing immunity against COVID-19: using a potent systemic priming with an mRNA-LNP-based vaccine followed by an intranasal (IN) boost with either unadjuvanted spike protein or immunologically silencing complexes encapsulating mRNA.

[0118] To evaluate the potential of IN non-adjuvanted subunit vaccine boosting for the development of respiratory mucosal immunity, K18-hACE2 mice were vaccinated with 1 μg of mRNA-LNP (Comirnaty) by IM injection (prime), and then 14 days later were vaccinated with 1 μg of recombinant non-adjuvanted spike protein by IN administration (prime and spike). Other control groups included: K18-hACE2 mice that received IM prime only, and mice that received IN spike only at the time of boost. Mice were euthanized on day 21 or 28 (7 or 14 days after boost), and the development of mucosal humoral immunity was evaluated ( Figure 1A ). Anti-SARS-CoV-2 Spike S1 IgG and IgA were assessed in nasal washes, bronchoalveolar lavage fluid (BALF), and serum. Only mice that received prime and spike produced high levels of anti-SARS-CoV-2 IgA and IgG in nasal washes and BALF ( Figure 1B (BE)). Neither IM priming alone nor IN spike alone was sufficient to generate mucosal antibodies. In serum, IM priming alone was sufficient to induce low levels of IgA and IgG; however, priming plus spike resulted in a significant systemic boost in anti-Spike S1 IgA and IgG ( Figure 1B (F,G)). The increase in antibody levels was associated with an increase in neutralizing titers in BALF and serum ( Figure 1B (HK)). These results suggest that a single dose of unadjuvanted intranasal spike is not immunogenic when used alone and that the potent mucosal and systemic antibody responses induced by the unadjuvanted spike require prior systemic priming, in this case with mRNA-LNPs.

[0119] Using intravenous (IV) CD45 labeling combined with B cell tetramers specific for the receptor binding domain (RBD) of the spike protein, we found that priming and spiking resulted in increased antigen-specific B cells (IV - CD19 + B220 + Tetramer + )( Figure 1D (L)). Given that tetramers only assess RBD binding, polyclonal tissue responses were also investigated, which may represent a more complete set of B cells within lung tissue that respond to the entire spike. Increased class-switched antibody-secreting cells (ASCs) were found in lung tissue expressing IgA or IgG (IV - CD19 + / - CD138 + )( Figure 1D (M,N)), and increased expression of IgA or IgG class switching B was found RM (IV - CD19 + B220+ IgD - IgM - CD38 + )( Figure 1D (O, P)). These results are consistent with increased mucosal antibody production, indicating that priming and spiking elicit local B cell responses in the lungs.

[0120] Similar to the above, the CD45 IV marker used to distinguish circulating cells from immune cells in lung tissue binds to the major histocompatibility complex (MHC) class I tetramer on the conserved Sarcoma coronavirus spike epitope (VNFNFNGL). Figure 2B (BD)) Inner and lower airway BALF ( Figure 2B (EG)) and upper airway turbinate ( Figure 2C (HJ))Medium Spike IV - Tetramer + CD8 + Significant induction of T cells, which express T RM Typical markers of + and CD103 + In addition, in lung tissue ( Figure 2C (KM)) and the lower airway recovered from BALF ( Figure 2C (NP)) was found to have significantly increased antigen-experienced CD4 + T cells (IV - CD44 + CD4 + ), many of which also express T RM CD69 + and CD103 + These results suggest that priming and spiking not only induce humoral mucosal responses but also strongly induce lung parenchymal and airway CD8 + T RM and CD4 + T RM .

[0121] After 84 days, K18-hACE2 mice that had received a 1 μg IM prime were boosted with IN spikes. We collected samples of mucosal humoral and cellular immune responses on days 91 (7 days after boost) and 140 (56 days after boost). We found that delayed IN spikes were sufficient to induce CD8 + T RM , and lasted for at least 56 days. At 7 days after the boost, early induction of CD4 + T RM ; however, their lifespan appeared to be reduced by 56 days, at least in polyclonal populations. +T RM The responses were similar, with not only adequate humoral responses to delayed boosts, but also robust and increased mucosal IgA and IgG in BALF, and robust and increased serum IgA and IgG 56 days after boost. These results suggest that priming and spiking, given at a 3-month interval between doses, is sufficient to elicit persistent mucosal and systemic humoral and cellular immune responses.

[0122] In another aspect, the present invention relates to a method of enhancing an immune response to SARS-CoV-2 in a human in need thereof; the method comprising administering an effective amount of a pharmaceutical composition comprising at least one mRNA to a mucosal site of a subject, wherein the human has previously been vaccinated against SARS-CoV-2 or has been infected with SARS-CoV-2. In some embodiments, the mRNA encodes the spike protein of SARS-CoV-2 or a fragment thereof.

[0123] In some embodiments, the human has previously been vaccinated with one or more COVID-19 vaccines selected from the group consisting of: BNT162b2 (Pfizer / BioNTech), mRNA-1273 (Moderna), AZD1222 / ChAdOxl (AstraZeneca / Oxford University), Ad5 vectored COVID-19 vaccine (CanSino Biologies), CoronaVac (Sinovac), NVX-CoV2373 (Novavax), and combinations thereof.

[0124] In some embodiments, the human has elevated IgG antibodies resulting from prior vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0125] In some embodiments, the human has elevated IgM antibodies resulting from prior vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0126] In some embodiments, the human has elevated IgA antibodies resulting from prior vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0127] In some embodiments, the human has elevated IgG antibodies resulting from prior infection with MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0128] In some embodiments, the human has elevated IgM antibodies resulting from prior infection with MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0129] In some embodiments, the human has elevated IgA antibodies resulting from prior infection with MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0130] In some embodiments, the elevated IgG is in the range of about 100 to 150, about 100 to 200, about 100 to 300, about 100 to 400, about 150 to 200, about 150 to 250, about 150 to 300, about 150 to 400, about 200 to 250, about 200 to 300, about 200 to 350, or about 200 to 400 BAU / ml.

[0131] In some embodiments, the elevated IgG is about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 280, 290, 295, or 300 BAU / ml.

[0132] In some embodiments, the elevated IgM is in the range of about 25 to 100, about 25 to 150, about 25 to 200, about 25 to 300, about 50 to 100, about 50 to 150, about 50 to 200, about 50 to 300, about 75 to 100, about 75 to 150, about 75 to 200, about 75 to 300, about 100 to 150, about 100 to 200, about 100 to 300, about 125 to 200, about 125 to 300, about 150 to 200, about 150 to 300, about 200 to 300, about 250 to 300 AU / ml.

[0133] In some embodiments, the elevated IgM is about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 180, 190, 195, or 200 AU / ml.

[0134] In some embodiments, the elevated IgA is in the range of about 10 to 100, about 10 to 150, about 10 to 200, about 25 to 100, about 25 to 150, about 25 to 200, about 50 to 100, about 50 to 150, about 50 to 200, about 75 to 100, about 75 to 150, about 75 to 200, about 100 to 150, about 100 to 200, about 125 to 150, about 125 to 200, about 150 to 200 AU / ml.

[0135] In some embodiments, the elevated IgA is about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 AU / ml.

[0136] In some embodiments, at least one mRNA encodes the spike protein of SARS-CoV-2, a variant thereof, or a fragment thereof. In some embodiments, at least one mRNA is a multivalent antigen. In some embodiments, the pharmaceutical composition comprises two or more different mRNAs. Two or more mRNAs encode two or more different proteins to induce a multivalent response against SARS-CoV-2. In some embodiments, the mRNA is an mRNA modified with N1-methyl-pseudouridine. In some embodiments, the mRNA is an mRNA modified with pseudouridine.

[0137] In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0138] In some embodiments, the pharmaceutical composition comprises an adjuvant.

[0139] In some embodiments, the pharmaceutical composition further comprises a lipid nanoparticle (LNP).In some embodiments, the lipid nanoparticle (LNP) comprises a poly(amine-co-ester) (PACE) polymer.

[0140] In some embodiments, at least one mRNA is encapsulated within a lipid nanoparticle (LNP).

[0141] In some embodiments, the lipid nanoparticle (LNP) comprises at least one cationic lipid.

[0142] In some embodiments, the at least one cationic lipid comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0143] In some embodiments, the lipid nanoparticle (LNP) further comprises at least one phospholipid.

[0144] In some embodiments, the at least one phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0145] In some embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 1000 nm.

[0146] In some embodiments, the average diameter of the lipid nanoparticles is in the range of about 50 nm to about 400 nm, about 50 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, or about 300 nm to about 600 nm.

[0147] In some embodiments, the immune response is a mucosal immune response.

[0148] In some embodiments, the mucosal immune response is antigen-specific IgA antibody production.

[0149] In some embodiments, the human was vaccinated against the virus or was infected with the virus about a week ago, two weeks ago, three weeks ago, one month ago, two months ago, three months ago, four months ago, five months ago, six months ago, seven months ago, eight months ago, nine months ago, ten months ago, eleven months ago, or twelve months ago.

[0150] In some embodiments, the pharmaceutical compositions described herein comprise polypeptides as antigens for vaccinating human subjects against SARS-CoV-2 and its immunogenic variants. In some embodiments, the polypeptide is a coronavirus spike (S) protein, an immunogenic variant thereof, or an antigenic fragment thereof. In some embodiments, the polypeptide has an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any known S protein, its subunit or fragment. In some embodiments, the polypeptide has an amino acid sequence having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any known S protein, its subunit or fragment. In some embodiments, the variant is a SARS-CoV-2 spike protein variant found in different SARS-CoV-2 strains. Variants include, but are not limited to, spike proteins from α, β, or δ variants of SARS-CoV-2, B.1.1.7 strain, B.1.351 strain, P.1 strain, CAL20 strain, or any combination thereof.

[0151] In some embodiments, SARS-CoV-2 variants include but are not limited to α (Alpha) (B.1.1.7 and Q lineages), β (Beta) (B.1.351 and descendant lineages), γ (Gamma) (P.1 and descendant lineages), δ (Delta) (B.1.617.2 and AY lineages), ε (Epsilon) (B.1.427 and B.1.429), η (Eta) (B.1.525), ι (I ota)(B.1.526), ​​κ(Kappa)(B.1.617.1), 1.617.3, μ(Mu)(B.1.621, B.1.621.1), ζ(Zeta)(P.2), μ (Mu)(B.1.621, B.1.621.1), ο(Omicron)(Pango pedigree B.1.1.529, BA.1, BA.1.1, BA.2, BA.3) and their combinations.

[0152] The DNA sequence encoding the spike protein of SARS-CoV-2 isolated in Wuhan is SEQ ID NO: 1.

[0153] Table 1 lists exemplary variants of the spike protein from different strains.

[0154] Table 1: List of amino acid positions and relative amino acid changes of different variants in the spike protein relative to the spike protein of the strain isolated in Wuhan (SEQ ID NO: 2).

[0155] In some embodiments, the polypeptide comprises other amino acid sequences of strains of SARS-CoV-2 S protein, including any sequence disclosed in Deng (2020) Science, 8:eabb9263 and Taboada (2020) J. Virol. 94:e01056. However, it is highly likely that other SARS-CoV-2 strains will exhibit immunological properties substantially the same as the α variant S protein, its fragments and its subunits from such strains. In some embodiments, the polypeptide is selected from the M protein, E protein, N protein or variants thereof and combinations thereof of SARS-CoV-2.

[0156] In some embodiments, the S protein variants described herein include mutations at positions corresponding to position 501N in the α variant. In some embodiments, the amino acid corresponding to position 501N is replaced by Y. In some embodiments, the S protein variants described herein that include mutations at positions corresponding to position 501N may include one or more further mutations. Such one or more further mutations may be selected from one or more of the mutations corresponding to the following amino acid positions (at positions relative to the α variant): 18L, 69H, 70V, 80D, 144Y, 215D, 246R, 242L, 243A and 244L, 417K, 484E, 570A, 614D, 681P, 701A, 716T, 982S and 1118D. In some embodiments, the amino acid corresponding to position 69H in the α variant is deleted. In some embodiments, the amino acid corresponding to position 70V is deleted. In some embodiments, the amino acid corresponding to position 144Y is deleted. In some embodiments, the amino acid corresponding to position 570A is D. In some embodiments, the amino acid corresponding to position 614D is G. In some embodiments, the amino acid corresponding to position 681P is H. In some embodiments, the amino acid corresponding to position 716T is I. In some embodiments, the amino acid corresponding to position 982S is A. In some embodiments, the amino acid corresponding to position 1118D is H. In some embodiments, the amino acid corresponding to position 80D is A. In some embodiments, the amino acid corresponding to position 215D is G. In some embodiments, the amino acid corresponding to position 484E is K. In some embodiments, the amino acid corresponding to position 701A is V. In some embodiments, the amino acid corresponding to position 18L is F. In some embodiments, the amino acid corresponding to position 246R is I. In some embodiments, the amino acid corresponding to position 417K is N. In some embodiments, the amino acid corresponding to position 242L is deleted. In some embodiments, the amino acid corresponding to position 243A is deleted. In some embodiments, the amino acid corresponding to position 244L is deleted.

[0157] In some embodiments, the S protein variant described herein is the S protein of SARS-CoV-2δ. In some embodiments, relative to the α variant, the S protein of SARS-CoV-2δ has the following spike protein substitutions: T19R, V70F, T95I, G142D, 156E deletion, 157F deletion, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N.

[0158] In some embodiments, the S protein variant described herein is the S protein of SARS-CoV-2ο. In some embodiments, relative to the alpha variant, the S protein of SARS-CoV-2o has the following spike protein substitutions: A67V, deletion of amino acids 69-70, T95I, deletion of amino acids 142-144, Y145D, deletion of amino acid 211, L212I, insertion of amino acid EPE at position 214, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F.

[0159] In some embodiments, administration of the pharmaceutical compositions described herein can be performed via a single administration, or boosted via multiple administrations.

[0160] In some embodiments, the pharmaceutical compositions described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally or intramuscularly. In some embodiments, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration can include enteral administration or parenteral administration, and enteral administration involves absorption through the gastrointestinal tract. As used herein, "parenteral administration" refers to administration in any manner other than through the gastrointestinal tract, such as by intravenous injection.

[0161] In some embodiments, the pharmaceutical compositions described herein can be administered intranasally.

[0162] In some embodiments, the amount of the polypeptide described herein can be applied per dose of 0.1 μg to 300 μg, 0.5 μg to 200 μg or 1 μg to 100 μg, such as about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg or about 100 μg. In some embodiments, the present invention contemplates the use of a single dose. In some embodiments, the present invention contemplates the use of a primary immunization dose, followed by one or more booster doses. A booster dose or the first booster dose can be applied about one week, about two weeks, about three weeks, about four weeks or about five weeks after the primary immunization dose is applied. In some embodiments, a booster dose or the first booster dose can be applied about one month, about two months, about three months, about four months or about five months, about six months, about seven months, about eight months, about nine months, about ten months, about eleven months or about twelve months after the primary immunization dose is applied.

[0163] In some embodiments, the amount of a polypeptide described herein may be administered at 60 μg or less, 50 μg or less, 40 μg or less, 30 μg or less, 20 μg or less, 10 μg or less, 5 μg or less, 2.5 μg or less, or 1 μg or less per dose.

[0164] In some embodiments, the amount of a polypeptide described herein can be administered at least 0.25 μg, at least 0.5 μg, at least 1 μg, at least 2 μg, at least 3 μg, at least 4 μg, at least 5 μg, at least 10 μg, at least 20 μg, at least 30 μg, or at least 40 μg per dose.

[0165] In some embodiments, the amount of a polypeptide described herein may be administered at 0.25 μg to 60 μg, 0.5 μg to 55 μg, 1 μg to 50 μg, 5 μg to 40 μg, or 10 μg to 30 μg per dose.

[0166] Pharmaceutical compositions and products described herein can be provided as frozen concentrates of injection solutions, such as frozen concentrates with a concentration of 0.50 mg / mL. In some embodiments, in order to prepare injection solutions, the drug product is thawed and diluted with isotonic sodium chloride solution (e.g., 0.9% NaCl, saline), such as by a one-step dilution process. In some embodiments, bacteriostatic sodium chloride solutions (e.g., 0.9% NaCl, saline) cannot be used as diluents. In some embodiments, the diluted drug product is an off-white suspension. The concentration of the final injection solution depends on the corresponding dosage level to be administered.

[0167] The present invention also encompasses a kit comprising the pharmaceutical compositions and modes of administration described herein. In some embodiments, the kit comprises a nasal spray device for intranasal administration. Nasal spray devices are well known in the art and are described in Djupesland, Drug Deliv. Transl. Res. (2013) 3(1):42–62, which is incorporated herein by reference in its entirety. The kit can facilitate self-administration for vaccination against SARS-CoV-2 or its variants.

[0168] In the kit of the present invention, the pharmaceutical composition comprises 0.5 to 75 μg of the polypeptide, such as 0.5 to 50 μg of the polypeptide, or 5 to 50 μg of the polypeptide. Example

[0169] Having generally described the present teachings, it will be more readily understood by reference to the following examples, which are provided for the purpose of illustrating certain aspects and embodiments of the disclosure. Methods and Materials:

[0170] All procedures were performed in a BSL-3 facility (for SARS-CoV-2-infected mice) and were approved by the Yale Institutional Animal Care and Use Committee and the Yale Environmental Health and Safety Department. Cells and viruses

[0171] Vero E6 cells overexpressing hACE2 and TMPRSS2 (kindly provided by Barney Graham NIH-VRC) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% sodium pyruvate and 5% fetal bovine serum (FBS) at 37°C and 5% CO2. SARS-CoV-2 isolate hCOV-19 / USA-WA1 / 2020 (NR-52281) was obtained from BEI Resources and amplified in Vero E6 cells overexpressing hACE2 and TMPRSS2. Cells were infected at an MOI of 0.01 for two to three days to produce working stocks, and the supernatant was clarified by centrifugation (500g×5 minutes) after incubation, filtered through a 0.45μm filter and stored at -80°C. Virus titers were measured by standard plaque assay using Vero E6 cells overexpressing hACE2 and TMPRSS2. Mouse

[0172] B6.Cg-Tg(K18-ACE2)2Prlmn / J(K18-hACE2) mice were purchased from Jackson Laboratory and then bred and maintained at Yale University. Female mice aged eight to twelve weeks were used for immunization experiments. All procedures used in this study (sex-matched, age-matched) were in accordance with federal guidelines and the institutional policies of the Yale School of Medicine Animal Care and Use Committee. Example 1: SARS-CoV-2 infection

[0173] Mice were anesthetized using 30% v / v isoflurane diluted in propylene glycol. Using a pipette, 6x10 5 50 μL intranasal delivery of PFU SARS-CoV-2. Example 2: Extraction of mRNA from Fubitai mRNA-LNP

[0174] mRNA was extracted from the vaccine preparation using the TRIzol / chloroform separation method described herein. In short, an aliquot of the vaccine was dissolved in TRIzol LS (Thermo Fisher Scientific) at a volume ratio of 1:6.6 vaccine to TRIzol. After incubation for 15 minutes (37°C, shaking), 0.2 mL of chloroform was added to each 1 mL of TRIzol. The solution was shaken vigorously for 1 minute and then incubated at room temperature for 3 minutes. The solution was centrifuged at 12,000 x g for 8 minutes at 4°C. The aqueous layer containing the isolated mRNA was further purified according to the manufacturer's instructions using the RNeasy Maxi kit purchased from Qiagen (Germantown, Maryland, USA). In the final step, RNA was eluted from the column with sodium acetate buffer (25 mM, pH 5.8) heated to 37°C. The concentration and purity of the extracted mRNA were analyzed by NanoDrop measuring absorbance at 260, 280, and 230 nm, and purity was assessed as A260 / A280>2 and A260 / A230>2. Agarose gel electrophoresis was used to determine the length and verify whether the mRNA remained intact. The extracted mRNA was loaded onto a 1% agarose gel containing 1:100 SYBR Safe stain (Thermo Fisher Scientific) and run at 75V in TAE buffer containing 1:5000 SYBR Safe stain. Example 3: Formulation and characterization of PACE complex

[0175] The synthesis and characterization of PACE polymers were as described previously. All complexes were prepared at a weight ratio of 50:1 polymer to mRNA. PACE polymers were dissolved in DMSO at a concentration of 100 mg / mL overnight (37°C, shaking). Prior to preparing the complexes, the optimal PACE polymer mixture was prepared by mixing PACE polymer solutions containing end group modifications and polyethylene glycol tails. mRNA and polymer were diluted into equal volumes of sodium acetate buffer (25 mM, pH 5.8). The polymer dilution was then vortexed for 15 seconds, mixed with the mRNA dilution, and vortexed for another 25 seconds. The complexes were incubated at room temperature for 10 minutes before use. Example 4: Vaccination

[0176] The vials of the Fifty-Nil vaccine used were obtained from Yale Health Pharmacy within 24 hours of opening and stored at 4°C. The vials contained residual vaccine (diluted to 100 μg / mL according to the manufacturer's instructions), which was removed and pooled with a spinal syringe. The pooled residual vaccine was aliquoted and stored at -80°C. Mice were anesthetized by intraperitoneal injection using a mixture of ketamine (50 mg / kg) and xylazine (5 mg / kg). The vaccine was diluted in sterile PBS, and 10 μL or 20 μL was injected into the left quadriceps muscle with a 31 g syringe, with a final dose of 1 μg or 0.05 μg (as indicated). For intranasal vaccination with SARS-CoV-2 stable spike protein (ACRObiosystems, SPN-C52H9) or SARS-CoV-1 spike protein (ACRO biosystems, SPN-S52H6), reconstituted in sterile endotoxin-free water according to the manufacturer's instructions, then diluted in sterile PBS and stored at -80°C. Mice were anesthetized using 30% v / v isoflurane diluted in propylene glycol and 1 μg or 5 μg (as indicated) were administered in 50 μL by IN route. For IN mRNA-PACE, 50 μL of the complexes in solution were administered at the indicated doses. Example 5: Virus titer analysis

[0177] At the indicated time points, mice were euthanized in 100% isoflurane. Approximately 50% of the total lungs were placed in a microbead homogenizer tube with 1 mL of PBS (containing 2% FBS and 2% antibiotics / antimycotics (Gibco)) and stored at -80°C. Debris in the lung homogenate was removed by centrifugation (3900 rpm, 10 minutes). The infection titer of SARS-CoV-2 was determined by plaque assay in Vero E6 cells overexpressing hACE2 and TMPRSS2 in DMEM supplemented with NaHCO3, 2% FBS and 0.6% Avicel RC-581. 40-42 hours after infection, the plaques were resolved for 30 minutes by fixing in 10% neutral buffered formalin for 1 hour and then staining in 0.5% crystal violet in 20% ethanol for 1 hour. The plate was rinsed with water to make the plaques visible. Example 6: SARS-CoV-2 specific antibody measurement

[0178] ELISA was performed as previously described, but modifications are noted and reproduced here for convenience. 96-well MaxiSorp plates (Thermo Scientific #442404) were coated with 50 μL / well of recombinant SARS-CoV-2 S1 protein (ACRO Biosystems S1NC52H3) or SARS-CoV-1 S1 protein (ACRO Biosystems S1N-S52H5) at a concentration of 2 μg / mL (in PBS) and incubated overnight at 4°C. The coating buffer was removed and the plates were incubated with 250 μL of blocking solution (PBS with 0.1% Tween-20, 5% milk powder) for 1 hour at room temperature. Serum or bronchoalveolar lavage fluid (BALF) was diluted in diluent solution (PBS with 0.1% Tween-20 and 2% milk powder), 100 μL of diluted serum or BALF was added, and incubated for 2 hours at room temperature. Using an automated plate washer, wash the plate five times (250 μL each time) with PBS-T (PBS containing 0.05% Tween-20), and add 50 μL of HRP anti-mouse IgG (Cell Signaling Technology #7076, 1:3,000) or HRP anti-mouse IgA (Southern Biotech #1040-05, 1:1,000) diluted in diluent solution to each well. After incubation for 1 hour at room temperature, wash the plate three times with PBS-T in an automated plate washer. Develop the plate with 50 μL of TMB substrate reagent kit (BD Biosciences #555214), and after 15 minutes, stop the reaction by adding 50 μL of 2N sulfuric acid. The plate is then read at wavelengths of 450 nm and 570 nm, and the difference is reported. Example 7: Immunohistochemistry and pathological analysis

[0179] Yale Pathology embedded, sectioned, and H&E stained lung tissues. A pulmonary pathologist blindly reviewed the slides and determined immune cell infiltration and other relevant pathology. Scores of 1–4 were as follows: (1) mild patchy mononuclear infiltration, parenchymal and perivascular, with alveolar cells and stromal reactions of varying reactivity; (2) moderate patchy mononuclear infiltration, parenchymal and perivascular, with alveolar cells and stromal reactions of varying reactivity; (3) mild, dense mixed infiltration, including monocytes and granulocytes / neutrophils; (4) moderate, dense mixed infiltration, including monocytes and granulocytes / neutrophils. Example 8: Intravascular labeling, cell isolation and flow cytometry

[0180] To distinguish intravascular and extravascular cells, mice were anesthetized with 30% isoflurane and injected intravenously with APC / Fire750CD45 Ab (30-F11, AB_2572116, BioLegend, #103154). After 3 minutes of labeling, mice were euthanized. The lungs were minced with scissors and incubated in a digestion mixture containing 1 mg / mL collagenase A (Roche) and 30 μg / mL DNase I (Sigma-Aldrich) in RPMI for 45 minutes at 37°C. The tissue was then filtered through a 70 μm filter. The cells were treated with potassium ammonium chloride buffer and resuspended in PBS containing 1% BSA. The single cell suspension was incubated with FC blocking agent and Aqua cell viability dye for 20 minutes at 4°C. The cells were washed once with PBS before surface staining. For T cell analysis, cells were incubated at 4 °C with anti-CD103 (BV421, 2E7, AB_2562901, BioLegend #121422), anti-CD3 (BV605, 17A2, AB_2562039, BioLegend #100237), anti-CD44 (BV711, IM7, AB_2564214, BioLegend #103057), anti-CD62L (FITC, mEL-14, AB_313093, BioLegend #104406), anti-CD8a ((PerC Cells were stained with anti-CD4 (AF700, GK1.5, AB_493699, BioLegend#100430) and PESARS-CoV-2S 539-546 MHC class I tetramer (H-2K(b)) for 30 min.For B cell analysis, cells were incubated at 4 °C with ant-GL7 (Pacific Blue, GL7, AB_2563292, BioLegend #144614), anti-IgM (BV605, RMM-1, AB_2563358, BioLegend #406523), anti-CD138 (BV711, 281-2, AB_2562571, BioLegend #142519), anti-CD19 (BV785, 6D5, AB_11218994, BioLegend #115543), anti-IgA (FITC, polyclonal, AB_2794370, Southern Biology, Biotechnology, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Bioengineering, Cells were stained with anti-SARS-CoV-2RBD tetramer, anti-CD38 (PE / Cy7,90,AB_2275531,BioLegend#102718), APC-SARS-CoV-2RBD tetramer and anti-IgD (AF700,11-26c.2a,AB_2563341,BioLegend#405730) for 30 minutes. After washing with PBS, cells were fixed with 4% paraformaldehyde. Cell population data were obtained on an Attune NxT flow cytometer and analyzed using FlowJo software (10.5.3; Tree Star). Example 9: Generation of SARS-CoV-2 receptor binding domain B cell tetramers

[0181] Recombinant SARS-CoV-2 spike RBD His biotin protein CF (R&D / BT10500-050) was incubated with streptavidin-PE (Prozyme PJRS25) or streptavidin-APC (Prozyme PJ27S) at a molar ratio of 4:1 for 30 minutes at 4°C. The mixture was then purified, concentrated in an Amicon Ultra (50kDA MWCO) spin column, and washed once with sterile cold PBS. The concentration was determined on a Nanodrop and using fluorophore-specific absorbance, the tetramer was diluted to 1.0 μM in PBS and stored at 4°C. Example 10: Production of pseudoviruses

[0182] VSV-based pseudotyped viruses were produced as described previously. The vector pCAGGS containing the SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein gene was produced under HHSN272201400008C and obtained through BEI Resources (NR-52310). The sequence of the Wuhan-Hu-1 isolate spike glycoprotein was identical to that of the USA-WA1 / 2020 isolate. The SARS-CoV-1 spike encoding plasmid was kindly provided by Dr. Vincent Munster (as described previously). 293T cells were transfected with either spike plasmid and then inoculated with replication-defective VSV expressing Renilla luciferase for 1 h at 37°C. The viral inoculum was then removed and the cells were washed three times with warm PBS. At 24 and 48 h after inoculation, the supernatant containing the pseudovirus was collected, clarified by centrifugation, concentrated using an Amicon Ultra centrifugal filter unit (100 kDa), and stored in aliquots at −80°C. Pseudovirus was titrated in Huh7.5 cells to give a relative light unit signal of -600 times the background of the cell-only control. Example 11: Pseudovirus Neutralization Test

[0183] One day before infection, Vero E6 cells overexpressing hACE2 and TMPRSS2 ( Figures 1A-1D ) or Huh7.5 cells ( Figures 5A-5C ) were inoculated into each well of a 96-well plate (3×10 4 On the day of infection, serum and BALF were heat-inactivated at 56°C for 30 min. Figures 1A-1D Serum was tested at a starting dilution of 1:50 and BALF samples were tested at a starting dilution of 1:4, both with eight two-fold serial dilutions. Figures 5A-5C Sera were tested at a starting dilution of 1:40 and eight three-fold serial dilutions were performed. Serial dilutions were mixed with pseudovirus at 1:1 and incubated at 37°C for 1 hour. The growth medium was then aspirated from the cells and replaced with 100 μL of the serum / virus mixture. At 24 hours, the infection medium was removed and the plates were snap frozen at -80°C. 30 μg of passive lysis buffer (Promega) was added to each well and the plates were incubated at room temperature for 15 minutes. 30 μg of Renilla-Glo luciferase assay system substrate (Promega) was added to each well and incubated at room temperature for 15 minutes. Luminescence was measured on a microplate reader (SpectraMax i3, Molecular Devices). Nonlinear regression calculations of IC50 were performed using Prism 9 (GraphPad Software). Example 12: IN boost with unadjuvanted SARS-CoV-2 spike induces mucosal humoral immunity.

[0184] To evaluate the potential of IN unadjuvanted subunit vaccines to boost respiratory mucosal immunity, we decided to exploit the potent systemic immunogenicity of mRNA-LNPs. We also benefited from extensive SARS-CoV-2 spike engineering, which helped stabilize the protein during prefusion validation by incorporating a C-terminal T4 fibrin trimerization motif, six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P), and alanine substitutions (R683A and R685A) at the furin cleavage site. These sets of mutations have been shown to significantly enhance immunogenicity and increase protein stability, some of which are used in current vaccines.

[0185] We vaccinated K18-hACE2 mice with 1 μg of mRNA-LNP (Fubitai) by IM injection (prime), and 14 days later, 1 μg of recombinant unadjuvanted spike protein was administered IN (prime and spike). Other control groups included K18-hACE2 mice that received only IM prime and K18-hACE2 mice that received IN spike only at boost. Mice were euthanized on day 21 or 28 (7 or 14 days after boost), and the development of mucosal humoral immunity was evaluated ( Figure 1A ).

[0186] First, we evaluated anti-SARS-CoV-2 spike S1 IgG and IgA in nasal washes, bronchoalveolar lavage fluid (BALF), and serum. We found that only mice that received prime and spike produced high levels of anti-SARS-CoV-2 IgA and IgG in nasal washes and BALF ( Figure 1B (BE)). Neither IM priming alone nor IN spike alone was sufficient to generate mucosal antibodies. In serum, IM priming alone was sufficient to induce low levels of IgA and IgG; however, priming plus spike resulted in a significant systemic boost in anti-Spike S1 IgA and IgG ( Figure 1B (F,G)). The increase in antibody levels was associated with an increase in neutralizing titers in BALF and serum ( Figure 1C (HK)). These results suggest that a single dose of unadjuvanted intranasal spike is not immunogenic when used alone and that the potent mucosal and systemic antibody responses induced by the unadjuvanted spike require prior systemic priming, in this case with mRNA-LNPs.

[0187] Tissue-resident memory B cells in the lung RM) have been shown to contribute to the rapid recall response of antibody-secreting B cells after secondary heterologous challenge in a mouse influenza model and may be an important local immune effector for protection against SARS-CoV-2. Using intravenous (IV) CD45 labeling coupled to B cell tetramers against the receptor binding domain (RBD) of the spike protein, we found that priming and spiking resulted in an increase in antigen-specific B cells in lung tissue (IV - CD19 + B220 + Tetramer + )( Figure 1D (L)). Given that only RBD binding was assessed for tetramers, we also investigated polyclonal tissue responses, which may represent a more complete set of B cells within lung tissue that respond to the entire spike. We found increased class-switched antibody-secreting cells (ASCs) in lung tissue expressing either IgA or IgG (IV - CD19 + / - CD138 + )( Figure 1D (M,N)), and we found increased expression of IgA or IgG class switching B RM (IV - CD19 + B220 + IgD - IgM - CD38 + )( Figure 1D (O, P)). These results are consistent with increased mucosal antibody production, indicating that priming and spiking elicit local B cell responses in the lungs. Example 13: Prime and spike to induce mucosal T cell immunity.

[0188] Given our finding that priming and spiking induce mucosal humoral memory responses in the respiratory tract, we next wanted to assess the role of lung tissue-resident memory T cells (T RM ) induction. Although subunit vaccines are not strong inducers of conventional antigen-specific T cell responses, we hypothesized that the immune memory generated by mRNA-LNP priming (which has been shown to be sufficient to induce T cell memory responses in animal models and humans) would achieve subunit-mediated T cell boosting responses. Similar to the above, we combined the CD45 IV marker used to distinguish circulating cells from immune cells in lung tissue with the major histocompatibility complex (MHC) class I tetramer on the conserved Sabeucoronavirus spike epitope (VNFNFNGL). We found that in lung tissue ( Figure 2B (BD)) Inner and lower airway BALF ( Figure 2B (EG)) and upper airway turbinate ( Figure 2C (HJ))Medium Spike IV -Tetramer + CD8 + Significant induction of T cells, which express T RM Typical markers of + and CD103 + In addition, in lung tissue ( Figure 2C (KM)) and the lower airway recovered from BALF ( Figure 2C (NP)), we found a significant increase in antigen-experienced CD4 + T cells (IVCD44 + CD4 + ), many of which also express T RM CD69 + and CD103 + These results suggest that priming and spiking not only induce humoral mucosal responses but also strongly induce lung parenchymal and airway CD8 + T RM and CD4 + T RM . Example 14: Delayed-interval priming and spiking are sufficient to induce mucosal immunity.

[0189] Although we showed that priming and spiking resulted in significant induction of mucosal humoral and cellular immune memory responses with a 14-day interval between priming and boosting, we wondered whether delayed boosting would also induce significant mucosal humoral and cellular responses. To test this question, K18-hACE2 mice that had received a 1 μg IM prime were boosted with IN spikes 84 days later. We collected samples of mucosal humoral and cellular immune responses on days 91 (7 days after boost) and 140 (56 days after boost) ( Fig. 6A We found that delayed IN spikes are sufficient to induce CD8 + T RM , and lasts for at least 56 days ( Figure 6B (BD)). 7 days after boost, early induced CD4 + T RM ; however, their lifespan appeared to be reduced by 56 days, at least in polyclonal populations. + T RM Similar responses were observed, in that we found not only a full humoral response to the delayed boost, but also robust and increased mucosal IgA and IgG in the BALF ( Figure 6C (H,I)), and robust and increased serum IgA and IgG 56 days after boost ( Figure 6C (J, K)). These results suggest that priming and spiking given with a 3-month interval between doses is sufficient to elicit durable mucosal and systemic humoral and cellular immune responses. Example 15: IN delivery of mRNA complexes also mediates mucosal enhancement.

[0190] We next evaluated the capabilities of alternative platforms for IN spike boosting. Poly(amine-coester) (PACE) is a biodegradable terpolymer that has been developed to encapsulate and deliver nucleic acids (e.g., mRNA or DNA) to specific tissues in the body (depending on the properties of the polymer). Recent studies have shown that mRNA-LNP delivered to the respiratory tract is dose-dependently lethal to mice. In contrast, PACE materials have been developed to be relatively immunologically silent, making it possible to administer to sites that are more susceptible to immunopathology (e.g., the respiratory tract). To evaluate the safety and efficacy of PACE-encapsulated mRNA encoding the spike protein, mRNA was extracted from Fubitai and encapsulated in PACE complexes. For vaccination, K18-hACE2 mice were injected with 1 μg IM prime (mRNA-LNP), and 14 days later K18-hACE2 mice received 1 μg of mRNA encapsulated in PACE and administered IN (PACE-spike). Other control groups included: PACE-spike alone, and IM prime + extracted mRNA without PACE encapsulation (naked mRNA) ( Figure 3A Similar to what we found with priming and spikes, priming and PACE-spike induced the expression of typical tissue resident markers (CD69 + and CD103 + ) antigen-specific CD8 + T RM (IV-tetramer + )( Figure 3B ). In addition, PACE-Spike-boosted mice developed high levels of BALF anti-SARS-CoV-2 IgA; BALF IgG and serum IgA and IgG levels were similar to those of mice primed with IM only ( Figure 3C IM priming followed by IN naked mRNA failed to induce mucosal or systemic immune responses above IM priming alone, suggesting that PACE-encapsulated mRNA is necessary for mucosal immune boosting. Furthermore, a single dose of IN PACE-Spike was not sufficient to elicit any detectable mucosal or systemic antibody responses at that dose when used alone. Example 16: Prime plus spike or prime plus PACE-spike protects against lethal SARS-CoV-2 challenge in the setting of mRNA-LNP immunity attenuation.

[0191] While current vaccines are initially highly effective in eliciting protective immunity, waning antibody levels and immune evasion will necessitate boosters against SARS-CoV-2 for the foreseeable future; however, the optimal method of boosting remains a question. To test whether IN administration would provide an alternative protective boost, we utilized a low-dose mRNA LNP vaccine challenge model to mimic waning immunity; we immunized with a single dose of 0.05 μg mRNA-LNP. While mice vaccinated with these low-dose mRNA-LNP vaccines consistently generated systemic antibody responses, we have previously shown that this dose is insufficient to protect against SARS-CoV-2 challenge. Fourteen days after priming, mice received IN spike (1 μg of unadjuvanted spike protein). Similar to the 1 μg IM primed mice we previously described, 0.05 μg IM primed mice boosted with IN spike generated antigen-specific CD8 + T RM and a significant increase in IgA and IgG in BALF ( Fig. 7A ). These data also demonstrate that even very low levels of immune memory generated by low-dose mRNA-LNP priming can be effectively boosted by unadjuvanted IN spike to induce mucosal and systemic humoral and cellular memory.

[0192] Then use 6x10 4 Naive mice, low-dose primed mice, and low-dose primed and spiked mice were challenged with PFU of homologous / ancestral WA1 strain SARS-CoV-2. Mice were euthanized at 2 DPI and viral load in nasal turbinates and lungs was assessed by plaque assay, euthanized at 5 DPI and lung pathology was assessed, or monitored for weight loss and mortality for 14 days ( Figure 4A All mice that received the prime and spike did not lose weight or die, but naive mice and mice that received only the low-dose prime were unable to resist the virus attack ( Figure 4B (BD)). In addition, mice that received prime and spike had significantly improved morbidity and mortality, with reduced viral loads in the upper respiratory tract (nasal concha) and lower respiratory tract (lungs) ( Figure 4B (E,F)). In addition, priming and spiking significantly protected against lung pathology, with only 1 of 6 mice developing limited mononuclear cell infiltration at 5 DPI, while the remaining mice were fully protected and their lung structures were similar to those of uninfected mice ( Figure 4B (G), Figure 4C To evaluate the protective capacity of mRNA-PACE IN boost, we again utilized mice primed with a low dose of mRNA-LNP and boosted IN with 10 μg of mRNA-PACE. We found that priming and PACE-spike significantly protected against morbidity and mortality ( Figure 4D , Figure 4E ). These data demonstrate that both IN unadjuvanted spike and mRNA-PACE-encoded spike are sufficient to boost mucosal immunity and protect against lung disease and death such as COVID-19 in preclinical mouse models. These results also highlight the robustness, versatility, and safety of this vaccine strategy, as an intranasal boost of systemic mRNA-LNP priming by either modality was sufficient to induce mucosal immunity and provide protection against lethal SARS-CoV-2 challenge. Example 17: Prime plus spike induces potent systemic immunity similar to parenteral mRNA-LNP-based boost.

[0193] IM injection of mRNA-LNP-based vaccines is currently the standard boost strategy recommended by many countries, as immunogenicity and vaccine efficacy studies have focused on this boost method. To compare prime and spike with IM mRNA-LNP prime / boost, we primed K18-hACE2 mice with 1 μg of mRNA-LNP and 14 days later with either 1 μg IN spike or 1 μg IM mRNA-LNP. 31 days after boost, mice were euthanized and antigen-specific CD8 + T RM , ELISA was used to evaluate antibodies in BALF and serum, and VSV pseudovirus neutralization assay was performed to evaluate serum antibody neutralization response ( Figure 8A-8C We found that both IM mRNA-LNP-boosted and IN spike-boosted animals produced extravascular (IV - )Tetramer + CD8 + T cells increased; however, only IN spike-boosted animals developed CD69-expressing + and CD103 + CD8 + TRM( Figure 8B ). By ELISA, we found that only IN spike-boosted animals produced anti-SARS-CoV-2 IgA in the BALF. BALF IgG levels were similar in mice boosted with IM mRNA-LNP and IN spike, which may indicate transcytosis caused by increased systemic antibody levels in mice boosted with IM mRNA-LNP. We similarly found that serum levels of anti-SARS-CoV-2 IgA and IgG were comparable in mice boosted with IM mRNA-LNP and IN spike. Serum neutralization assays also showed similar IC50s for IM mRNA-LNP and IN spike boosts. These data suggest that priming and spiking induces systemic neutralizing antibody levels similar to those of IM mRNA-LNP boost, which has been shown to be associated with protection and uniquely elicits mucosal IgA and CD8+ T RM . Example 18: Heterologous spikes strongly induce cross-reactive immunity in the absence of any trace of the original antigen.

[0194] The above experiments clearly demonstrate that boosting at different anatomical locations (in this case, the respiratory mucosa) by either unadjuvanted subunit spikes or by PACE-spike-encoded spikes can form new mucosal immune memory at the newly boosted site and enhance systemic immunity to the antigen. In both unadjuvanted subunit spikes and mRNA PACE, the boosting antigen is homologous to the systemic priming antigen (mRNA-LNP). The spike protein sequence and structure of current circulating strains of SARS-CoV-2 (especially δ and ο) have changed significantly. δ contains T19R, G142D, Δ156-157, R158G, Δ213-214, L452R, T478K, D614G, P681R, and D950N mutations, while o contains A67V, Δ69-70, T95I, G142D, Δ143-145, N211I, L212V, ins213-214RE, V215P, R216E, G339D, S 371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F mutations. These mutations allow δ and ο to spread more rapidly and evade pre-existing humoral immunity, and future variants may show greater divergence, suggesting that a booster strategy to induce broadly reactive immunity to neutralize future variants is necessary. To test the ability of unadjuvanted heterologous spike proteins for IN boosting, we primed K18-hACE2 mice with 1 μg of mRNA-LNP and boosted 14 days later with 5 μg of SARS-CoV-1 spike containing trimer-stabilizing mutations (R667A, K968P, V969P) or prime and spike X ( Figure 5A ). Although SARS-CoV-1 is a related Sarcoma coronavirus, its spike protein has only 76% homology to the original SARS-CoV-2 spike sequence encoded by the currently used mRNA-LNP vaccine. For comparison, we also boosted the mRNA-LNP-primed mice with 1 μg IM mRNA-LNP. 31 days after boost, we found that the typical T cells expressed RM CD69 marker + and CD103 + IV- Tetramer + CD8 + T cells increased significantly ( Figure 5B ). As previously mentioned, this MHC I tetramer sequence is highly conserved among the SabinCoV family, to which both SARS-CoV-1 and SARS-CoV-2 belong. Next, we assessed the production of anti-SARS-CoV-1 antibodies in BALF and serum and found significant increases in anti-SARS-CoV-1 IgA and IgG in the respiratory mucosa and circulation with prime plus Spike X compared to IM mRNA-LNP prime / boost. Consistent with previous studies, we found that two doses of SARS-CoV-2 mRNA LNPs were sufficient to induce detectable antibodies that bind to the SARS-CoV-1 Spike. Next, we assessed anti-SARS-CoV-2 antibodies in BALF and serum. We found that prime plus Spike X induced higher levels of BALF IgA than IM SARS-CoV-2 mRNA-LNP prime / boost. We found similar levels of anti-SARS-CoV-2 IgG in BALF, which may represent the increase in serum IgG we found ( Figure 5C Next, using a VSV-based pseudovirus neutralization assay, we found that sera from primed and spiked mice had higher neutralization titers against SARS-CoV-1 than sera from mice boosted with I SARS-CoV-2 mRNA-LNP ( Figure 5C (M,N)). Similarly, consistent with serum IgG levels, the neutralization titers of SARS-CoV-2 against SARS-CoV-2 in IM SARS-CoV-2 mRNA-LNP prime / boost mice were significantly higher than those in prime and spike-X mice ( Figure 5C (O,P)). Taken together, these data demonstrate that IN boosting with unadjuvanted heterologous Spike protein can induce robust mucosal cellular and humoral memory to a significantly differentiated Spike protein in the absence of any trace of the original antigen. Example 19: Prime and spike to induce mucosal immunity to SARS-CoV-2

[0195] Vaccines containing modified mRNA encapsulated in lipid nanoparticles (LNPs) have been highly effective during the SARS-CoV-2 pandemic over the past two years. Phase 3 clinical trials and subsequent post-marketing vaccine efficacy studies initially showed >90% efficacy of the vaccine against symptomatic disease. Unfortunately, recent studies have shown that vaccine effectiveness in asymptomatic infection as well as symptomatic and severe infection decreases starting approximately 4 months after the second dose with an mRNA-LNP-based regimen. This study was designed to analyze the effect of priming and stimulating in patients who had been previously vaccinated with a SARS-CoV-2 vaccine or infected with SARS-CoV-2. For this purpose, patients who had been vaccinated with a SARS-CoV-2 vaccine or infected with SARS-CoV-2 were equally divided into two groups. One group, a control group, was administered an intranasal formulation containing a placebo; the other group was administered an intranasal formulation containing an FDA-approved mRNA vaccine. Administration was performed 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after infection or vaccination. To assess the effectiveness of the prime and spike, CD8 + T cells, CD4 + T cells, memory T cells (T RM ), B cells (B RM ) as well as mucosal IgG and dimeric IgA. Example 20: Prime and spike to induce mucosal immunity to human papillomavirus (HPV)

[0196] The aim of this study was to analyze the effects of priming and spiking on patients who had previously received the HPV vaccine or were already infected with HPV. For this purpose, patients who had previously received the HPV vaccine or were already infected with HPV were equally divided into two groups. One group was the control group, which was administered a vaginal preparation containing a placebo; the other group was administered a vaginal preparation containing the HPV vaccine. The administration was performed 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the patient was infected or vaccinated. To evaluate the effectiveness of priming and spiking, CD8 + T cells, CD4 + T cells, memory T cells (T RM ), B cells (B RM ) as well as mucosal IgG and dimeric IgA. Example 21: Prime and spike to induce mucosal immunity to rotavirus

[0197] The aim of this study was to analyze the effects of priming and spiking on patients who had previously received the rotavirus vaccine or were already infected with rotavirus. For this purpose, patients who had previously received the rotavirus vaccine or were already infected with rotavirus were divided equally into two groups. One group was the control group, which was administered an oral formulation containing a placebo; the other group was administered an oral formulation containing the rotavirus vaccine. The administration was performed 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months after the patient was infected or vaccinated. To evaluate the effectiveness of priming and spiking, CD8 + T cells, CD4 + T cells, memory T cells (T RM ), B cells (B RM ) as well as mucosal IgG and dimeric IgA.

Claims

1. A method for enhancing an immune response to an antigen in a human in need thereof, the method comprising administering to a mucosal site of a subject an effective amount of a pharmaceutical composition comprising the antigen or a nucleic acid encoding the antigen, wherein: The human has been previously vaccinated against the virus or has been infected with the virus.

2. The method according to claim 1, wherein: The humans have been vaccinated parenterally against the virus.

3. The method according to claim 1, wherein: The antigen is a multivalent antigen.

4. A method of enhancing an immune response to an antigen in a human in need thereof, the method comprising administering to a subject an effective amount of a pharmaceutical composition comprising the antigen at a mucosal site, wherein: The humans have elevated antibodies, memory B cells, effector CD4 - and / or CD8 + T cells.

5. The method according to claim 4, wherein: The elevated antibodies, memory B cells, effector CD4 + and / or CD8 + The T cells are elicited by previous vaccination against the virus.

6. The method according to claim 4, wherein: The elevated antibodies, memory B cells, effector CD4 + and CD8 + T cells are primed by a previous viral infection.

7. The method according to claim 5 or claim 6, wherein: The elevated antibodies are immunoglobulin G (IgG), IgM, IgA or a combination thereof.

8. The method according to any one of claims 1 to 7, wherein: The mucosal part is the nasal mucosal part.

9. The method according to any one of claims 1 to 8, wherein: The antigen comprises at least one protein or polypeptide.

10. The method according to claim 1, wherein: The nucleic acid is DNA or RNA.

11. The method according to claim 1, wherein: The nucleic acid is mRNA.

12. The method according to claim 11, wherein: The mRNA is mRNA modified by N1-methyl-pseudouridine.

13. The method according to claim 11, wherein: The mRNA is mRNA modified with pseudouridine.

14. The method according to any one of claims 1 to 13, wherein: The antigen is derived from a microbial pathogen.

15. The method according to claim 14, wherein: The microbial pathogen is mycobacterium, bacteria, fungi, viruses, parasites or prions.

16. The method according to any one of claims 1 to 15, wherein: The virus is selected from the group consisting of rotavirus, norovirus, adenovirus, astrovirus, variants thereof, and any combination of these.

17. The method according to any one of claims 1 to 15, wherein: The virus is selected from influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, variants thereof, and any combination of these.

18. The method according to any one of claims 1 to 15, wherein: The virus is selected from the group consisting of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human papillomavirus (HPV), variants thereof, and any combination of these.

19. The method according to any one of claims 1 to 15, wherein: The virus is selected from human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes virus, adenovirus, poliovirus, Japanese encephalitis virus, smallpox virus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, human papillomavirus (HPV), molluscum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV-2, Henoch-Schönlein purpura (HSP) virus, RNA virus, DNA virus, variants thereof, and any combination thereof.

20. The method according to claim 19, wherein: The RNA virus is selected from the group consisting of common cold virus, influenza virus, SARS virus, MERS virus, Covid-19 virus, dengue virus, hepatitis C virus, hepatitis E virus, West Nile virus, Ebola virus disease virus, rabies virus, polio virus, mumps virus, measles virus, variants thereof, and any combination thereof.

21. The method according to claim 19, wherein: The DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella zoster virus, Epstein-Barr virus, roseola virus, human herpes virus-7, Kaposi's sarcoma-associated virus, variants thereof, and any combination thereof.

22. The method according to any one of claims 1 to 21, wherein: The antigen is administered by mucosal delivery.

23. The method according to any one of claims 1 to 22, wherein: The antigen is administered to a mucosal tissue of a human subject.

24. The method according to claim 23, wherein: The mucosal tissue is selected from the group consisting of anterior nares, sinuses, rectum, vagina, esophagus, urethra, sublingual and buccal mucosal tissue.

25. The method according to any one of claims 1 to 24, wherein: The pharmaceutical composition is administered intranasally.

26. The method according to any one of claims 1 to 24, wherein: The pharmaceutical composition does not contain an adjuvant.

27. The method according to any one of claims 1 to 24, wherein: The pharmaceutical composition comprises an adjuvant.

28. The method according to any one of claims 1 to 27, wherein: The pharmaceutical composition comprises lipid nanoparticles (LNPs).

29. The method according to claim 28, wherein: The antigen is encapsulated within lipid nanoparticles (LNPs).

30. The method of claim 28, wherein: The lipid nanoparticle (LNP) comprises at least one cationic lipid.

31. The method according to claim 30, wherein: The at least one cationic lipid comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

32. The method of claim 28, wherein: The lipid nanoparticles (LNPs) comprise poly(amine-co-ester) (PACE) polymers.

33. The method of claim 28, wherein: The lipid nanoparticle (LNP) further comprises at least one phospholipid.

34. The method of claim 33, wherein: The at least one phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

35. The method according to any one of claims 1 to 34, wherein: The human was vaccinated against the virus or was infected with the virus about a week ago, two weeks ago, three weeks ago, one month ago, two months ago, three months ago, four months ago, five months ago, six months ago, seven months ago, eight months ago, nine months ago, ten months ago, eleven months ago or twelve months ago.

36. A method of enhancing an immune response to SARS-CoV-2 in a human in need thereof, the method comprising administering to a mucosal site of a subject an effective amount of a pharmaceutical composition comprising at least one mRNA, wherein: The human has been previously vaccinated against the virus or has been infected with the virus.

37. The method of claim 36, wherein: The human has previously been vaccinated with one or more COVID-19 vaccines selected from the following: BNT162b2 (Pfizer / BioNTech), mRNA-1273 (Moderna), AZD1222 / ChAdOxl (AstraZeneca / Oxford University), Ad5 vector COVID-19 vaccine (CanSino Biologics), CoronaVac (Sinovac), NVX-CoV2373 (Novavax), and combinations thereof.

38. The method of claim 36, wherein: The at least one mRNA encodes the spike protein of SARS-CoV-2, a variant thereof, or a fragment thereof.

39. The method of claim 36, wherein: The pharmaceutical composition comprises mRNA encoding two or more different antigens.

40. The method of claim 36, wherein: The two or more antigens are the spike protein of SARS-CoV-2, its variants or fragments thereof.

41. The method of claim 40, wherein: The two or more antigens comprise at least one mutation listed in Table 1.

42. The method according to any one of claims 36 to 41, wherein: The pharmaceutical composition does not contain an adjuvant.

43. The method according to any one of claims 36 to 41, wherein: The pharmaceutical composition comprises an adjuvant.

44. The method according to any one of claims 36 to 43, wherein: The pharmaceutical composition also comprises lipid nanoparticles (LNPs).

45. The method of claim 44, wherein: The at least one mRNA is encapsulated within lipid nanoparticles (LNPs).

46. ​​The method of claim 45, wherein: The lipid nanoparticle (LNP) comprises at least one cationic lipid.

47. The method of claim 46, wherein: The at least one cationic lipid comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

48. The method of claim 45, wherein: The lipid nanoparticles (LNPs) comprise poly(amine-co-ester) (PACE) polymers.

49. The method of claim 45, wherein: The lipid nanoparticle (LNP) further comprises at least one phospholipid.

50. The method of claim 49, wherein: The at least one phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and / or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

51. The method according to any one of claims 36 to 50, wherein: The average diameter of the lipid nanoparticles is in the range of about 50 nm to about 1000 nm.

52. The method according to any one of claims 36 to 50, wherein: The lipid nanoparticles have an average diameter ranging from about 50 nm to about 400 nm, about 50 nm to about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, or about 300 nm to about 600 nm.

53. The method according to any one of claims 1 to 52, wherein: The immune response is a mucosal immune response.

54. The method of claim 53, wherein: The mucosal immune response is the production of antigen-specific IgA antibodies.

55. The method of claim 53, wherein: The mucosal immune response is the production of antigen-specific IgG antibodies.

56. The method of claim 53, wherein: The mucosal immune response is the production of antigen-specific IgM antibodies.

57. The method of claim 36, wherein: The human has elevated neutralizing antibody levels resulting from prior vaccination against a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

58. The method of claim 36, wherein: The human has elevated neutralizing antibody levels resulting from prior infection with a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

59. The method of claim 57 or claim 58, wherein: The elevated neutralizing antibodies are IgG, IgM, IgA or a combination thereof.

60. The method of claim 59, wherein: The human has elevated IgG antibodies resulting from prior vaccination against a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

61. The method of claim 59, wherein: The human has elevated IgM antibodies resulting from previous vaccination against a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

62. The method of claim 59, wherein: The human has elevated IgA antibodies resulting from previous vaccination against a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

63. The method of claim 59, wherein: The human has elevated IgG antibodies caused by previous infection with a virus selected from MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

64. The method of claim 59, wherein: The human has elevated IgM antibodies caused by previous infection with a virus selected from MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

65. The method of claim 59, wherein: The human has elevated IgA antibodies caused by previous infection with a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-Cov-2, and variants thereof.

66. The method according to any one of claims 36 to 65, wherein: The human was vaccinated against the virus or was infected with the virus about a week ago, two weeks ago, three weeks ago, one month ago, two months ago, three months ago, four months ago, five months ago, six months ago, seven months ago, eight months ago, nine months ago, ten months ago, eleven months ago or twelve months ago.

67. The method according to any one of claims 35 to 65, wherein: The SARS-CoV-2 variants are selected from α (B.1.1.7 and Q lineages), β (B.1.351 and descendant lineages), γ (P.1 and descendant lineages), δ (B.1.617.2 and AY lineages), ε (B.1.427 and B.1.429), η (B.1.525), ι (B.1.526), ​​κ (B.1.617.1), 1.617.3, μ (B.1.621, B.1.621.1), ζ (P.2), μ (B.1.621, B.1.621.1), ο (Pango lineage B.1.1.529, BA.1, BA.1.1, BA.2, BA.3) and combinations thereof.

68. The method according to any one of claims 36 to 67, wherein: The at least one mRNA is an mRNA modified with N1-methyl-pseudouridine.

69. The method according to any one of claims 36 to 67, wherein: The at least one mRNA is a pseudouridine-modified mRNA.

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