Method for enhancing immunity

Abstract

The present invention relates to a method for enhancing immunity.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 336,499, filed on April 29, 2022, entitled "METHOD FOR ENHANCING IMMUNITY", and U.S. Provisional Patent Application No. 63 / 648,451, filed on June 2, 2022, entitled "METHOD FOR ENHANCING IMMUNITY", each of which is hereby incorporated by reference in its entirety.

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

[0003] Sequence Listing This application contains a sequence listing submitted electronically in XML file format, which is hereby incorporated by reference in its entirety. The sequence listing of this application is entitled "130481 - 5002 - WO.XML", which was created on April 25, 2023 and is 8,500 bytes in size.

Background Art

[0004] mRNA - based vaccines for SARS - CoV - 2 have shown the untapped potential of mRNA therapeutics for safe and effective use in the general population. However, more recent studies have shown that the efficacy of the vaccines decreases not only for symptomatic and severe infections starting approximately 4 months after the second dose in an mRNA - lipid nanoparticle (LNP) - based regimen, but also for asymptomatic infections. Furthermore, the continued evolution of the virus, particularly with concerning variants of concern (VOCs) such as beta (B.1.351), delta (B.1.617.2), and currently omicron (B.1.529), which particularly increase immune evasion, has also contributed to the decline in vaccine efficacy against COVID - 19. Current vaccines not only have a reduced effect in preventing SARS - CoV - 2 infection, but they also have a reduced ability to prevent viral transmission.

[0005] Therefore, it is necessary to enhance immunity against COVID-19. The present invention meets such a need.

Brief Description of the Drawings

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Summary of the Invention

[0007] The present invention encompasses a method for enhancing an immune response against 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 the antigen or a nucleic acid encoding the antigen, wherein the human has been previously vaccinated against or infected with a virus. In some embodiments, the human has elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells. In some embodiments, the elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells are induced by a previous vaccination against a virus.

[0008] In some embodiments, the elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells are induced by a previous viral infection.

[0009] In some embodiments, the mucosal site is selected from the group consisting of rectal, vaginal, bladder, ocular, oral, sublingual, esophageal, nasal, gastrointestinal, pulmonary, and oral mucosal sites.

[0010] In some embodiments, the antigen comprises a protein or polypeptide.

[0011] In some embodiments, the antigen is a multivalent antigen.

[0012] In some embodiments, the antigen comprises a nucleic acid encoding a protein or polypeptide.

[0013] In some embodiments, the nucleic acid is DNA or RNA.

[0014] In some embodiments, the nucleic acid is mRNA.

[0015] In some embodiments, the antigen is derived from a microbial pathogen.

[0016] In some embodiments, the microbial pathogen is Mycobacterium, bacteria, fungi, virus, parasite, or prion.

[0017] In some embodiments, the virus is selected from the group consisting of rotavirus, norovirus, adenovirus, astrovirus, variants thereof, and any combination thereof.

[0018] In some embodiments, the virus is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, variants thereof, and any combination thereof.

[0019] 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 thereof.

[0020] In some embodiments, the virus is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpes virus, adenovirus, polio, Japanese encephalitis, smallpox, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, paralytic virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, human papillomavirus (HPV), molluscum contagiosum virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV-2, Henoch-Schönlein purpura (HSP), RNA virus, DNA virus, variants thereof, and any combination thereof.

[0021] In some embodiments, the RNA virus is selected from the group consisting of cold, influenza, SARS, MERS, Covid-19, dengue virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, rubella, variants thereof, and any combination thereof.

[0022] In some embodiments, the DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus, roseolovirus, human herpesvirus-7, Kaposi's sarcoma-associated virus, variants thereof, and any combination thereof.

[0023] In some embodiments, the pharmaceutical composition is administered by mucosal delivery.

[0024] In some embodiments, the mucosal delivery is selected from the group consisting of rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, intranasal delivery, vaginal delivery, and oral delivery.

[0025] In some embodiments, the pharmaceutical composition is administered to the mucosal tissue of a human subject.

[0026] In some embodiments, the mucosal tissue is selected from the group consisting of the anterior external nares, nasal cavity, rectum, vagina, esophagus, urethra, sublingual, and buccal.

[0027] In some embodiments, the pharmaceutical composition is administered orally, intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0028] In some embodiments, the pharmaceutical composition is administered by intranasal spray.

[0029] In some embodiments, the pharmaceutical composition does not contain an adjuvant.

[0030] In some embodiments, the pharmaceutical composition contains an adjuvant.

[0031] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNP).

[0032] In some embodiments, the antigen is encapsulated within lipid nanoparticles (LNP).

[0033] In another aspect, the present invention relates to a method of enhancing an immune response against SARS-CoV-2 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 at least one mRNA, wherein the human has been previously vaccinated against SARS-CoV-2 or is infected with SARS-CoV-2. In some embodiments, the mRNA encodes the spike protein of SARS-CoV-2, or a fragment thereof.

[0034] In some embodiments, the pharmaceutical composition does not contain an adjuvant.

[0035] In some embodiments, the pharmaceutical composition contains an adjuvant.

[0036] In some embodiments, the pharmaceutical composition further comprises lipid nanoparticles (LNP).

[0037] In some embodiments, the mRNA is encapsulated within lipid nanoparticles (LNP).

[0038] In some embodiments, the lipid nanoparticles (LNP) comprise at least one cationic lipid.

[0039] In some embodiments, the at least one cationic lipid comprises 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), and / or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

[0040] In some embodiments, the lipid nanoparticles (LNPs) further comprise at least one phospholipid.

[0041] 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).

[0042] In some embodiments, the lipid nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm.

[0043] In some embodiments, the lipid nanoparticles have an average diameter 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.

[0044] In some embodiments, the immune response is a mucosal immune response.

[0045] In some embodiments, the mucosal immune response is antigen-specific IgA antibody production.

[0046] In some embodiments, the mucosal immune response is antigen-specific IgG antibody production.

[0047] In some embodiments, the human has elevated IgA antibodies.

[0048] In some embodiments, the human has elevated IgG antibodies.

[0049]

Mode for Carrying Out the Invention

[0050] Definition As used in this specification and the appended claims, the articles "a" and "an" are used for the purpose of indicating that the grammatical object of the article is one or more (i.e., at least one), unless the context clearly indicates otherwise. By way of example, "an element" means one element or more than one element.

[0051] The term "variant" means a polypeptide or nucleotide that contains changes, i.e., substitutions, insertions, and / or deletions, at one or more (e.g., several) positions. In some embodiments, the term "variant" refers to a SARS-CoV-2 virus variant.

[0052] The terms "spike", "boost", or "booster" are used synonymously.

[0053] As used herein, the term "immunogenic agent" encompasses any substance, composition of substances, or composition of organic materials, such as, for example, a suspension of cells or cell components, and an immunogenic agent, when administered in an appropriate amount and mixed with an appropriate substance, can elicit a substantial immune response against coronavirus in a human subject.

[0054] The term "immunologically equivalent" means that a polypeptide is functionally equivalent to a polypeptide having the amino acid sequence of any S protein with respect to its ability to induce an immune response.

[0055] As used herein, the term "polypeptide" encompasses short peptides of 2 to 10 amino acid residues, oligopeptides (11 to 100 amino acid residues), and long peptides (the normal interpretation of polypeptides, i.e., more than 100 amino acid residues), as well as proteins (functional entities containing at least one peptide, oligopeptide, or polypeptide that can be chemically modified by glycosylation or conjugated to other chemical groups). The definition of polypeptide also includes the native form of polypeptides or proteins in SARS-CoV-2, as well as recombinant proteins or peptides in any type of expression vector that transforms any type of host, and furthermore, chemically synthesized peptides.

[0056] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single-stranded or double-stranded form, including DNA, RNA, and their hybrids. DNA can be in the form of antisense molecules, plasmid DNA, cDNA, PCR products, or vectors. RNA can be in the form of 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 linkages, which are synthetic, natural, and non-natural and have binding properties similar to those of reference nucleic acids.

[0057] The present invention relates to a method of vaccinating a human subject against a virus, where the human subject has previously been systemically vaccinated against the virus or is infected with the virus. The method comprises administering to a mucosal site of the subject an effective amount of a pharmaceutical composition comprising an antigen. This method is also referred to as "prime and spike" or "prime and boost". In some embodiments, the prime and spike method utilizes a non - adjuvant intranasal spike boost that exploits existing immunity generated by a primary systemic vaccination to induce mucosal immune memory in the respiratory tract. Further, using diverse spike proteins, prime and spike enables the induction of cross - reactive immunity against sarbecoviruses. In some embodiments, prime and spike enables a polyvalent response against sarbecoviruses such as MERS - CoV, SARS - CoV - 1, SARS - Cov - 2, or variants thereof.

[0058] In one aspect, the present invention encompasses a method of enhancing, in a human in need of enhancing an immune response to an antigen, the method comprising administering to a mucosal site of the subject an effective amount of a pharmaceutical composition comprising the antigen or a nucleic acid encoding the antigen, wherein the human has previously been vaccinated against a virus or is infected with a virus. In some embodiments, the human has elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells. In some embodiments, the elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells are caused by a previous vaccination against the virus. In some embodiments, the previous vaccination was administered parenterally.

[0059] In some embodiments, the elevated antibodies, memory B cells, and effector CD4 + and CD8 + T cells are caused by a previous viral infection. In some embodiments, the elevated antibodies are immunoglobulin G (IgG), IgM, and IgA.

[0060] In some embodiments, the mucosal site is selected from the group consisting of rectal, vaginal, bladder, ocular, oral, sublingual, esophageal, nasal, gastrointestinal, pulmonary, and oral mucosal sites.

[0061] In some embodiments, the antigen comprises a protein or polypeptide.

[0062] In some embodiments, the antigen comprises at least one nucleic acid encoding a protein or polypeptide.

[0063] In some embodiments, the nucleic acid is DNA or RNA.

[0064] In some embodiments, the nucleic acid is mRNA. In some embodiments, the mRNA is N1-methyl-pseudouridine modified mRNA. In some embodiments, the mRNA is pseudouridine modified mRNA. 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 polyvalent response.

[0065] In some embodiments, the antigen is derived from a microbial pathogen.

[0066] In some embodiments, the microbial pathogen is Mycobacterium, bacterium, fungus, virus, parasite, or prion.

[0067] In some embodiments, the virus is selected from the group consisting of rotavirus, norovirus, adenovirus, astrovirus, variants thereof, and any combination thereof.

[0068] In some embodiments, the virus is selected from the group consisting of influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, variants thereof, and any combination thereof.

[0069] 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 thereof.

[0070] In some embodiments, the virus is selected from the group consisting of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpes virus, adenovirus, polio, Japanese encephalitis, smallpox, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus, respiratory syncytial virus (RSV), mumps virus, rotavirus, paralytic 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), RNA virus, DNA virus, variants thereof, and any combination thereof.

[0071] In some embodiments, the RNA virus is selected from the group consisting of cold, influenza, SARS, MERS, Covid-19, dengue virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, rubella, variants thereof, and any combination thereof.

[0072] In some embodiments, the DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus, roseolovirus, human herpesvirus-7, Kaposi's sarcoma-associated virus, variants thereof, and any combination thereof.

[0073] In some embodiments, the pharmaceutical composition is administered by mucosal delivery.

[0074] In some embodiments, the mucosal delivery is selected from the group consisting of rectal delivery, buccal delivery, pulmonary delivery, ocular delivery, nasal delivery, intranasal delivery, vaginal delivery, and oral delivery.

[0075] In some embodiments, the pharmaceutical composition is administered to the mucosal tissue of a human subject.

[0076] In some embodiments, the mucosal tissue is selected from the group consisting of the anterior external nares, nasal cavity, rectum, vagina, esophagus, urethra, sublingual, and buccal.

[0077] In some embodiments, the pharmaceutical composition is administered orally, intravenously, intramuscularly, intradermally, subcutaneously, intranasally, or by inhalation.

[0078] In some embodiments, the pharmaceutical composition is administered by intranasal spray.

[0079] In some embodiments, the pharmaceutical composition does not contain an adjuvant.

[0080] In some embodiments, the pharmaceutical composition contains an adjuvant.

[0081] In some embodiments, the pharmaceutical composition comprises lipid nanoparticles (LNP). In some embodiments, the lipid nanoparticles (LNP) comprise a poly(amine-co-ester) (PACE) polymer. In some embodiments, the PACE polymer is 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 by reference in their entirety.

[0082] In some embodiments, the antigen is encapsulated within the lipid nanoparticles (LNP).

[0083] In some embodiments, the human was vaccinated against the virus or infected with the virus about 1 week ago, 2 weeks ago, 3 weeks ago, 1 month ago, 2 months ago, 3 months ago, 4 months ago, 5 months ago, 6 months ago, 7 months ago, 8 months ago, 9 months ago, 10 months ago, 11 months ago, or 12 months ago.

[0084] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is one or more selected from small molecule RNA, ribozyme, small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (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, the nucleic acid once administered to a human subject is ultimately translated into a protein, and the protein provides a therapeutic function or vaccination.

[0085] In some embodiments, the nanoparticles comprise 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 by reference in their entirety.

[0086] In some embodiments, the nanoparticles comprise one or more compounds 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 by reference in their entirety.

[0087] In some embodiments, the particles have an average particle size of from about 100 nm to about 300 nm, preferably from about 150 nm to about 275 nm. In some embodiments, the polymer:polypeptide weight:weight ratio is from about 25:1 to 250:1.

[0088] 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 + T cells in animal models and humans. However, parenteral vaccines do not induce high levels of potent antiviral immune memory at the site of infection, such as tissue-resident memory T cells (T RM ) and B cells (B RM ), as well as mucosal IgG and dimeric IgA. This is in contrast to SARS-CoV-2 infection in humans and mice, in which CD8 + T RM is strongly induced. Vaccines targeting the respiratory mucosa may address the drawbacks of parenteral vaccination, as demonstrated by the recent preclinical evaluation of intranasally delivered SARS-CoV-2 spike encoding an adenovirus vector, which showed impressive mucosal immunogenicity and protection and reduced viral shedding in mice, hamsters, and non-human primates. Preclinical mucosal influenza vaccine trials have also shown that mucosal immunity can enhance protection against heterosubtypic challenge via CD8 + T RM or dimeric IgA and can improve the durability of immunity.

[0089] Primary respiratory administration of a vaccine induces a strong mucosal immune response and, after systemic priming, boosting intranasally results in similar systemic immunity but with the added benefit of improved mucosal immunity.

[0090] Recent studies have shown that the decline in the effectiveness of the FDA-approved mRNA vaccine against COVID-19 begins approximately four months after the second dose, with respect to asymptomatic as well as symptomatic and severe infections. In such settings of immune decline from parenteral vaccination regimens, the present disclosure describes a method of enhancing immunity against COVID-19, the method utilizing an intranasal boost (IN) with either a non-adjuvant spike protein or an immunologically silent polyplex encapsulating mRNA, after strong systemic priming with an mRNA-LNP-based vaccine.

[0091] To evaluate the potential of an IN non - adjuvant subunit vaccine boost for the development of respiratory mucosal immunity, K18 - hACE2 mice were vaccinated with 1 μg of mRNA - LNP (Comirnaty) by IM injection (prime), and 14 days later, 1 μg of recombinant non - adjuvant spike protein (prime and spike) was inoculated by IN administration. Additional control groups included K18 - hACE2 mice that received only the IM prime, and mice that received only IN spike at the boost. Mice were euthanized on day 21 or 28 (7 or 14 days after boost) and evaluated for the development of mucosal humoral immunity (Figure 1A). Anti - SARS - CoV - 2 spike S1 IgG and IgA in nasal washes, bronchoalveolar lavage fluid (BALF), and serum were evaluated. Only mice that received prime and spike developed high levels of anti - SARS - CoV - 2 IgA and IgG in nasal washes and BALF (Figure 1B (B - E)). Neither the IM prime alone nor the IN spike alone was sufficient for the development of mucosal antibodies. In serum, the IM prime alone was sufficient to induce low levels of IgA and IgG. However, prime and spike resulted in a significant systemic boost of both anti - spike S1 IgA and IgG (Figure 1B (F, G)). These increases in antibody levels correlated with increases in neutralizing titers in both BALF and serum (Figure 1B (H - K)). These results suggest that a single dose of non - adjuvant intranasal spike alone is not immunogenic, and that prior systemic priming (in this case with mRNA - LNP) is required for the induction of a strong mucosal and systemic antibody response by non - adjuvant spike.

[0092] Using intravenous (IV) CD45 labeling combined with a B - cell tetramer specific for the receptor - binding domain (RBD) of the spike protein, it was found that prime and spike resulted in an increase in antigen - specific B cells in lung tissue (IV - CD19 + B220 + tetramer +)(Figure 1D (L)). Since the tetramers were evaluated only for RBD binding, we also examined the polyclonal tissue response, which is likely to represent a more complete set of B cells reactive to the whole spike in lung tissue. Class switch antibody-secreting cells (ASCs) (IV - CD19 + / - CD138 + ) that express IgA or IgG were increased (Figure 1D (M, N)), and class switch B RM (IV - CD19 + B220 + IgD - IgM - CD38 + ) that express IgA or IgG were increased (Figure 1D (O, P)). These results are consistent with the increased mucosal antibody production and indicate that prime and spike induce a local B cell response in the lung.

[0093] Similarly, CD45 IV labeling to distinguish immune cells from circulation in lung tissue was combined with major histocompatibility complex (MHC) class I tetramers (VNFNFNGL) against the conserved sarbecovirus spike epitope. CD69 + and CD103 + were expressed in the spike IV RM tetramer - containing + CD8 + T cells, and significant induction of these T cells was found in lung tissue (Figure 2B (B - D)), lower airway BALF (Figure 2B (E - G)), and upper airway turbinates (Figure 2C (H - J)). Furthermore, a significant increase in antigen-experienced CD4 + T cells (IVCD44 + CD4 + ) was found, and many of them also expressed the markers of T RM CD69 + and CD103 + both in lung tissue (Figure 2C (K - M)) and in the lower airway recovered from BALF (Figure 2C (N - P)). These results indicate that prime and spike not only induce a humoral mucosal response but also CD8 in the lung parenchyma and airways.+ T RM and CD4 + T RM has been shown to strongly induce.

[0094] K18-hACE2 mice administered an IM prime of 1 μg were boosted with IN spike 84 days later. Humoral and cellular mucosal immune responses were sampled on day 91 (7 days after boost) and day 140 (56 days after boost). Delayed IN spike was sufficient to induce CD8 + T RM which was found to persist for at least 56 days. CD4 + T RM was induced early at 7 days after boost, but its lifespan appeared to decline to at least 56 days, at least polyclonally. CD8 + T RM Similar to the CD8

[0095] In another aspect, the present invention relates to a method of enhancing an immune response against SARS-CoV-2 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 at least one mRNA, wherein the human has been previously vaccinated against SARS-CoV-2 or is infected with SARS-CoV-2. In some embodiments, the mRNA encodes the spike protein of SARS-CoV-2, or a fragment thereof.

[0096] In some embodiments, the human has 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 Univ), Ad5-vectored COVID-19 vaccine (CanSino Biologies), CoronaVac (Sinovac), NVX-CoV2373 (Novavax), and combinations thereof.

[0097] In some embodiments, the human has elevated IgG antibodies caused by previous vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0098] In some embodiments, the human has elevated IgM antibodies caused by previous vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0099] In some embodiments, the human has elevated IgA antibodies caused by previous vaccination against MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0100] In some embodiments, the human has elevated IgG antibodies caused by previous infection with MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0101] In some embodiments, the human has elevated IgM antibodies caused by previous infection with MERS-CoV, SARS-CoV-1, SARS-Cov-2, or variants thereof.

[0102] In some embodiments, the human has elevated IgA antibodies caused by previous infection with MERS-CoV, SARS-CoV-1, SARS-CoV-2, or variants thereof.

[0103] In some embodiments, the elevated IgG is in the range of about 100 - 150, about 100 - 200, about 100 - 300, about 100 - 400, about 150 - 200, about 150 - 250, about 150 - 300, about 150 - 400, about 200 - 250, about 200 - 300, about 200 - 350, or about 200 - 400 BAU / ml.

[0104] 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.

[0105] In some embodiments, the elevated IgM is in the range of about 25 - 100, about 25 - 150, about 25 - 200, about 25 - 300, about 50 - 100, about 50 - 150, about 50 - 200, about 50 - 300, about 75 - 100, about 75 - 150, about 75 - 200, about 75 - 300, about 100 - 150, about 100 - 200, about 100 - 300, about 125 - 200, about 125 - 300, about 150 - 200, about 150 - 300, about 200 - 300, about 250 - 300 AU / ml.

[0106] 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.

[0107] In some embodiments, the elevated IgA is in the range of about 10 - 100, about 10 - 150, about 10 - 200, about 25 - 100, about 25 - 150, about 25 - 200, about 50 - 100, about 50 - 150, about 50 - 200, about 75 - 100, about 75 - 150, about 75 - 200, about 100 - 150, about 100 - 200, about 125 - 150, about 125 - 200, about 150 - 200 AU / ml.

[0108] 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.

[0109] In some embodiments, at least one mRNA encodes a spike protein of SARS-CoV-2, or 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. The 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 N1-methyl-pseudouridine modified mRNA. In some embodiments, the mRNA is pseudouridine modified mRNA.

[0110] In some embodiments, the pharmaceutical composition does not contain an adjuvant.

[0111] In some embodiments, the pharmaceutical composition contains an adjuvant.

[0112] In some embodiments, the pharmaceutical composition further comprises lipid nanoparticles (LNP). In some embodiments, the lipid nanoparticles (LNP) comprise a poly(amine-co-ester) (PACE) polymer.

[0113] In some embodiments, at least one mRNA is encapsulated within lipid nanoparticles (LNP).

[0114] In some embodiments, the lipid nanoparticles (LNP) comprise at least one cationic lipid.

[0115] 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).

[0116] In some embodiments, the lipid nanoparticles (LNP) further comprise at least one phospholipid.

[0117] 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).

[0118] In some embodiments, the lipid nanoparticles have an average diameter in the range of about 50 nm to about 1000 nm.

[0119] In some embodiments, the lipid nanoparticles have an average diameter 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.

[0120] In some embodiments, the immune response is a mucosal immune response.

[0121] In some embodiments, the mucosal immune response is antigen-specific IgA antibody production.

[0122] In some embodiments, the human was vaccinated against the virus or infected with the virus about 1 week ago, 2 weeks ago, 3 weeks ago, 1 month ago, 2 months ago, 3 months ago, 4 months ago, 5 months ago, 6 months ago, 7 months ago, 8 months ago, 9 months ago, 10 months ago, 11 months ago, or 12 months ago.

[0123] In some embodiments, the pharmaceutical composition described herein comprises a polypeptide as an antigen for vaccinating a human subject against SARS-CoV-2 and its immunogenic variants. In some embodiments, the polypeptide is a coronavirus spike (S) protein, its immunogenic variant, or an antigenic fragment thereof. In some embodiments, the polypeptide has an amino acid sequence having a degree of sequence identity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with any known S protein, or a subunit thereof, or a fragment thereof. In some embodiments, the polypeptide has an amino acid sequence having a degree of sequence identity of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% with any known S protein, or a subunit thereof, or a fragment thereof. In some embodiments, the variant is a SARS-CoV-2 spike protein variant found in different strains of SARS-CoV-2. Variants include, but are not limited to, spike proteins derived from the alpha, beta, or delta variants of SARS-CoV-2, B.1.1.7 strain, B.1.351 strain, P.1 strain, CAL20 strain, or any combination thereof.

[0124] In some embodiments, the SARS-CoV-2 variants include, but are not limited to, alpha (B.1.1.7 and Q lineages), beta (B.1.351 and descendant lineages), gamma (P.1 and descendant lineages), delta (B.1.617.2 and AY lineages), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1), zeta (P.2), Mu (B.1.621, B.1.621.1), Omicron (Pango lineage B.1.1.529, BA.1, BA.1.1, BA.2, BA.3), and combinations thereof.

[0125] The DNA sequence encoding the spike protein of SARS-CoV-2 isolated in Wuhan is listed as SEQ ID NO: 1.

[0126] Exemplary variants of the spike protein from different strains are shown in Table 1.

[0127] [Table 1]

[0128] In some embodiments, the polypeptide includes other amino acid sequences of strains of the SARS-CoV-2 S protein, including any disclosed in Deng (2020) Science, 8: eabb9263 and Taboada (2020) J. Virol. 94: e01056. However, other SARS-CoV-2 strains are likely to exhibit substantially the same immunological properties as the alpha variant S protein, fragments, and subunits derived from such strains. In some embodiments, the polypeptide is selected from the group consisting of the M protein, E protein, N protein, and combinations thereof, derived from SARS-CoV-2 or their variants.

[0129] In some embodiments, the S protein variants described herein include a mutation at a position corresponding to position 501N of the alpha variant. In some embodiments, the amino acid corresponding to position 501N is replaced with Y. In some embodiments, the S protein variants described herein that include a mutation at a position corresponding to 501N may include one or more additional mutations. Such one or more additional mutations may be one or more selected from mutations at positions corresponding to the following amino acids at positions relative to the alpha 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 of the alpha 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.

[0130] In some embodiments, the S protein variant described herein is the S protein of SARS-CoV-2 Delta. In some embodiments, the S protein of SARS-CoV-2 Delta has the following spike protein substitutions relative to the Alpha variant: T19R, V70F, T95I, G142D, deletion of 156E, deletion of 157F, R158G, A222V, W258L, K417N, L452R, T478K, D614G, P681R, and D950N.

[0131] In some embodiments, the S protein variant described herein is the S protein of SARS-CoV-2 Omicron. In some embodiments, the S protein of SARS-CoV-2 Omicron has the following spike protein substitutions relative to the Alpha variant: A67V, deletion of amino acids 69-70, T95I, deletion of amino acids 142-144, Y145D, amino acids 211, L212I, insertion of EPE at amino acid 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.

[0132] In some embodiments, administration of the pharmaceutical composition described herein is performed by a single administration or can be boosted by multiple administrations.

[0133] In some embodiments, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In some embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration can include enteral administration with absorption through the gastrointestinal tract or parenteral administration. As used herein, "parenteral administration" refers to administration by any method other than through the gastrointestinal tract, such as intravenous injection.

[0134] In some embodiments, the pharmaceutical compositions described herein can be administered intranasally.

[0135] In some embodiments, an amount of the polypeptide described herein from 0.1 μg to 300 μg, 0.5 μg to 200 μg, or 1 μg to 100 μg per dose can be administered, for example, 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 a single-dose administration. In some embodiments, the present invention contemplates an administration of a priming dose followed by one or more booster doses. The booster dose or the first booster dose can be administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about 5 weeks after the administration of the priming dose. In some embodiments, the booster dose or the first booster dose can be administered about 1 month, about 2 months, about 3 months, about 4 months, or about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after the administration of the priming dose.

[0136] In some embodiments, an amount of the polypeptide described herein of 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 can be administered.

[0137] In some embodiments, an amount of the polypeptide described herein of 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 can be administered.

[0138] In some embodiments, an amount of the polypeptide described herein from 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 can be administered.

[0139] The pharmaceutical compositions and products described herein can be provided, for example, as a frozen concentrate for an injectable solution at a concentration of 0.50 mg / mL. In some embodiments, for the preparation of the injectable solution, the pharmaceutical is thawed and diluted with an isotonic sodium chloride solution (e.g., 0.9% NaCl saline), for example, by a one-step dilution process. In some embodiments, a bacteriostatic sodium chloride solution (e.g., 0.9% NaCl saline) cannot be used as a diluent. In some embodiments, the diluted pharmaceutical is an off-white suspension. The concentration of the final solution for injection varies depending on each dosage level administered.

[0140] The invention also encompasses a kit comprising the pharmaceutical compositions and means of administration described herein. In some embodiments, the kit includes a nasal spray device for nasal 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 by reference in its entirety. The kit can be convenient for self-administration for vaccination against SARS-CoV-2, or variants thereof.

[0141] In the kit according to the invention, the pharmaceutical composition comprises 0.5 to 75 μg of a polypeptide, for example, 0.5 to 50 μg of a polypeptide, or 5 to 50 μg of a polypeptide.

Examples

[0142] The teachings generally described herein are more readily understood by reference to the following examples, which are included for purposes of illustration only of specific aspects and embodiments of the present disclosure.

[0143] Methods and Materials: All procedures were performed in a BSL-3 facility (for SARS-CoV-2-infected mice) with approval from the Yale Institutional Animal Care and Use Committee and Yale Environmental Health and Safety.

[0144] Cells and Viruses Vero E6 cells overexpressing hACE2 and TMPRSS2 (provided by Barney Graham NIH-VRC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% sodium pyruvate and 5% fetal bovine serum (FBS) at 37 °C and 5% CO2. The 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 2 - 3 days to generate a working stock. After incubation, the supernatant was clarified by centrifugation (500 g × 5 min), filtered through a 0.45 micron filter, and stored at -80 °C. Virus titers were measured by a standard plaque assay using Vero E6 cells overexpressing hACE2 and TMPRSS2.

[0145] Mice B6.Cg-Tg(K18-ACE2)2Prlmn / J (K18-hACE2) mice were purchased from the Jackson Laboratory and then bred and housed at Yale University. Female mice aged 8 - 12 weeks were used for the immunization experiments. All procedures used in this study (matched for sex and age) were in accordance with federal guidelines and the Yale School of Medicine Animal Care and Use Committee facility guidelines.

[0146] Example 1: SARS-CoV-2 Infection Mice were anesthetized using 30% v / v isoflurane diluted in propylene glycol. Using a pipette, 6×10 550 μL containing PFU of SARS-CoV-2 was delivered intranasally.

[0147] Example 2: mRNA Extraction from Comirnaty mRNA-LNP mRNA was extracted from the vaccine formulation using the Trizol / chloroform separation method described herein. Briefly, an aliquot of the vaccine was dissolved in Trizol LS (Thermo Fisher Scientific) at a vaccine to Trizol volume ratio of 1:6.6. After incubation for 15 minutes (37 °C, shaking), 0.2 mL of chloroform was added per 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×g for 8 minutes at 4 °C. The aqueous layer containing the isolated mRNA was further purified using the RNeasy Maxi Kit purchased from Qiagen (Germantown, MD, USA) according to the manufacturer's protocol. The RNA was eluted from the column in the final step using sodium acetate buffer (25 mM, pH 5.8) warmed to 37 °C. The extracted mRNA was analyzed for concentration and purity by NanoDrop measurement of absorbance at 260, 280, and 230 nm, and the purity was evaluated as A260 / A280 > 2 and A260 / A230 > 2. The length was determined using agarose gel electrophoresis to verify that the mRNA remained intact. The extracted mRNA containing 1:100 SYBR Safe stain (Thermo Fisher Scientific) was loaded onto a 1% agarose gel and electrophoresed at 75 V using TAE buffer containing 1:5000 SYBR Safe stain.

[0148] Example 3: Formulation and Characterization of PACE Polyplexes The PACE polymer was synthesized and characterized as described above. All polyplexes were formulated at a polymer to mRNA weight ratio of 50:1. The PACE polymer was dissolved overnight at 100 mg / mL in DMSO (37 °C, shaking). Prior to the production of the polyplex, an optimal PACE polymer blend was generated by mixing a solution of the PACE polymer containing end group modification and polyethylene glycol tails. The mRNA and polymer were diluted in an equal volume 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 an additional 25 seconds. The polyplex was incubated at room temperature for 10 minutes before use.

[0149] Example 4: Vaccination Used vials of the Comirnaty vaccine were obtained from the Yale Health Pharmacy within 24 hours of opening and stored at 4 °C. The vials contained the remaining vaccine (diluted to 100 μg / mL according to the manufacturer's instructions), which was removed with a spinal syringe and pooled. The pooled remaining vaccine was aliquoted and stored at -80 °C. Mice were anesthetized by intraperitoneal injection with a mixture of ketamine (50 mg / kg) and xylazine (5 mg / kg). The vaccine was diluted in sterile PBS and injected into the left quadriceps muscle using a 31-g syringe at a final dose of 1 μg or 0.05 μg as indicated in 10 μL or 20 μL. For intranasal vaccination with SARS-CoV-2 stabilized spike (ACRO biosystems, SPN-C52H9) or SARS-CoV-1 spike (ACRO biosystems, SPN-S52H6), it was reconstituted with endotoxin-free sterile water according to the manufacturer's protocol, then diluted in sterile PBS and stored at -80 °C. Mice were anesthetized using 30% v / v isoflurane diluted in propylene glycol and administered 1 μg or 5 μg (as indicated) in 50 μL via the IN route. For IN mRNA-PACE, 50 μL of the polyplex in solution was given at the indicated dose.

[0150] Example 5: Virus Titer Analysis Mice were euthanized in 100% isoflurane at the indicated time points. Approximately 50% of the whole lungs were placed in a bead homogenizer tube containing 1 mL of PBS with 2% FBS and 2% antibiotics / antifungal agents (Gibco) and stored at -80°C. Debris was removed from the lung homogenate by centrifugation (3900 rpm, 10 minutes). The infectivity 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. Plaques were lysed by fixing in 10% neutral buffered formalin for 1 hour at 40 - 42 hours post-infection, and then stained with 0.5% crystal violet in 20% ethanol for 30 minutes. The plates were rinsed with water and the plaques were visualized.

[0151] Example 6: Measurement of SARS-CoV-2-specific antibodies ELISA was performed as previously described, with the modifications described for convenience and reproduced here. A 96-well MaxiSorp plate (Thermo Scientific #442404) was coated with 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, 50 μL / well, and incubated overnight at 4°C. The coating buffer was removed, and the plate was incubated with 250 μL of blocking solution (PBS containing 0.1% Tween-20 and 5% milk powder) for 1 hour at room temperature. Serum or bronchoalveolar lavage fluid (BALF) was diluted in a dilution solution (PBS containing 0.1% Tween-20 and 2% milk powder), 100 μL of diluted serum or BALF was added, and the plate was incubated for 2 hours at room temperature. The plate was washed 5 times with PBS-T (PBS containing 0.05% Tween-20) using an automated plate washer (250 μL per cycle), and 50 μL of HRP-conjugated anti-mouse IgG (Cell Signaling Technology #7076, 1:3,000) or HRP-conjugated anti-mouse IgA (Southern Biotech #1040-05, 1:1,000) diluted in the dilution solution was added to each well. After incubation for 1 hour at room temperature, the plate was washed 3 times with PBS-T using an automated plate washer. The plate was developed with 50 μL of TMB substrate reagent set (BD Biosciences #555214), and the reaction was stopped after 15 minutes by adding 50 μL of 2N sulfuric acid. The plate was then read at wavelengths of 450 nm and 570 nm, and the difference was reported.

[0152] Example 7: Immunohistochemistry and pathological analysis Yale pathologists performed embedding, sectioning, and H&E staining of lung tissue. A lung pathologist blinded and examined the slides to identify immune cell infiltration and other related pathologies. Scoring was done as follows from 1 - 4: (1) mild patchy mononuclear infiltration, parenchymal and perivascular, variable reactive pneumocytes and interstitial reaction; (2) moderate patchy mononuclear infiltration, parenchymal and perivascular, variable reactive pneumocytes and interstitial reaction; (3) mild, high density mixed infiltrate containing mononuclear cells and granulocytes / neutrophils; (4) moderate, high density mixed infiltrate containing mononuclear cells and granulocytes / neutrophils.

[0153] Example 8: Intravascular Labeling, Cell Isolation, and Flow Cytometry To distinguish intravascular cells from extravascular cells, mice were anesthetized with 30% isoflurane, and APC / Fire 750 CD45 Ab (30-F11, AB_2572116, BioLegend, #103154) was injected iv. After 3 minutes of labeling, the mice were euthanized. Lungs were excised with scissors and incubated in a digestion cocktail containing 1 mg / mL of collagenase A (Roche) and 30 μg / mL of DNase I (Sigma-Aldrich) in RPMI at 37 °C for 45 minutes. Next, the tissue was filtered through a 70 μm filter. Cells were treated with ammonium chloride-potassium buffer and resuspended in PBS containing 1% BSA. The single-cell suspension was incubated with Fc block and Aqua cell viability dye at 4 °C for 20 minutes. After washing the cells once with PBS, surface staining was performed. For T cell analysis, cells were stained 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 (PerCP / Cy5.5, 16-10A1, AB_2566491, BioLegend #305232), anti-CD69 (PE / Cy7, H1.2F3, AB_493564, BioLegend #104512), anti-CD183 (CXCR3) (APC, CXCR3-173, AB_1088993, BioLegend #126512), anti-CD4 (AF700, GK 1.5, AB_493699, BioLegend #100430), and PESARS-CoV-2 S539~546 MHC class I tetramer (H-2K(b)) at 4 °C for 30 minutes.For B cell analysis, cells were stained with anti-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 Biotech #1040-02), anti-B220 (PerCP / Cy5.5, RA3-6B2, AB_893354, BioLegend #103236), PE-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) at 4°C for 30 minutes. After washing with PBS, cells were fixed using 4% paraformaldehyde. Cell population data were acquired with an Attune NxT Flow Cytometer and analyzed using FlowJo Software (10.5.3, Tree Star).

[0154] Example 9: Production of SARS-CoV-2 Receptor-Binding Domain B Cell Tetramers Recombinant SARS-CoV-2 spike RBD His biotin protein, CF (R&D / BT10500-050), was incubated with either streptavidin-PE (Prozyme PJRS25) or streptavidin-APC (Prozyme PJ27S) at a molar ratio of 4:1 at 4°C for 30 minutes. The mixture was then purified with an Amicon Ultra (50kDA MWCO) spin column, concentrated, and washed once with sterile cold PBS. Concentration was determined using the specific absorbance of the fluorophore with a Nanodrop, and the tetramer was diluted to 1.0 μM in PBS and stored at 4°C.

[0155] Example 10: Production of Pseudovirus VSV-based pseudoviruses were generated as described above. The vector pCAGGS containing the SARS-CoV-2 Wuhan-Hu-1 spike glycoprotein gene was produced under HHSN272201400008C and obtained from BEI Resources (NR-52310). The sequence of the Wuhan-Hu-1 isolate spike glycoprotein is identical to the sequence of the USA-WA1 / 2020 isolate. The plasmid encoding SARS-CoV-1 spike was provided by Dr. Vincent Munster and has been described previously. 293T cells were transfected with any of the spike plasmids and then inoculated with replication-deficient VSV expressing Renilla luciferase at 37 °C for 1 hour. Then, the virus inoculum was removed and the cells were washed three times with warm PBS. Supernatants containing pseudoviruses were collected 24 and 48 hours after inoculation, clarified by centrifugation, concentrated using an Amicon Ultra centrifugal filter unit (100 kDa), and stored in aliquots at -80 °C. Pseudoviruses were titrated on Huh7.5 cells, achieving a relative light unit signal approximately 600-fold that of the cell-alone control background.

[0156] Example 11: Pseudovirus Neutralization Assay Vero E6 (Figs. 1A - 1D) or Huh7.5 cells (Figs. 5A - 5C) overexpressing hACE2 and TMPRSS2 were seeded into each well of a 96-well plate (3×10 4)。On the day of infection, sera and BALF were heat-inactivated at 56°C for 30 minutes. The sera in Figures 1A - 1D were tested at an initial dilution of 1:50, and the BALF samples were tested at an initial dilution of 1:4 (both with 8 two-fold serial dilutions). The sera in Figures 5A - 5C were tested at an initial dilution of 1:40 with 8 three-fold serial dilutions. The serial dilutions were mixed 1:1 with the pseudovirus and incubated at 37°C for 1 hour. Subsequently, the growth medium was aspirated from the cells and replaced with 100 μL of the serum / virus mixture. After 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 using a microplate reader (SpectraMax i3, Molecular Devices). The IC50 was calculated using Prism 9 (GraphPad Software) non-linear regression.

[0157] Example 12: IN boosting with non-adjuvant SARS-CoV-2 spike induces mucosal humoral immunity. To evaluate the potential of boosting IN non-adjuvant subunit vaccines for the development of respiratory mucosal immunity, the inventors determined to utilize the strong systemic immunogenicity of mRNA-LNP. The inventors further benefited from extensive SARS-CoV-2 spike engineering that serves to stabilize the protein in pre-fusion conformation by the addition of a C-terminal T4 fibritin trimerization motif, six proline substitutions (F817P, A892P, A899P, A942P, K986P, V987P), and alanine substitutions (R683A and R685A) at the furin cleavage site. This series of mutations has been shown to significantly enhance immunogenicity and increase protein stability, and some of these are used in current vaccines.

[0158] K18-hACE2 mice were vaccinated with 1 μg of mRNA-LNP (Comirnaty) by IM injection (prime), and 14 days later, 1 μg of recombinant non-adjuvant spike protein was inoculated by IN administration (prime and spike). Additional control groups included K18-hACE2 mice that received only the IM prime, and mice that received only the IN spike at the time of boosting. Mice were euthanized on day 21 or 28 (7 or 14 days after boost) to evaluate the development of mucosal humoral immunity (Figure 1A).

[0159] First, anti-SARS-CoV-2 spike S1 IgG and IgA in nasal wash, bronchoalveolar lavage fluid (BALF), and serum were evaluated. It was found that only mice administered prime and spike developed high levels of anti-SARS-CoV-2 IgA and IgG in nasal wash and BALF (Figure 1B (B-E)). Neither IM prime alone nor IN spike alone was sufficient for the development of mucosal antibodies. In serum, IM prime alone was sufficient to induce low levels of IgA and IgG. However, prime and spike resulted in a significant systemic boost of both anti-spike S1 IgA and IgG (Figure 1B (F, G)). These increases in antibody levels correlated with increases in neutralizing titers in both BALF and serum (Figure 1C (H-K)). These results suggest that a single dose of non-adjuvant intranasal spike alone is not immunogenic, and that prior systemic priming (in this case with mRNA-LNP) is required for the induction of a strong mucosal and systemic antibody response by non-adjuvant spike.

[0160] Tissue-resident memory B cells in the lung (B RM) has been shown to assist the rapid recall response of antibodies secreted by B cells during secondary heterologous administration in the mouse influenza model and may be an important local immune effector in protecting against SARS-CoV-2. Using intravenous (IV) CD45 labeling combined with a B cell tetramer specific for the receptor-binding domain (RBD) of the spike protein, we found that prime and spike resulted in an increase in antigen-specific B cells in lung tissue (IV - CD19 + B220 + tetramer + )(Figure 1D (L)). Since the tetramer was only evaluated for RBD binding, we also examined the polyclonal tissue response, which is likely to represent a more complete set of B cells reactive to the entire spike in lung tissue. We found an increase in class-switched antibody-secreting cells (ASCs) (IV CD19 + / - CD138 + ) expressing IgA or IgG in lung tissue (Figure 1D (M, N)), and an increase in class-switched B RM (IV - CD19 + B220 + IgD - IgM - CD38 + ) expressing IgA or IgG (Figure 1D (O, P)). These results are consistent with an increase in mucosal antibody production and indicate that prime and spike induce a local B cell response in the lung.

[0161] Example 13: Prime and spike induce mucosal T cell immunity. Since it was found that prime and spike induce a mucosal humoral memory response in the airway, next, lung tissue resident memory T cells (T RMIt was decided to evaluate the induction of (). Conventional subunit vaccines are not strong inducers of antigen-specific T cell responses, but the inventors hypothesized that the immune memory generated by mRNA-LNP priming, which has been shown to be sufficient for the induction of T cell memory responses in both animal models and humans, enables a subunit-mediated T cell boost response. Similar to the above, CD45 IV labeling for distinguishing immune cells and circulation in lung tissue was combined with major histocompatibility complex (MHC) class I tetramers (VNFNFNGL) against the conserved SARS-CoV spike epitope. CD69 + and CD103 + A spike IV RM expressing standard markers of T - tetramer + CD8 + A significant induction of T cells was found. Furthermore, a significant increase in antigen-experienced CD4 + T cells (IVCD44 + CD4 + ) was found, and many of them also expressed the markers of T RM CD69 + and CD103 + in both lung tissue (Figure 2C (K - M)) and the lower airways recovered from BALF (Figure 2C (N - P)). These results indicate that prime and spike not only induce a humoral mucosal response, but also strongly induce CD8 + T RM and CD4 + T RM in the lung parenchyma and airways.

[0162] Example 14: Delayed interval prime and spike are sufficient to induce mucosal immunity. Prime and spike with a 14-day interval between priming and boosting were shown to significantly induce mucosal humoral and cellular immune memory responses, while there was interest in whether a delayed boost could also induce significant humoral and cellular responses. To test this question, K18-hACE2 mice administered 1 μg of IM prime were boosted with IN spike 84 days later. Humoral and cellular mucosal immune responses were sampled on day 91 (7 days after boost) and day 140 (56 days after boost) (Figure 6A). Delayed IN spike was found to be sufficient to induce CD8 + T RM which persisted for at least 56 days (Figure 6B (B-D)). CD4 + T RM was induced early at 7 days after boost, but its lifespan appeared to decrease to at least 56 days, at least polyclonally (Figure 6B (E-G)). Similar to the CD8 + T RM response, not only an appropriate humoral response to the delayed boost, but also mucosal IgA and IgG in BALF were strong and increased (Figure 6C (H, I)), and serum IgA and IgG were strong and increased at 56 days after boost (Figure 6C (J, K)). These results indicate that prime and spike administered at a 3-month dose interval are sufficient to elicit long-term mucosal and systemic humoral and cellular immune responses.

[0163] Example 15: IN delivery of mRNA polyplexes also mediates mucosal boosting. Next, the ability of an alternative platform for IN spike boost was evaluated. Poly(amine-co-ester) (PACE) is a biodegradable terpolymer developed to encapsulate nucleic acids such as mRNA or DNA depending on the polymer's properties and deliver them to specific tissues in vivo. Recent studies have shown that mRNA-LNPs delivered to the airways are lethally dose-dependent in mice. In contrast, PACE materials are developed to be relatively immunologically silent, allowing administration to sites susceptible to immune lesions such as the respiratory tract. To evaluate the safety and efficacy of PACE encapsulating mRNA encoding the spike protein, mRNA was extracted from comminati and encapsulated into PACE polyplexes. For vaccination, K18-hACE2 mice were injected with 1 μg of IM prime (mRNA-LNP), and 14 days later, 1 μg of mRNA encapsulated in PACE was given and administered by IN (PACE-spike). Additional control groups included PACE-spike only, and extracted mRNA (naked mRNA) without IM prime + PACE encapsulation (Figure 3A). Similar to what was found with prime and spike, prime and PACE-spike induced antigen-specific CD8 + and CD103 + expressing antigen-specific CD8 + T RM (IV - tetramer + ) (Figure 3B). Furthermore, PACE-spike boost mice developed high levels of BALF anti-SARS-CoV-2 IgA, and the levels of BALF IgG and serum IgA and IgG were similar to those of mice with IM prime only (Figure 3C). IM prime followed by IN naked mRNA was unable to induce a mucosal or systemic immune response beyond that of IM prime alone, indicating that encapsulation of mRNA by PACE is required for mucosal boost. Furthermore, a single dose of IN PACE-spike alone was insufficient to induce a detectable mucosal or systemic antibody response at this dose.

[0164] Example 16: Prime and Spike, or Prime and PACE-Spike in the context of reduced mRNA-LNP immunity protects from lethal SARS-CoV-2 challenge. Current vaccines were initially highly effective at inducing protective immunity, but antibody level decline and immune escape will likely require boosters against SARS-CoV-2 for the foreseeable future. However, the best method for boosting remains a question. To test whether IN administration provides an alternative protective boost, a low-dose mRNA-LNP vaccine challenge model was utilized to mimic waning immunity. Single-dose immunizations were performed using 0.05 μg of mRNA-LNP. These low-dose mRNA-LNP vaccinated mice uniformly developed a systemic antibody response, but we have previously shown that this dose is not sufficient to protect from SARS-CoV-2 challenge. Fourteen days after priming, mice were given IN Spike (1 μg of non-adjuvanted Spike protein). Similar to the 1 μg IM-primed mice described above, 0.05 μg IM-primed mice boosted with IN Spike developed significant increases in lung antigen-specific CD8 + T RM as well as IgA and IgG in the BALF at 42 days post-boost (Figure 7A). These data also show that even the very low levels of immune memory generated by low-dose mRNA-LNP priming can be effectively enhanced by non-adjuvanted IN Spike to induce mucosal and systemic humoral and cellular memory.

[0165] Next, naive, low-dose prime alone, and low-dose prime and Spike mice were challenged with 6×10 4Challenged with the homologous / ancestral WA1 strain of PFU SARS-CoV-2. Mice were euthanized at 2 DPI and the viral load was evaluated by plaque assay from the turbinates and lungs, or euthanized at 5 DPI and the lungs were evaluated for pathology, or body weight loss and mortality were observed for 14 days (Figure 4A). All mice administered prime and spike were completely protected from body weight loss or death, while naive mice and mice with low-dose prime alone were not protected from viral infection (Figure 4B(BD)). Furthermore, this significant improvement in morbidity and mortality in mice administered prime and spike was accompanied by a decrease in viral load in both the upper airway (turbinates) and the lower airway (lungs) (Figure 4B(E,F)). Additionally, prime and spike protected significantly from lung lesions, with only 1 out of 6 mice developing limited mononuclear infiltration at 5 DPI and the remaining mice being completely protected with lung pathology similar to that seen in uninfected mice (Figure 4B(G), Figure 4C). To evaluate the protective ability of the mRNA-PACE IN boost, the inventors reused the low-dose prime mRNA-LNP mice and boosted them IN with 10 μg of mRNA-PACE. Prime and PACE-spike were found to significantly protect from morbidity and mortality (Figure 4D, Figure 4E). These data suggest that either IN non-adjuvant spike or spike encoding mRNA-PACE is sufficient to boost mucosal immunity that protects from COVID-19, as well as lung disease and mortality in a preclinical mouse model. These results also highlight the robustness, versatility, and safety of this vaccine strategy, as intranasal boosting of systemic mRNA-LNP priming by either modality is sufficient to induce mucosal immunity and protect from lethal SARS-CoV-2 challenge.

[0166] Example 17: Prime and spike induce similarly strong systemic immunity as parenteral mRNA-LNP-based boost. mRNA-LNP-based vaccines administered by IM injection are the current standard boosting strategy recommended in many countries because research on immunogenicity and vaccine efficacy has focused most on this boosting method. To compare prime and spike with IM mRNA-LNP prime / boost, K18-hACE2 mice were primed with 1 μg of mRNA-LNP, followed 14 days later by boosting with either 1 μg of IN spike or 1 μg of IM mRNA-LNP. Mice were euthanized 31 days after boosting, and antigen-specific CD8 + T RM cells were evaluated by flow cytometry, antibodies from BALF and serum were evaluated by ELISA, and a VSV pseudovirus neutralization assay was performed to evaluate serum antibody neutralization responses (Figures 8A–8C). We found that both IM mRNA-LNP-boosted animals and IN spike-boosted animals had elevated levels of extravascular (IV - ) tetramer + CD8 + T cells. However, only IN spike-boosted animals developed CD8 + T RM cells and expressed CD69 + and CD103 + in the lungs (Figure 8B). By ELISA, we found that only IN spike-boosted animals developed anti-SARS-CoV-2 IgA in the BALF. The BALF IgG levels were similar between IM mRNA-LNP and IN spike-boosted mice, which may represent transcytosis due to elevated systemic antibody levels in IM mRNA-LNP-boosted mice. Similarly, comparable serum levels of anti-SARS-CoV-2 IgA and IgG were found in IM mRNA-LNP and IN spike-boosted mice. Neutralization assays from serum also showed similar IC50s between IM mRNA-LNP boosting and IN spike boosting. These data indicate that prime and spike induce systemic neutralizing antibody levels similar to those of IM mRNA-LNP boosting, which has been shown to be correlated with protection, and mucosal IgA and CD8 + T RMDemonstrate that it is induced independently.

[0167] Example 18: Heterologous spikes robustly induce cross-reactive immunity without original antigenic sin. The above experiments clearly demonstrate that boosting at a distinct anatomical location, in this case the respiratory mucosa, by either non - adjuvant subunit spike or spikes encoding PACE - spike enables the formation of new mucosal immune memory at the newly boosted site and improves systemic immunity to that antigen. In both non - adjuvant subunit spike and mRNA PACE, the boost antigen is homologous to the systemic priming antigen (mRNA - LNP). Currently circulating strains of SARS - CoV - 2, particularly Delta and Omicron, have significant changes in the sequence and structure of the spike protein. Delta has mutations of T19R, G142D, Δ156 - 157, R158G, Δ213 - 214, L452R, T478K, D614G, P681R, and D950N, while Omicron has mutations of A67V, Δ69 - 70, T95I, G142D, Δ143 - 145, N211I, L212V, ins213 - 214RE, V215P, R216E, 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. Due to these mutations, both Delta and Omicron are transmitted more rapidly and existing humoral immunity is evaded, and future variants may further diverge, suggesting that a boosting strategy that broadly induces reactive immunity is required to neutralize future variants. To test the ability of non - adjuvant heterologous spike proteins for IN boost, K18 - hACE2 mice were primed 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). SARS - CoV - 1 is a related sarbecovirus, but its spike protein shares only 76% homology with the original SARS - CoV - 2 spike sequence encoded by currently used mRNA - LNP vaccines.For comparison, mRNA-LNP-primed mice were also boosted with 1 μg of IM mRNA-LNP. 31 days after the boost, standard T cells were labeled with CD45 IV. RM marker CD69 + and CD103 + expressing IV - tetramers + CD8 +We found a significant increase in T cells (Figure 5B). As mentioned before, this MHC I tetramer sequence is highly conserved within the sarbecovirus family, of which both SARS-CoV-1 and SARS-CoV-2 are part. We then evaluated the development of anti-SARS-CoV-1 antibodies in BALF and serum and found a significant increase in anti-SARS-CoV-1 IgA and IgG in both the respiratory mucosa and circulation compared to IM mRNA-LNP prime / boost. Consistent with previous studies, we found that two doses of SARS-CoV-2 mRNALNP were sufficient to induce detectable antibodies that bound to the SARS-CoV-1 spike. We then evaluated anti-SARS-CoV-2 antibodies in BALF and serum. We found that prime and spike-X induced higher BALF IgA than IM SARS-CoV-2 mRNA-LNP prime / boost. Similar levels of anti-SARS-CoV-2 IgG were found in the BALF, which may represent the increased serum IgG we found (Figure 5C(I-L)). Next, using a VSV-based pseudovirus neutralization assay, we show that sera from prime and spike X mice generate higher neutralizing titers against SARS-CoV-1 than mice boosted with I SARS-CoV-2 mRNA-LNP (Figure 5C(M,N)). Similarly, consistent with serum IgG levels, IM SARS-CoV-2 mRNA-LNP prime / boost mice have significantly higher neutralizing titers against SARS-CoV-2 than prime and spike X mice (Figure 5C(O,P)). Taken together, these data indicate that IN boosting with non-adjuvanted heterologous spike protein can induce strong mucosal cellular and humoral memory against a significantly diverged spike protein in the absence of antigenic original sin.

[0168] Example 19: Prime and Spike induce mucosal immunity against SARS-CoV-2 During the SARS-CoV-2 pandemic over the past two years, vaccines containing modified mRNA encapsulated in lipid nanoparticles (LNPs) have been highly effective. In phase 3 clinical trials and subsequent post-marketing tests of vaccine efficacy, initially, vaccine efficacy of over 90% was shown against symptomatic disease. Unfortunately, more recent studies have shown that the efficacy of the vaccine decreases not only for symptomatic and severe infections but also for asymptomatic infections starting approximately four months after the second dose in mRNA-LNP-based regimens. This trial is to analyze the effect of prime and spike on patients previously vaccinated against SARS-CoV-2 or patients infected with SARS-CoV-2. For this purpose, patients previously vaccinated against SARS-CoV-2 or patients infected with SARS-CoV-2 were equally divided into two groups. One group was the control group and was administered an intranasal formulation containing placebo, and the other group was administered an intranasal formulation containing an mRNA vaccine approved by the FDA. 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's infection or vaccination. To evaluate the effectiveness of prime and spike, CD8 + T cells, CD4 + T cells, memory T cells (T RM ) and B cells (B RM ), as well as mucosal IgG and dimeric IgA, are measured at the mucosal sites where the vaccine is administered.

[0169] Example 20: Prime and spike induce mucosal immunity against human papillomavirus (HPV) This test is for analyzing the effects of prime and spike on patients who were previously vaccinated against HPV or patients infected with HPV. For this purpose, patients who were previously vaccinated against HPV or patients infected with HPV were equally divided into two groups. One group was the control group, which was administered an intravaginal preparation containing placebo, and the other group was administered an intravaginal preparation containing a vaccine against HPV. The administration was carried out 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's infection or vaccination. To evaluate the effectiveness of prime and spike, CD8 + T cells, CD4 + T cells, memory T cells (T RM ) and B cells (B RM ), as well as mucosal IgG and dimeric IgA, were measured at the mucosal site where the vaccine was administered.

[0170] Example 21: Prime and spike induce mucosal immunity against rotavirus This test is for analyzing the effects of prime and spike on patients who were previously vaccinated against rotavirus or patients infected with rotavirus. For this purpose, patients who were previously vaccinated against HPV or patients infected with rotavirus were equally divided into two groups. One group was the control group, which was administered an oral preparation containing placebo, and the other group was administered an oral preparation containing a vaccine against rotavirus. The administration was carried out 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's infection or vaccination. To evaluate the effectiveness of prime and spike, CD8 + T cells, CD4 + T cells, memory T cells (T RM ) and B cells (B RM ), as well as mucosal IgG and dimeric IgA, were measured at the mucosal site where the vaccine was administered.

Claims

1. A pharmaceutical composition for use in enhancing an immune response to an antigen in a human being requiring enhancement of such enhancement in a mucosal site, wherein the pharmaceutical composition comprises the antigen or a nucleic acid encoding the antigen, the human being has been previously vaccinated against or infected with the virus, and the pharmaceutical composition further comprises lipid nanoparticles (LNPs) containing a poly(amine-co-ester) (PACE) polymer, or lipid nanoparticles (LNPs) containing a cationic lipid selected from 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), and / or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), wherein the antigen is encapsulated within the lipid nanoparticles (LNPs).

2. The pharmaceutical composition for use according to claim 1, wherein the human being is parenterally vaccinated against the virus.

3. The pharmaceutical composition for use according to claim 1, wherein the antigen is a polyvalent antigen.

4. An effective amount of a pharmaceutical composition for use in enhancing an immune response to an antigen in a human being who requires enhancement of such response in a mucosal area, wherein the pharmaceutical composition comprises the antigen or a nucleic acid encoding the antigen, and the human being has elevated antibodies, memory B cells, effector CD4 cells, etc., caused by prior vaccination against a virus or prior viral infection. ＋ and / or CD8 ＋ A pharmaceutical composition for use, comprising T cells, wherein the elevated antibody is immunoglobulin G (IgG), IgM, IgA, or a combination thereof.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the mucous membrane portion is the nasal cavity.

6. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the antigen comprises at least a protein or polypeptide.

7. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the nucleic acid is DNA or RNA.

8. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the nucleic acid is mRNA.

9. The pharmaceutical composition for use according to claim 8, wherein the mRNA is N1-methyl-psoidouridine modified mRNA or pseudoouridine modified mRNA.

10. The aforementioned antigen is derived from Mycobacterium, bacteria, fungi, viruses, parasites, or prions. The aforementioned viruses include rotavirus, norovirus, adenovirus, astrovirus, influenza virus, respiratory syncytial virus, parainfluenza virus, metapneumovirus, rhinovirus, coronavirus, adenovirus, bocavirus, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), human papillomavirus (HPV), human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, herpesvirus, adenovirus, polio, Japanese encephalitis, smallpox, influenza virus, flavivirus, and echovirus. Selected from the group consisting of rhinovirus, coxsackievirus, coronavirus, respiratory mixed virus (RSV), mumps virus, rotavirus, paralytic virus, rubella virus, parvovirus, vaccinia virus, human T-lymphotropic virus (HTLV), dengue virus, human papillomavirus (HPV), ointment virus, poliovirus, rabies virus, JC virus, arboviral encephalitis virus, SARS-CoV-2, Henoch-Schönlein purpura (HSP), RNA viruses, DNA viruses, their variants, and any combination thereof, The RNA virus is selected from the group consisting of the common cold, influenza, SARS, MERS, Covid-19, dengue virus, hepatitis C, hepatitis E, West Nile fever, Ebola virus disease, rabies, polio, mumps, rubella, their variants, and any combination thereof. The DNA virus is selected from the group consisting of herpes simplex virus, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus, roseolovirus, human herpesvirus-7, Kaposi's sarcoma-associated virus, their variants, and any combination thereof. A pharmaceutical composition for use according to any one of claims 1 to 4.

11. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the mucosal area is selected from the group consisting of the anterior external nostrils, nasal cavity, rectum, vagina, esophagus, urethra, sublingual area, and buccal area.

12. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the pharmaceutical composition does not contain an adjuvant.

13. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the pharmaceutical composition comprises an adjuvant.

14. A pharmaceutical composition for use according to any one of claims 1 to 4, wherein the lipid nanoparticles (LNPs) further comprise at least one phospholipid selected from 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).

15. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the person has been vaccinated against the virus or has been infected with the virus about one week, two weeks, three weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or twelve months prior to the use of the pharmaceutical composition on the mucous membrane site.

16. The antigen is SARS-CoV-2, The aforementioned 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 Univ), Ad5 vectorized COVID-19 vaccine (CanSino Biologies), CoronaVac (Sinovac), NVX-CoV2373 (Novavax), and combinations thereof. The mRNA encodes the SARS-CoV-2 spike protein, or a variant thereof, or a fragment thereof. A pharmaceutical composition for use according to any one of claims 1 to 4.

17. The pharmaceutical composition comprises mRNA encoding two or more different antigens, The two or more antigens are the SARS-CoV-2 spike protein, a variant thereof, or a fragment thereof. The two or more antigens include at least one mutation listed in Table 1. A pharmaceutical composition for use according to any one of claims 1 to 4.

18. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the lipid nanoparticles have an average diameter 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.

19. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the immune response is a mucosal immune response selected from antigen-specific IgA antibody production, antigen-specific IgG antibody production, and antigen-specific IgM antibody production.

20. The pharmaceutical composition for use according to claim 16, wherein the human has elevated neutralizing antibody levels caused by prior vaccination against a virus selected from the group consisting of MERS-CoV, SARS-CoV-1, SARS-CoV-2, and their variants, or by prior infection with such viruses, and the elevated neutralizing antibody is IgG, IgM, IgA, or a combination thereof.

21. The pharmaceutical composition for use according to claim 16, wherein the SARS-CoV-2 variant is selected from the group consisting of alpha (B.1.1.7 and Q lineage), beta (B.1.351 and progeny lineage), gamma (P.1 and progeny lineage), delta (B.1.617.2 and AY lineage), epsilon (B.1.427 and B.1.429), eta (B.1.525), iota (B.1.526), ​​kappa (B.1.617.1), 1.617.3, Mu (B.1.621, B.1.621.1), zeta (P.2), Mu (B.1.621, B.1.621.1), O micron (Pango lineage B.1.1.529, BA.1, BA.1.1, BA.2, BA.3), and combinations thereof.

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