Serial vaccinations using mannan adjuvants

Abstract

Provided herein are serial vaccination methods to induce an immune response against a pathogen (e.g., Beta coronavirus) comprising administering a first composition comprising a nucleic acid encoding a first antigen (e.g., a first Beta coronavirus antigen) and administering a second composition comprising a nucleic acid encoding an second antigen (e.g., a second Beta coronavirus antigen), and wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan (e.g., a plant mannan or a fungal mannan).

Description

[0001] SERIAL VACCINATIONS USING MANNAN ADJUVANTS

[0002] RELATED APPLICATIONS

[0003] This Application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 709138, filed on October 18, 2024, the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (C123370291WO00-SEQ-ACZ.xml; Size: 20,540 bytes; and Date of Creation: October 17, 2025) is herein incorporated by reference in their entirety.

[0006] FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under Grant no. AI165505, awarded by The National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND OF INVENTION

[0009] Serial vaccination is a strategy used to maintain or enhance immunity against a pathogen over time, especially against pathogens that frequently mutate (e.g., a Beta coronavirus). However, serial vaccination has been linked a phenomenon termed “antigenic sin” in which the immune system responds more strongly to antigens from the original strain rather than a new variant, reducing the effectiveness of vaccines targeting new strains.

[0010] SUMMARY OF INVENTION

[0011] Adjuvants can be used for the enhancement of vaccine immunogenicity, broadening the response of the immunogen to other antigens, particularly in situations where antigenic sin may limit the immune response to newer antigens and / or in non- or less-responding individuals. However, the immunological benefit of adding adjuvants to vaccine formulations and their utility in serial vaccination strategies to overcome antigenic sin is not always replicated in clinical studies. The inventors of this disclosure surprisingly discovered that the antigenic sin can be reduced and / or eliminated using a mannan adjuvant in the vaccine composition.

[0012] Accordingly, some aspects of the present disclosure provide a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising: (i)

[0013] 12413624.1 administering to the subject a first composition comprising a nucleic acid encoding a first antigen; and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen; wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan. In some embodiments, the first composition comprises the adjuvantation system comprising the mannan. In some embodiments, the second composition comprises the adjuvantation system comprising the mannan. In some embodiments, the first composition and the second composition comprise an adjuvantation system comprising a mannan.

[0014] In some embodiments, the method further comprises administering to the subject a third composition comprising a nucleic acid encoding a third antigen. In some embodiments, the third composition is administered to the subject prior to administering the second composition. In some embodiments, the third composition comprises an adjuvantation system comprising a mannan.

[0015] In some embodiments, the mannan is a plant mannan or a fungal mannan. In some embodiments, the mannan is a glucomannan, a galactomannan, or a galactoglucomannan. In some embodiments, the plant mannan is konjac mannan, aloe vera mannan, salep mannan, porang mannan, ivory nut mannan, cassia gum mannan, locust bean gum mannan, guar gum mannan, tar gum mannan, fenugreek gum mannan, or Norway spruce mannan. In some embodiments, the plant mannan is a konjac glucomannan. In some embodiments, the fungal mannan is a yeast mannan. In some embodiments, the fungal mannan is a Candida albicans mannan.

[0016] In some embodiments, the adjuvantation system further comprises an aluminum salt. In some embodiments, the aluminum salt is aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is between 1:1 and 1:100. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or 1:100.

[0017] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the RNA is a messenger RNA (mRNA).

[0018] In some embodiments, the first antigen is a bacterial antigen, a viral antigen, or a fungal antigen. In some embodiments, the viral antigen comprises a Beta coronavirus protein or polypeptide. In some embodiments, the Beta coronavirus protein or polypeptide comprises a

[0019] 12413624.1 Beta coronavirus spike protein or spike protein receptor binding domain (RBD). In some embodiments, the Beta coronavirus spike protein is a MERS-CoV spike protein, a SARS-CoV-1 spike protein, or a SARS-CoV-2 spike protein. In some embodiments, the Beta coronavirus spike protein RBD is a MERS-CoV spike protein RBD, a SARS-CoV-1 spike protein RBD, or a SAS-CoV-2 spike protein RBD.

[0020] In some embodiments, the second antigen is a variant of the first antigen. In some embodiments, the third antigen and the first antigen are the same.

[0021] In some embodiments, the ratio of the nucleic acid to the adjuvantation system is between 1:1 and 1:100. In some embodiments, the ratio of the nucleic acid to the adjuvantation system is 1:1, 1:10, or 1:100. In some embodiments, the adjuvantation system and the nucleic acid are admixed.

[0022] In some embodiments, the second composition is administered between 1 week and 6 weeks after administration of the first composition. In some embodiments, the second composition is administered about 2 weeks after administration of the first composition. In some embodiments, the third composition is administered between 6 weeks and 10 weeks after administration of the first composition. In some embodiments, the third composition is administered about 8 weeks after administration of the first composition.

[0023] In some embodiments, the adjuvantation system increases recruitment of immune cells in the subject, compared to when the nucleic acid is administered alone. In some embodiments, the immune cells are B cells or T cells.

[0024] In some embodiments, the subject is a human. In some embodiments, the subject is a human neonate, a human infant, an adult human, or an elderly human. In some embodiments, the subject is a companion animal or a research animal. In some embodiments, the subject is immune-compromised, has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease.

[0025] In some embodiments, the administration is intramuscular administration, intradermal administration, oral administration, intravenous administration, topical administration, intranasal administration, or sublingual administration. In some embodiments, the administration is intramuscular administration. In some embodiments, the administration is prophylactic.

[0026] In some embodiments, the first composition and the second composition comprise different mannans. In some embodiments, the first composition and the second composition comprise different amounts of the same mannan.

[0027] 12413624.1 In some aspects, this disclosure provides a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising: (i) administering to the subject a first composition comprising a nucleic acid encoding a first SARS-CoV-2 spike protein; and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second SARS-CoV-2 spike protein and an adjuvantation system comprising a mannan and an aluminum salt.

[0028] BRIEF DESCRIPTION OF DRAWINGS

[0029] The accompanying drawings are not intended to be drawn to scale. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0030] FIG. 1 is a schematic of a vaccine administration schedule where mice were injected with Ancestral Comirnaty (BNT162b2 mRNA, PfizerO-BioNTech®) on days 0 and 14, followed by a dose of Updated Comirnaty XBB.1.5 (Pfizer®-BioNTech®) with or without alum-mannan on day 56.

[0031] FIG. 2 shows neutralization titers of a SARS-CoV-2 pseudovirus expressing the Spike protein of SARS-CoV-2 BAA from mouse sera collected on days 70 and 84 post-initial immunization, ns = not significant; ** p < 0.01.

[0032] FIG. 3 shows INF-y levels in mouse sera collected on days 70 and 84 post-initial immunization, ns = not significant; *** p< 0.001; **** p < 0.0001.

[0033] FIG. 4 shows germinal center B cell counts in mouse draining lymph nodes collected up to 84 days post-initial immunization that recognize the SARS-Cov-2 WAI Spike protein (left panel), the SARS-CoV-2 BA.4 / 5 Spike protein (middle panel), or the SARS-CoV-2 XBB.1.5 Spike protein (right panel), ns = not significant; * p< 0.05.

[0034] FIG. 5 shows neutralization titers of a SARS-CoV-2 pseudovirus expressing the Spike protein of SARS-CoV-2 XBB.1.5 (left) or a SARS-CoV-2 pseudovirus expressing the Spike protein of SARS-CoV-2 BA.5 (right) from mouse sera collected on days 70 post-initial immunization, ns = not significant; * p < 0.1; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0035] DETAILED DESCRIPTION OF INVENTION

[0036] Antigenic sin refers to a phenomenon in which the immune system preferentially relies on memory responses from an encounter with a pathogen, even when it encounters a slightly

[0037] 12413624.1 different strain of that pathogen later on, resulting in a situation where the immune system may not be optimal for targeting the new, slightly different strain. This can influence the effectiveness of a vaccine or immunogenic composition, especially when the target pathogen mutates frequently (e.g., Beta coronavirus). For example, booster shots designed to target new variants of a pathogen may not be as effective if the immune system favors the initial memory response. There are a number of strategies that have been employed to overcome antigenic sin, such as the development of broadly neutralizing vaccines or multivalent vaccines or the use of sequential and heterologous vaccination strategies. However, such vaccines can be challenging to develop and keep pace with mutations, and some individuals may retain cross-reactive immune memory from earlier variants that can interfere with their response to new strains.

[0038] Adjuvants can be used for the enhancement of vaccine immunogenicity, amplifying the body’s response to an antigen by stimulating various pathways in the immune system, increasing antigen presentation, and promoting a more robust immune memory. However, the immunological benefit of adding adjuvants to vaccine formulations is not always replicated in clinical studies.

[0039] Accordingly, aspects of this disclosure provide a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising (i) administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., of the pathogen); and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen (e.g., of the pathogen), wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan.

[0040] A ’’pathogen” is an agent that can cause disease (e.g., SARS-CoV2) in its host (e.g., a human). Pathogens include bacteria, viruses, fungi, parasites, and prions that are spread through various means, such as direct contact, contaminated food or water, airborne transmission, or through vectors. When a pathogen enters the body, antigens present on its surface are recognized by the host’s immune system as “non-self,” triggering an immune response. Not all pathogens cause disease when they infect a host. A disease state outcome is dependent on a number of factors, such as the pathogen’s virulence and the host’s immune response.

[0041] In some embodiments, the pathogen is a bacterium. A “bacterium” is a single-celled microorganism that lacks a true nucleus and other membrane-bound organelles. Non-limiting examples of bacteria include Campylobacter spp., Clostridioides difficile, Haemophilus ducreyi,

[0042] 12413624.1 Clostridium perfringens, Escherichia coli, Klebsiella granulomatis, Haemophilus influenzae, Legionella spp., Leptospira spp., Listeria monocytogenes, Borrelia burgdorfei, Borrelia mayonii, Neisseria meningitidis, Yersinia pestis, Chlamydia psittaci, Coxiella burnetii, Ricinus communis, Rickettsia rickettsia, Salmonella spp., Shigella spp., Staphylococcus aureus, Streptococcus spp., Clostridium tetani, Mycobacterium tuberculosis, Francisella tularensis, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, or Yersinia enterocolitica.

[0043] In some embodiments, the pathogen is a fungus. A “fungus” is a eukaryotic organism that has a defined nucleus and membrane-bound organelles. Non-limiting examples of fungi include Coccidioides spp., Blastomyces spp., Cryptococcus gattii, Histoplasma spp., Paracoccidioides spp., Pneumocystis jirovecii, Candida spp., Cryptococcus neoformans, Talaromyces spp., or Aspergillus spp.

[0044] In some embodiments, the pathogen is a virus. A “virus” is an microscopic agent that comprises genetic material (e.g., DNA or RNA) enclosed in a protein coat. Unlike bacteria and fungi, viruses lack the machinery necessary for metabolism and reproduction on their own. Nonlimiting examples of viruses include Alpha coronavirus, Beta coronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2), Alphavirus (e.g., Barmah Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Getah virus, Sagiyama virus, Mayaro virus, O’nyong-nyong virus, Ross river virus, Semliki forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), Arenavirus (e.g., Junin arenavirus, Lassa virus, Lymphocytic choriomeningitis virus, Machupo virus), Bandavirus (e.g., Severe fever with thrombocytopenia syndrome virus), Bocaparvovirus (e.g., Human bocavirus), Cardiovirus (e.g., Encephalomyocarditis virus), Coltivirus (e.g., Colorado tick fever virus, California hare coltivirus, Eyach virus), Cytomegalovirus (e.g., Human cytomegalovirus), Deltaretrovirus (e.g., Human T-lymphotropic virus), Delta virus (e.g., Hepatitis delta virus), Ebolavirus, Enterovirus (e.g., Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Coxsackievirus A, Coxsackievirus B, Echovirus, Human enterovirus D68, Poliovirus), Erythrovirus (e.g., Human parvovirus B19), Flavivirus (e.g., Murray valley encephalitis virus, Dengue virus, Saint Louis encephalitis virus, Tick-borne encephalitis virus, Powassan virus, Kyasanur Forest disease virus, Alkhurma virus, Omsk hemorrhagic fever virus, West Nile virus, Yellow fever virus, Zika virus), Hantavirus (e.g., Puumala virus, Saaremaa virus, Dobrava virus), Henipavirus (e.g., Hendra virus, Langya virus, Nipah virus), Hepacivirus (e.g., Hepatitis C virus), Hepatovirus (e.g., Hepatitis A virus), Hepevirus (e.g., Hepatitis E virus), Influenzavirus A, Influenzavirus B,

[0045] 12413624.1 Influenzavirus C, Lentivirus (e.g., Human immunodeficiency virus 1, Human immunodeficiency virus 2), Lymphocryptovirus (e.g., Epstein-Barr virus), Lyssavirus (e.g., Mokola virus, Lagos bat virus, Shimoni bat virus, Rabies virus, Aravan virus, Duvenhage virus, Australian bat lyssavirus, European bat lyssavirus, Irkut virus, Khujand virus), Mamastrovirus (e.g., Human astrovirus), Marburgvirus (e.g., Lake Victoria Marburgvirus), Mastadenovirus (e.g., Human adenovirus), Metapneumovirus (e.g., Human metapneumovirus), Molluscipoxvirus (e.g., Molluscum contagiosum virus), Morbilivirus (e.g., Measles virus), Norovirus, Orthobunyavirus (e.g., Oropouche virus, Batai virus, Bunyamwera virus, Cache Valley virus, Ngari virus), Orthoflavivirus (e.g., Japanese encephalitis virus), Orthohantavirus (e.g., Hantaan virus, Sin Nombre virus), Orthohepadnavirus (e.g., Hepatitis B virus), Orthonairovirus (e.g., Dugbe virus, Crimean-Congo hemorrhagic fever virus), Orthopneumovirus (e.g., Respiratory syncytial virus), Orthopoxvirus (e.g., Monkeypox virus, Cowpox virus, Small pox virus, Tanapox virus, Yaba- like disease virus, Vaccinia virus, Yaba monkey tumor virus), Orthorubulavirus (e.g., Human parainfluenzavirus 2, Human parainfluenzavirus 4), Papillomaviridae (e.g., Human papillomavirus), Parapoxvirus (e.g., Orf virus), Phlebovirus (e.g., Punta toro phlebovirus, Rift valley fever virus, Sandfly fever Naples phlebovirus, Uukuniemi virus), Polyomavirus (e.g., BK polyomavirus, JC polyomavirus, KI polyomavirus, Merkel cell polyomavirus, WU polyomavirus), Respirovirus (e.g., Human parainfluenzavirus 1, Human parainfluenzavirus 3), Rhadinovirus (e.g., Human herpesvirus 8), Rosavirus (e.g., Rosavirus A), Rotavirus (e.g., Rotavirus A, Rotavirus B, Rotavirus C), Rubivirus (e.g., Rubella virus), Rubulavirus (e.g., Mumms virus), Salivirus (e.g., Salivirus A), Sapovirus (e.g., Sapporo virus), Seadomavirus (e.g., Banna virus), Simplexvirus (e.g., Human herpesvirus 1, Human herpesvirus 2, Macacine alphaherpesvirus), Thogotovirus (e.g., Dhori virus, Batken virus, Bourbon virus, Thogotobirus), Torovirus (e.g., Human torovirus), or Varicellovirus (e.g., Varicella-zoster virus), Vesiculovirus (e.g., Chandipura virus, Indiana vesiculovirus, Isfahan virus).

[0046] In some embodiments, a composition of the present disclosure comprises a nucleic acid encoding an antigen e.g., of the pathogen. An “antigen” refers to an entity that is bound by an antibody or receptor, or an entity that induces the production of the antibody (e.g., in a subject). In some embodiments, an antigen increases the production of antibodies that specifically bind the antigen. In some embodiments, an antigen comprises a protein or polypeptide. In some embodiments, the antigen is from a microbial pathogen. In some embodiments, the antigen is a bacterial antigen, a fungal antigen, or a viral antigen. In some embodiments, the antigen may

[0047] 12413624.1 comprise parts (e.g., coats, capsules, cell walls, flagella, fimbriae, toxins) of bacteria, viruses, fungi, and other microorganisms.

[0048] In some embodiments, the antigen is a bacterial antigen. A “bacterial antigen” is an antigen that originates from bacteria or has a sequence that is identical or substantially similar (homologous) to an endogenous bacterial protein or bacterial polypeptide. In some embodiments, the bacterial antigen may comprise parts of Campylobacter spp., Clostridioides difficile, Haemophilus ducreyi, Clostridium perfringens, Escherichia coli, Klebsiella granulomatis, Haemophilus influenzae, Legionella spp., Leptospira spp., Listeria monocytogenes, Borrelia burgdorfei, Borrelia mayonii, Neisseria meningitidis, Yersinia pestis, Chlamydia psittaci, Coxiella burnetii, Ricinus communis, Rickettsia rickettsia, Salmonella spp., Shigella spp., Staphylococcus aureus, Streptococcus spp., Clostridium tetani, Mycobacterium tuberculosis, Francisella tularensis, Salmonella typhi, Salmonella paratyphi, Vibrio cholerae, or Yersinia enterocolitica.

[0049] In some embodiments, the antigen is a fungal antigen. A “fungal antigen” is an antigen that originates from fungi or has a sequence that is identical or substantially similar (homologous) to an endogenous fungal protein or fungal polypeptide. In some embodiments, the fungal antigen may comprise parts of Coccidioides spp., Blastomyces spp., Cryptococcus gattii, Histoplasma spp., Paracoccidioides spp., Pneumocystis jirovecii, Candida spp., Cryptococcus neoformans, Talaromyces spp., or Aspergillus spp.

[0050] In some embodiments, the antigen is a viral antigen. A “viral antigen” is an antigen that originates from a virus or has a sequence that is identical or substantially similar (homologous) to an endogenous viral protein or viral polypeptide. In some embodiments, the viral antigen may comprise parts of an Alpha coronavirus, a Beta coronavirus (e.g., MERS-CoV, SARS-CoV-1, SARS-CoV-2), Alphavirus (e.g., Barmah Forest virus, Chikungunya virus, Eastern equine encephalitis virus, Getah virus, Sagiyama virus, Mayaro virus, O’nyong-nyong virus, Ross river virus, Semliki forest virus, Sindbis virus, Venezuelan equine encephalitis virus, Western equine encephalitis virus), Arenavirus (e.g., Junin arenavirus, Lassa virus, Lymphocytic choriomeningitis virus, Machupo virus), Bandavirus (e.g., Severe fever with thrombocytopenia syndrome virus), Bocaparvovirus (e.g., Human bocavirus), Cardiovirus (e.g., Encephalomyocarditis virus), Coltivirus (e.g., Colorado tick fever virus, California hare coltivirus, Eyach virus), Cytomegalovirus (e.g., Human cytomegalovirus), Deltaretrovirus (e.g., Human T-lymphotropic virus), Delta virus (e.g., Hepatitis delta virus), Ebolavirus, Enterovirus

[0051] 12413624.1 (e.g., Human rhinovirus A, Human rhinovirus B, Human rhinovirus C, Coxsackievirus A, Coxsackievirus B, Echovirus, Human enterovirus D68, Poliovirus), Erythrovirus (e.g., Human parvovirus B19), Flavivirus (e.g., Murray valley encephalitis virus, Dengue virus, Saint Louis encephalitis virus, Tick-borne encephalitis virus, Powassan virus, Kyasanur Forest disease virus, Alkhurma virus, Omsk hemorrhagic fever virus, West Nile virus, Yellow fever virus, Zika virus), Hantavirus (e.g., Puumala virus, Saaremaa virus, Dobrava virus), Henipavirus (e.g., Hendra virus, Langya virus, Nipah virus), Hepacivirus (e.g., Hepatitis C virus), Hepatovirus (e.g., Hepatitis A virus), Hepevirus (e.g., Hepatitis E virus), Influenzavirus A, Influenzavirus B, Influenzavirus C, Lentivirus (e.g., Human immunodeficiency virus 1, Human immunodeficiency virus 2), Lymphocryptovirus (e.g., Epstein-Barr virus), Lyssavirus (e.g., Mokola virus, Lagos bat virus, Shimoni bat virus, Rabies virus, Aravan virus, Duvenhage virus, Australian bat lyssavirus, European bat lyssavirus, Irkut virus, Khujand virus), Mamastrovirus (e.g., Human astrovirus), Marburgvirus (e.g., Lake Victoria Marburgvirus), Mastadenovirus (e.g., Human adenovirus), Metapneumovirus (e.g., Human metapneumovirus), Molluscipoxvirus (e.g., Molluscum contagiosum virus), Morbilivirus (e.g., Measles virus), Norovirus, Orthobunyavirus (e.g., Oropouche virus, Batai virus, Bunyamwera virus, Cache Valley virus, Ngari virus), Orthoflavivirus (e.g., Japanese encephalitis virus), Orthohantavirus (e.g., Hantaan virus, Sin Nombre virus), Orthohepadnavirus (e.g., Hepatitis B virus), Orthonairovirus (e.g., Dugbe virus, Crimean-Congo hemorrhagic fever virus), Orthopneumovirus (e.g., Respiratory syncytial virus), Orthopoxvirus (e.g., Monkeypox virus, Cowpox virus, Small pox virus, Tanapox virus, Yaba- like disease virus, Vaccinia virus, Yaba monkey tumor virus), Orthorubulavirus (e.g., Human parainfluenzavirus 2, Human parainfluenzavirus 4), Papillomaviridae (e.g., Human papillomavirus), Parapoxvirus (e.g., Orf virus), Phlebovirus (e.g., Punta toro phlebovirus, Rift valley fever virus, Sandfly fever Naples phlebovirus, Uukuniemi virus), Polyomavirus (e.g., BK polyomavirus, JC polyomavirus, KI polyomavirus, Merkel cell polyomavirus, WU polyomavirus), Respirovirus (e.g., Human parainfluenzavirus 1, Human parainfluenzavirus 3), Rhadinovirus (e.g., Human herpesvirus 8), Rosavirus (e.g., Rosavirus A), Rotavirus (e.g., Rotavirus A, Rotavirus B, Rotavirus C), Rubivirus (e.g., Rubella virus), Rubulavirus (e.g., Mumms virus), Salivirus (e.g., Salivirus A), Sapovirus (e.g., Sapporo virus), Seadomavirus (e.g., Banna virus), Simplexvirus (e.g., Human herpesvirus 1, Human herpesvirus 2, Macacine alphaherpesvirus), Thogotovirus (e.g., Dhori virus, Batken virus, Bourbon virus, Thogotobirus), Torovirus (e.g., Human torovirus), or Varicellovirus (e.g., Varicella-zoster virus), Vesiculovirus

[0052] 12413624.1 (e.g., Chandipura virus, Indiana vesiculovirus, Isfahan virus). In some embodiments, the antigen comprises a nucleic acid derived from or encode components of Orthomyxoviruses, Hepatitis C Virus (HCV), Ebola disease, polio, measles, adult Human T-cell lymphotropic virus type 1 (HTLV-1), lymphocytic choriomeningitis virus, human immunodeficiency virus (HIV), rhinoviruses, Influenza, SARS, MERS, COVID- 19, Dengue virus, Hepatitis C, Hepatitis E, West Nile fever, Ebola virus, respiratory syncytial virus, Rabies, mumps virus, hantavirus, Marburg virus, Lassa virus, parainfluenza, Monkey pox or combinations thereof.

[0053] In some embodiments, the antigen comprises a Beta coronavirus protein or polypeptide. “Beta coronavirus” is one of four genera (Alpha-, Beta-, Gamma-, and Delta-) of coronaviruses. Beta coronaviruses belong to the subfamily Orthocoronavirinae in the family Coronaviridae, of the order Nidovirales. They are enveloped, positive-sense, single-stranded RNA viruses of zoonotic origin. Beta coronaviruses of the greatest clinical significance to humans include SARS-CoV-1 (which causes Severe Acute Respiratory Syndrome, SARS), SARS-CoV-2 (which causes the disease Coronavirus Disease 2019, COVID-19), and MERS-CoV (which causes Middle East Respiratory Syndrome, MERS). In some embodiments, the Beta coronavirus protein or polypeptide comprises a Beta coronavirus spike protein, a Beta coronavirus nucleocapsid protein, a Beta coronavirus membrane protein, a Beta coronavirus envelope protein, or an immunogenic fragment thereof (e.g., a Beta coronavirus spike protein receptor binding domain (RBD)). In some embodiments, the antigen comprises a MERS-CoV spike protein, a MERS-CoV nucleocapsid protein, a MERS-CoV membrane protein, a MERS-CoV envelope protein, or an immunogenic fragment thereof (e.g., a MERS-CoV spike protein RBD). In some embodiments, the antigen comprises a SARS-CoV-1 spike protein, a SARS-CoV-1 nucleocapsid protein, a SARS-CoV-1 membrane protein, a SARS-CoV-1 envelope protein, or an immunogenic fragment thereof (e.g., a SARS-CoV-1 spike protein RBD). In some embodiments, the antigen comprises a SARS-CoV-2 spike protein, a SARS-CoV-2 nucleocapsid protein, a SARS-CoV-2 membrane protein, a SARS-CoV-2 envelope protein, or an immunogenic fragment thereof (e.g., a SARS-CoV-2 spike protein RBD). In some embodiments, the viral antigen comprises an antigen from human papillomavirus. In some embodiments, the antigen from human papillomavirus is selected from LI antigen, E6 antigen, E7 antigen, or a combination thereof. In some embodiments, the viral antigen comprises an antigen from hepatitis B virus. In some embodiments, the antigen from hepatitis B virus is HBsAg. In some embodiments, the viral antigen comprises an antigen from Epstein-Barr virus.

[0054] 12413624.1 In some embodiments, the antigen from Epstein-Barr virus is Epstein-Barr nuclear antigen. In some embodiments, the viral antigen is SV40.

[0055] Amino acid sequences of example Beta coronavirus antigens comprised by the compositions described herein are provided in Table 1. Table 1. Beta coronavirus protein antigens.

[0056] 12413624.1

[0057] 12413624.1

[0058] In some embodiments, the antigen is a wild type (i.e., “native”) antigen. In some embodiments, the antigen is a wild type protein or a polypeptide antigen. In some embodiments, the antigen is a polypeptide variant to a wild type protein or polypeptide antigen. The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. In some embodiments, the polypeptide variant is a variant of high consequence (VOHC), a variant of concern (VOC), a variant of interest (VOI), or a variant being monitored (VBM). These four categories are used to identify the degree of risk a polypeptide variant poses to public health. For example, a variant of high consequence (VOHC) refers to a variant that is associated with severe consequences, such as significantly increased morbidity or mortality, and may have the potential to evade immunity or undermine public health measures, while a variant of concern (VOC) refers to a variant that exhibits increased transmissibility, virulence, reduced effectiveness of treatments, and / or decreased vaccine efficacy as compared to other circulating strains. In some embodiments, the polypeptide variant possesses at least 50%

[0059] 12413624.1 identity to a native or reference sequence. In some embodiments, the polypeptide variant shares at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identity with a native or reference sequence.

[0060] In some embodiments, the polypeptide variant comprises substitutions, insertions, and / or deletions. In some embodiments, the polypeptide variant encompasses covalent variants and derivatives. The term “derivative” is used synonymously with the term “variant” but generally refers to a molecule that has been modified and / or changed in any way relative to a reference molecule or starting molecule. In some embodiments, the polypeptide variants comprise at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. In some embodiments, the antigen is a polypeptide that includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions compared to a reference protein.

[0061] In some embodiments, the substitution is a conservative amino acids substitution. The term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a nonpolar residue.

[0062] In some embodiments, protein fragments, functional protein domains, and homologous proteins are used as antigens in accordance with the present disclosure. For example, an antigen may comprise any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference

[0063] 12413624.1 protein 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to a reference protein (e.g., a protein from a microbial pathogen) herein can be utilized in accordance with the disclosure.

[0064] In some embodiments, a composition of the present disclosure comprises a nucleic acid encoding an antigen (e.g., as described herein). In some embodiments, the antigen comprises a nucleic acid encoding an protein or polypeptide. The term “nucleic acid” or “polynucleotide,” in its broadest sense, includes any compound and / or substance that comprises a polymer of nucleotides. Nucleic acids encoding proteins or polypeptides typically comprise an open reading frame (ORF), and one or more regulatory sequences. Nucleic acids (also referred to as polynucleotides) may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino- LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

[0065] In some embodiments, the nucleic acid encoding an antigen (e.g., a SARS-CoV-2 spike protein) is DNA (e.g., an expression vector for an immunogenic protein or polypeptide). In some embodiments, the nucleic acid encoding an antigen (e.g., a SARS-CoV-2 spike protein) is RNA (e.g., a messenger RNA). A “messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly- A tail.

[0066] In some embodiments, the coding region of the nucleic acid (e.g., DNA or RNA) encoding an antigen (e.g., a SARS-CoV-2 spike protein) is codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or

[0067] 12413624.1 remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0068] In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild- type sequence (e.g., a naturally-occurring or wild- type mRNA sequence encoding an antigen). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally- occurring or wild-type mRNA sequence encoding an antigen).

[0069] In some embodiments, the nucleic acid encoding an antigen (e.g., a SARS-CoV-2 spike protein) comprises one or more chemical modifications. The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides in at least one of their position, pattern, percent or population.

[0070] In some embodiments, the nucleic acids are antigens or encode antigens. In some embodiments, the nucleic acids (e.g., DNA or RNA) comprise various (more than one) different modifications. In some embodiments, a particular region of a nucleic acid (e.g., DNA or RNA) contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified nucleic acid (e.g., DNA or RNA), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid. In some embodiments, a modified nucleic acid (e.g., DNA or RNA), introduced

[0071] 12413624.1 into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0072] Modified nucleic acids (e.g., DNA or RNA) may comprise modifications that are naturally-occurring and / or non-naturally-occurring. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an intemucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone). Modified nucleic acids (e.g., DNA or RNA), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on an internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

[0073] In some embodiments, a chemically modified nucleic acid comprises one or more modified nucleosides. A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside and a (one or more) phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0074] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uridine include 5-cyano uridine, and 4’ -thio uridine. In some embodiments, a modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0075] In some embodiments, a modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl- adenosine (mlA), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A). In some embodiments, a modified nucleobase is a modified guanine. Exemplary nucleobases and

[0076] 12413624.1 nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7- aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0077] In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a Beta coronavirus protein or polypeptide. In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprise a nucleic acid encoding an antigen, wherein the nucleic acid encodes a Beta coronavirus spike protein, a Beta coronavirus nucleocapsid protein, a Beta coronavirus membrane protein, a Beta coronavirus envelope protein, or an immunogenic fragment thereof (e.g., a Beta coronavirus spike protein receptor binding domain (RBD)). In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a MERS-CoV spike protein, a MERS-CoV nucleocapsid protein, a MERS-CoV membrane protein, a MERS-CoV envelope protein, or an immunogenic fragment thereof (e.g., a MERS-CoV spike protein RBD). In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a variant MERS-CoV spike protein, a variant MERS-CoV nucleocapsid protein, a variant MERS-CoV membrane protein, a variant MERS-CoV envelope protein, or an immunogenic fragment thereof (e.g., a variant MERS-CoV spike protein RBD). In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a SARS-CoV-1 spike protein, a SARS-CoV-1 nucleocapsid protein, a SARS-CoV-1 membrane protein, a SARS-CoV-1 envelope protein, or an immunogenic fragment thereof (e.g., a SARS-CoV-1 spike protein RBD). In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a variant SARS-CoV-1 spike protein, a variant SARS-CoV-1 nucleocapsid protein, a variant SARS-CoV-1 membrane protein, a variant SARS-CoV-1 envelope protein, or an immunogenic fragment thereof (e.g., a variant SARS-CoV-1 spike protein RBD). In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a SARS-CoV-2 spike protein, a SARS-CoV-2 nucleocapsid

[0078] 12413624.1 protein, a SARS-CoV-2 membrane protein, a SARS-CoV-2 envelope protein, or an immunogenic fragment thereof (e.g., a SARS-CoV-2 spike protein RBD).In some embodiments, a composition described herein (e.g., a first composition or a second composition) comprises a nucleic acid encoding an antigen, wherein the nucleic acid encodes a variant SARS-CoV-2 spike protein, a variant SARS-CoV-2 nucleocapsid protein, a variant SARS-CoV-2 membrane protein, a variant SARS-CoV-2 envelope protein, or an immunogenic fragment thereof (e.g., a variant SARS-CoV-2 spike protein RBD).

[0079] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with reference molecules (e.g., reference polypeptides or reference polynucleotides), for example, wild-type molecules. The term “identity” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between them as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al (1997), "Gapped BLAST and PSLBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. (1981) “Identification

[0080] 12413624.1 of common molecular subsequences.” J. Mol. Biol. 147:195-197.) A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453.). More recently a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has been developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm. Other tools are described herein, specifically in the definition of “identity” below.

[0081] The term “identity” refers to the overall relatedness between polymeric molecules, for example, between polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleic acid sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991. For example, the percent identity between two nucleic acid sequences can be determined using the algorithm of Meyers and Miller

[0082] 12413624.1 (CAB IOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleic acid sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988). Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).

[0083] In some embodiments, the nucleic acid encoding a Beta coronavirus antigen in a composition described herein comprises a nucleic acid (e.g., DNA or RNA, such as mRNA) encoding a protein having an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%) identical to any one of SEQ ID NOs: 1-15. In some embodiments, the nucleic acid encoding a Beta coronavirus antigen in a composition described herein comprises a nucleic acid (e.g., DNA or RNA, such as mRNA) encoding a protein having an amino acid sequence that is 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to any one of SEQ ID NOs: 1-15. In some embodiments, the nucleic acid encoding a Beta coronavirus antigen in a composition described herein comprises a nucleic acid (e.g., DNA or RNA, such as mRNA) encoding a protein comprising the amino acid sequence of any one of SEQ ID NO: ID NOs: 1-15. The first antigen may be any antigen, e.g., an antigen described herein. The second antigen may be any antigen, e.g., an antigen described herein.

[0084] In some aspects, this disclosure provides a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising (i) administering to the subject a first composition comprising a nucleic acid encoding a first antigen; and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen, wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan. In some embodiments, the first composition comprises the adjuvantation system comprising the mannan. In some embodiments, the second composition comprises the adjuvantation system comprising the mannan. In some embodiments,

[0085] 12413624.1 the first composition and the second composition comprise an adjuvantation system comprising a mannan.

[0086] The term “inducing an immune response” as used herein refers to a process by which a substance (e.g., a pathogen or an antigen of a pathogen) triggers the body’s immune system to recognize and react to it. For example, when an immune response is induced, the substance (e.g., a pathogen or an antigen of a pathogen) is identified by the immune system as “non-self,” immune cells (e.g., macrophages, dendritic cells, lymphocytes) are activated and antibodies targeting the substance (e.g., a pathogen or an antigen of a pathogen) are produced. The degree to which an immune response is induced can be evaluated and / or measured using, for example, antibody testing (e.g., enzyme-linked immunosorbent assay (ELISA), neutralization tests, titer testing), cell-mediated immunity testing (e.g., flow cytometry, T-cell proliferation assay, cytokine assay), functional assays (e.g., phagocytosis assay, killing assay), and / or biomarker testing. The degree to which an immune response is induced can be evaluated and / or measured against a control (e.g., from a sample obtained from a healthy subject or population of healthy subjects, or from a sample obtained from a subject or population of subjects that were not administered compositions according to methods described herein).

[0087] An “adjuvantation system” refers to a composition comprising one or more adjuvants. An “adjuvant” refers to a pharmacological or immunological agent that modifies the effect of other agents, for example, of a vaccine or vaccine components (e.g., antigen, mRNA, lipids). Adjuvants are typically included in vaccines to enhance the recipient subject’s immune response to an antigen. The use of adjuvants allows the induction of a greater immune response in a subject with the same dose of antigen, the induction of a similar level of immune response with a lower dose of antigen, and / or the induction of a prolonged immune response in a subject with the same dose of antigen. Adjuvants may also allow the induction of cross -reactive antibody responses against multiple variants, thus broadening the immune response. Adjuvants are thought to function in several ways, including by increasing the surface area of antigen, prolonging the retention of the antigen in the body thus allowing time for the lymphoid system to have access to the antigen, slowing the release of antigen, targeting antigen to macrophages, activating macrophages, activating leukocytes such as antigen-presenting cells (e.g., monocytes, macrophages, and / or dendritic cells), or otherwise eliciting broad activation of the cells of the immune system. The ability of an adjuvant to induce and increase a specific type of immune response and the identification of that ability is thus a key factor in the selection of particular

[0088] 12413624.1 adjuvants for vaccine use against a particular pathogen. Adjuvants that are known to those of skill in the art, include, without limitation: aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, aluminum potassium sulfate; collectively referred to herein as “alum”), liposomes, lipopolysaccharide (LPS) or derivatives thereof such as monophosphoryl lipid A (MPLA) and glycopyranosyl lipid A (GLA), molecular cages for antigen, endocytosed nucleic acids such as double- stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA. Typical adjuvants include water and oil emulsions, e.g., Freund's adjuvant and MF59, and chemical compounds such as alum. At present, currently licensed vaccines in the United States contain only a limited number of adjuvants, such as alum which enhances production of T helper type 2 (Th2) cells, and MPLA which activates innate immunity via Toll-like receptor 4 (TLR4).

[0089] In some embodiments, an adjuvantation system of the present disclosure comprises a mannan. Mannans are polysaccharides of D-mannose joined by P-(l,4) linkages that can be found in various natural sources, such as plants and fungi. A mannan may be a branched polysaccharide or a linear polysaccharide. A mannan may be a derivative mannan, such as, for instance, a shortened (i.e., lower molecular weight) or elongated (i.e., higher molecular weight) version of a mannan (e.g., a plant mannan or a fungal mannan). A mannan may be in native, oxidated, or reduced form of a mannan (e.g., a plant mannan or a fungal mannan). A mannan may be covalently conjugated to another chemical moiety, such as but not limited to a protein (e.g., a glycoprotein). In some embodiments, a mannan may be composed of one or more than one type of carbohydrate monomer covalently linked in such a way as to form a polysaccharide. In some embodiments, at least 30% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99%) of the mannose saccharides are connected by P-(l,4) linkages. In some embodiments, a mannan comprises P-l,4-linked backbone. In some embodiments, the P-l,4-linked backbone is substituted with side chains of a-l,6-linked galactose residues. A mannan (e.g., a plant mannan or a fungal mannan) may be soluble, partially soluble, or insoluble in solution, particularly in an aqueous solution. A mannan (e.g., a plant mannan or a fungal mannan) may have antigenic properties (i.e., activates an immune response in an animal or human subject). In some embodiments, the mannan is a glucomannan, a galactomannan, or a galactoglucomannan. In some embodiments, the mannan is a plant mannan or a fungal mannan.

[0090] In some embodiments, the mannan is a fungal mannan. A fungal mannan may be produced and secreted by a fungal cell or occur as a component of a fungal cell (e.g., as a

[0091] 12413624.1 structural component of a fungal cell wall). In some embodiments, the fungal mannan elicits an immune response in a subject. In some embodiments, the fungal mannan is Candida mannan. In some embodiments, the fungal mannan is from a pathogenic fungus that elicits an immune response, such as Candida albicans (C. albicans). In some embodiments, the fungal mannan is from C. albicans. In some embodiments, the fungal mannan is a C. albicans mannan.

[0092] In some embodiments, the mannan is a plant mannan. A plant mannan may be produced and secreted by a plant cell or occur as a component of a plant cell (e.g., as a structural component of a plant cell). For example, in some embodiments, the plant mannan is derived from Ceratonia siliqua, Phytelephas macrocarpa, Schizolobium amazonicum, Schizolobium parahybum, Carum carvi, Cyamopsis tetragonolobus, Amorphophallus konjac, Coffea arabica, Aloe barbadensis, and / or Cesalpinia spinosa. In some embodiments, the plant mannan is a glucomannan. A glucomannan is a water-soluble polysaccharide comprising P-(l,4)-linked D- mannose and D-glucose. The ratio of D-mannose to D-glucose in a glucomannan is typically about 1.6:1. In some embodiments, the plant mannan is from konjac, salep, or conifers (e.g., cedars, Douglas firs, cypresses, firs, junipers, kauri, larches, pines, hemlocks, redwoods, spruces, yews). In some embodiments, the plant mannan is a konjac mannan, a salep mannan, or a conifer mannan (e.g., a cedar mannan, a Douglas fir mannan, a cypress mannan, a fir mannan, a juniper mannan, a kauri mannan, a larch mannan, a pine mannan, a hemlock mannan, a redwood mannan, a spruce mannan, a yew mannan). In some embodiments, the plant mannan is a glucomannan from a konjac plant. In some embodiments, the plant mannan is a konjac glucomannan. In some embodiments, the plant mannan is a galactomannan. A galactomannan is a polysaccharide comprising P-(l,4)-linked D-mannose with (l,6)-linked D-galactose. The ratio of D-mannose to D-galactose may vary from about 1:1 to about 1:5. In some embodiments, the plant mannan is from fenugreek gum, guar gum, tara gum, locust bean gum, or cassia gum. In some embodiments, the plant mannan is a fenugreek gum mannan, a guar gum mannan, a tara gum mannan, a locust bean gum mannan, or a cassia gum mannan. In some embodiments, the plant mannan is a galactoglucomannan. A galactoglucomannan is a water-soluble polysaccharide comprising P-(l,4,)-linked D-mannose and D-glucose units with (l,6)-linked D- galactose units attached to D-mannose units. In some embodiments, the plant mannan is from a Norway spruce. In some embodiments, the plant mannan is a Norway spruce mannan.

[0093] In some embodiments, the adjuvantation system further comprises an aluminum salt. In some embodiments, the aluminum salt is aluminum hydroxide, aluminum phosphate, or

[0094] 12413624.1 aluminum hydroxyphosphate. In some embodiments, the aluminum salt is Alhydrogel® (InvivoGen, USA). In some embodiments, the adjuvantation system comprises a mannan (e.g., a plant mannan or a fungal mannan) and an aluminum salt. In some embodiments, the adjuvantation system comprises a plant mannan (e.g., a konjac glucomannan) and an aluminum salt. In some embodiments, the adjuvantation system comprises a konjac glucomannan and an aluminum salt. In some embodiments, the adjuvantation system comprises a fungal mannan (e.g., a C. albicans mannan) and an aluminum salt. In some embodiments, the adjuvantation system comprises a C. albicans mannan and an aluminum salt.

[0095] In some embodiments, the mannan (e.g., a fungal mannan or a plant mannan) is admixed with the aluminum salt. In some embodiments, in an adjuvantation system comprising a fungal mannan (e.g., a C. albicans mannan) and an aluminum salt, the fungal mannan is admixed with the aluminum salt. In some embodiments, in an adjuvantation system comprising a plant mannan (e.g., a konjac glucomannan) and an aluminum salt, the plant mannan is admixed with the aluminum salt. Where a mannan (e.g., a plant mannan or a fungal mannan) is admixed with an aluminum salt, the quantity of each component in the adjuvantation system may be varied according to the desired properties or effects in the final admixture. In some embodiments, the adjuvantation system comprises equal quantities of aluminum salt and mannan (e.g., having a 1:1 weight ratio of aluminum salt to mannan). In some embodiments, the adjuvantation comprises different quantities of aluminum salt and mannan. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is between 1:1 and 1:100. A “weight ratio” is a comparison of the weights of different substances in a mixture or a compound, expressed as the proportion of one substance to another based on their respective weights. For example, an adjuvantation system comprising a 1:1 weight ratio of aluminum salt to mannan may comprise 100 pg of aluminum salt and 100 pg of mannan, while an adjuvantation system comprising a 1:100 weight ratio of aluminum salt to mannan may comprise 1 pg of aluminum salt and 100 pg of mannan. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:1. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:2. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation is 1:3. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:4. In some embodiments, the weight ratio of aluminum salt to plant mannan (e.g., konjac glucomannan) in the adjuvantation system is 1:4. In some embodiments, the weight ratio of

[0096] 12413624.1 aluminum salt to konjac glucomannan in the adjuvantation system is 1:4. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:5. In some embodiments, the weight ratio of aluminum salt to fungal mannan (e.g., C. albicans mannan) in the adjuvantation system is 1:5. In some embodiments, the weight ratio of an aluminum salt to C. albicans mannan in the adjuvantation system is 1:5. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:10. In some embodiments, the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:100.

[0097] In some embodiments, an adjuvantation system of the present disclosure is admixed with a nucleic acid encoding an antigen. In some embodiments, the weight ratio of the nucleic acid to the adjuvantation system is between 1:1 and 1:100. In some embodiments, the weight ratio of the nucleic acid to the adjuvantation system is 1:10. In some embodiments, the weight ratio of the nucleic acid to the adjuvantation system is 1:100.

[0098] In some embodiments, a composition described herein comprises a mannan (e.g., a plant mannan or a fungal mannan), an aluminum salt, and a nucleic acid encoding an antigen. In some embodiments, a composition described herein comprises a plant mannan (e.g., a konjac glucomannan), an aluminum salt, and a nucleic acid encoding an antigen. In some embodiments, a composition described herein comprises a fungal mannan (e.g., a C. albicans mannan), an aluminum salt, and a nucleic acid encoding an antigen. In some embodiments, a composition described herein comprises a nucleic acid encoding an antigen and an adjuvantation system, wherein the adjuvantation system comprises aluminum salt and konjac glucomannan. In some embodiments, a composition described herein comprises a nucleic acid encoding an antigen and an adjuvantation system at a 1:60 weight ratio of nucleic acid to adjuvantation system, wherein the adjuvantation system comprises aluminum salt and konjac glucomannan at a 1:4 weight ratio of aluminum salt to konjac glucomannan. In some embodiments, a composition described herein comprises a nucleic acid encoding an antigen and an adjuvantation system at a 1:100 weight ratio of nucleic acid to adjuvantation system, wherein the adjuvantation system comprises aluminum salt and konjac glucomannan at a 1:4 weight ratio of aluminum salt to konjac glucomannan. In some embodiments, a composition described herein comprises a nucleic acid encoding an antigen and an adjuvantation system, wherein the adjuvantation system comprises aluminum salt and C. albicans mannan. In some embodiments, a composition described herein comprises a nucleic acid encoding an antigen and an adjuvantation system at a 1:100 weight ratio of nucleic acid to adjuvantation system, wherein the adjuvantation system comprises

[0099] 12413624.1 aluminum salt and C. albicans mannan at a 1:5 weight ratio of aluminum salt to C. albicans mannan.

[0100] Adjuvants and adjuvantation systems as described herein are used in compositions (e.g., a vaccine composition). The terms “vaccine composition” and “vaccine” are used interchangeably herein. Vaccine compositions are a type of immunogenic composition. An “immunogenic composition” is a composition that activates or enhances a subject’s immune response to an antigen after the vaccine is administered to the subject. In some embodiments, the present disclosure provides a vaccine comprising a composition described herein and a pharmaceutically acceptable excipient.

[0101] In some embodiments, a composition described herein further comprises a pharmaceutically-acceptable excipient. In some embodiments, a composition described herein is formulated for administration to a subject. In some embodiments, a composition described herein is formulated or administered in combination with one or more pharmaceutically- acceptable excipients. In some embodiments, a composition described herein is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and / or (6) alter the release profile of encoded protein (antigen) in vivo.

[0102] The phrase “pharmaceutically-acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The phrase “pharmaceutically-acceptable excipient” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body. Each excipient must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the patient (e.g., physiologically compatible, sterile, physiologic pH, etc.). The term “excipient” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the immunogenic compositions (e.g., vaccine composition) described herein also are capable of being co-mingled with the molecules

[0103] 12413624.1 of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Some examples of materials which can serve as pharmaceutically-acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (9) glycols, such as propylene glycol; (10) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (11) esters, such as ethyl oleate and ethyl laurate; (12) agar; (13) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (14) alginic acid; (15) pyrogen-free water; (16) isotonic saline; (17) Ringer's solution; (18) ethyl alcohol; (19) pH buffered solutions; (20) polyesters, polycarbonates and / or poly anhydrides; (21) bulking agents, such as polypeptides and amino acids (22) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core- shell nanoparticles, peptides, proteins, cells transfected with DNA or RNA vaccines (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[0104] In some embodiments, a composition described herein is formulated in an aqueous solution. In some embodiments, a composition described herein is formulated in a nanoparticle. In some embodiments, compositions described herein formulated in a lipid nanoparticle. In some embodiments, a composition described herein is formulated in a lipid-polycation complex, referred to as a lipid nanoparticle. In some embodiments, where a composition is formulated in a nanoparticle (e.g., a lipid nanoparticle), the nanoparticle has a diameter between 400 nm - 1600 nm. In some embodiments, where a composition is formulated in a nanoparticle (e.g., a lipid nanoparticle), the nanoparticle has a diameter between 200 nm - 500 nm, 400 nm - 500

[0105] 12413624.1 nm, 400 nm - 750 nm, 400 nm - 1000 nm, 400 nm - 1250 nm, 400- 1500 nm, 500 nm - 750 nm, 500 nm - 1000 nm, 500 nm - 1250 nm, 500 nm - 1600 nm, 750 nm - 1000 nm, 750 nm - 1250 nm, 750 nm - 1500 nm, 750 nm - 1600 nm, 1000 nm - 1250 nm, 1000 nm - 1600 nm, or 1250 nm - 1600 nm. “Between” as used in reference to a range refers to an inclusive range. In some embodiments, the nanoparticle is at least 200 nm in diameter. In some embodiments, the nanoparticle is at least 400 nm in diameter. In some embodiments, the nanoparticle is less than 1000 nm in diameter. In some embodiments, this disclosure provides a plurality of nanoparticles as described herein with an average diameter between 300 - 500 nm, 350 - 450 nm, 500 - 1000 nm or 500 - 700 nm.

[0106] The formation of the lipid nanoparticle may be accomplished by methods known in the art, for example as described in U.S. Pub. No. 20120178702. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyomithine and / or polyarginine and the cationic peptides described in International Pub. No. WO2012013326 or US Patent Pub. No. US20130142818. In some embodiments, a composition described herein is formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0107] In some embodiments, a composition described herein is a nanoparticle that comprises at least one lipid (termed a “lipid nanoparticle” or “LNP”). The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2- DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D- DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. As a nonlimiting example, the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- { [(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en- 1 -yloxy] -2- { [(9Z)-octadec-9-en- 1 -yloxy]methyl } propan- 1 -ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- [(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3- [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-{ [(9Z,12Z)-octadeca-9,12-dien-l- yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010)

[0108] 12413624.1 Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578.

[0109] In some embodiments, a composition described herein is formulated in a liposome. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. In some embodiments, a composition described herein, when formulated in a liposome, comprises two distinct particle sizes, wherein one size is associated with the liposome and the other size is associated with the adjuvantation system. In some embodiments, the liposomes comprise a nucleic acid. In some embodiments, the adjuvantation system comprises alum-mannan complex. In some embodiments, the particle size is analyzed using dynamic light scattering technic. In some embodiments, the liposomes remain intact and show minimal physicochemical interactions with the adjuvantation system.

[0110] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products. As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372. In some embodiments, a composition described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N-

[0111] 12413624.1 dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0112] In some embodiments, a composition described herein may be formulated in a water-in- oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is dispersed. As a non-limiting example, the emulsion may be made by the methods described in International Publication No. W0201087791.

[0113] In some embodiments, a composition described herein is formulated in nanoparticles, liposomes, emulsions, or combinations thereof. In some embodiments, the nucleic acid is outside the nanoparticles. In some embodiments, the nucleic acid in within the nanoparticles. In some embodiments, the nucleic acid is a DNA, an RNA or a combination thereof. In some embodiments, the mannan is within the nanoparticles. In some embodiments, the mannan is outside the nanoparticles. In some embodiments, the mannan comprises a linear polysaccharide or a branched polysaccharide. In some embodiments, the mannan is within lipid membrane of the lipid nanoparticle. In some embodiments, the mannan is within the lipid nanoparticles. In some embodiments, the nucleic acid is administered naked.

[0114] In some embodiments, a composition described herein comprise at least one additional active substance, such as, for example, a therapeutically-active substance, a prophylactically- active substance, or a combination of both. A composition of the present disclosure may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as compositions of the present disclosure, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

[0115] Formulations of a composition described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the composition into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0116] Relative amounts of the composition, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure

[0117] 12413624.1 will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1- 30%, between 5-80%, at least 80% (w / w) active ingredient.

[0118] A composition described herein may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. The term "unit dose" when used in reference to a composition described herein refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., excipient.

[0119] In some embodiments, a composition described herein is formulated for administration to a subject. In some embodiments, a composition described herein is formulated with a pharmaceutically-acceptable excipient for administration to a subject. The formulation of the compositions described herein may be dependent upon the route of administration. Injectable preparations suitable for parenteral administration, intralesional or perilesional administration include, for example, sterile injectable aqueous or oleaginous suspensions and may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0120] For topical administration, a composition described herein can be formulated into ointments, salves, gels, or creams, as is generally known in the art. Topical administration can utilize transdermal delivery systems well known in the art. An example is a dermal patch. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory

[0121] 12413624.1 agent. Other compositions suitable for oral administration include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.

[0122] Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the anti-inflammatory agent, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, poly orthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono- di- and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which the anti-inflammatory agent is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,832,253, and 3,854,480. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

[0123] Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. Long-term release, are used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

[0124] In some embodiments, a composition described herein used for administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. The cyclic Psap peptide and / or a composition described herein ordinarily will be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation. The pH of the preparations typically will be about from 6 to 8, although higher or lower pH values can also be appropriate in certain instances. The chimeric constructs of the

[0125] 12413624.1 present disclosure can be used as vaccines by conjugating to soluble immunogenic carrier molecules. Suitable carrier molecules include protein, including keyhole limpet hemocyanin, which is a preferred carrier protein. The chimeric construct can be conjugated to the carrier molecule using standard methods. (Hancock et al., “Synthesis of Peptides for Use as Immunogens,” in Methods in Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 23-32 (Humana Press 1992)).

[0126] In some embodiments, the compositions described herein are used in methods of vaccinating a subject by prophylactically administering to the subject an effective amount of compositions described herein. In some embodiments, the methods comprise administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., a Beta coronavirus antigen) and administering to the subject a second composition comprising a nucleic acid encoding a second antigen (e.g., a variant Beta coronavirus antigen), wherein the second antigen is a variant of the first antigen. In some embodiments, the methods comprise administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., a Beta coronavirus antigen) and administering to the subject a second composition comprising a nucleic acid encoding a second antigen, wherein the second antigen is a variant of the first antigen (e.g., a variant Beta coronavirus antigen), and wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan (e.g., a plant mannan or a fungal mannan). In some embodiments, the methods comprise administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., a Beta coronavirus antigen) and administering to the subject a second composition comprising a nucleic acid encoding a second antigen, wherein the second antigen is a variant of the first antigen (e.g., a variant Beta coronavirus antigen), and wherein the first composition and / or the second composition comprises an adjuvantation system comprising a plant mannan (e.g., a konjac glucomannan). In some embodiments, the methods comprise administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., a Beta coronavirus antigen) and administering to the subject a second composition comprising a nucleic acid encoding a second antigen, wherein the second antigen is a variant of the first antigen (e.g., a variant Beta coronavirus antigen), and wherein the first composition and / or the second composition comprises an adjuvantation system comprising a fungal mannan (e.g., a C. albicans mannan). In some embodiments, where the first composition and the second composition comprise an adjuvantation system comprising a mannan, the first composition and the second composition

[0127] 12413624.1 comprise different mannans. For example, the first composition may comprise a plant mannan (e.g., a konjac glucomannan) and the second composition may comprise a fungal mannan (e.g., a C. albicans mannan). In some embodiments, where the first composition and the second composition comprise an adjuvantation system comprising a mannan, the first composition and the second composition comprise different amounts of the same mannan (e.g., a plant mannan or a fungal mannan).

[0128] In some embodiments, the method further comprises administering to the subject a third composition prior to administering the second composition. In some embodiments, the third composition comprise an adjuvantation system comprising a mannan. In some embodiments, the third antigen and the first antigen are the same. In some embodiments, where the first composition and / or the second composition comprise an adjuvantation system comprising a mannan, the third composition may comprise a different mannan from the first composition and / or the second composition. . In some embodiments, where the first composition and / or the second composition comprise an adjuvantation system comprising a mannan, the third composition may comprise different amounts of the same mannan (e.g., a plant mannan or a fungal manna) as the first composition and / or the second composition.

[0129] Thus, in some embodiments, this disclosure provides a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising: (i) administering to the subject a first composition comprising a nucleic acid encoding a first antigen (e.g., a Beta coronavirus antigen); and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen and an adjuvantation system comprising a mannan and an aluminum salt, wherein the second antigen is a variant of the first antigen (e.g., varianta Beta coronavirus antigen). In some embodiments, this disclosure provides a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising: (i) administering to the subject a first composition comprising a nucleic acid encoding a Beta coronavirus antigen (e.g., a SARS-CoV-2 antigen); and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen and an adjuvantation system comprising a mannan and an aluminum salt, wherein the second antigen is a variant of the first antigen (e.g., a variant SARS-CoV-2 antigen). In some embodiments, this disclosure provides a method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising: (i) administering to the subject a first composition comprising a nucleic acid encoding a SARS-CoV-2 antigen (e.g., a SARS-CoV-2 antigen spike

[0130] 12413624.1 protein); and (ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen and an adjuvantation system comprising a mannan and an aluminum salt, wherein the second antigen is a variant of the first antigen (e.g., a variant SARS-CoV-2 spike protein). In some embodiments, the method further comprises administering to the subject a third composition prior to administering the second composition, wherein the third composition comprises a nucleic acid encoding a third antigen. In some embodiments, the method further comprises administering to the subject a third composition prior to administering the second composition, wherein the third composition comprises a nucleic acid encoding a third antigen, wherein the third antigen is the same as the first antigen.

[0131] Other aspects of this disclosure provide, a method of stimulating immune cell recruitment in a subject in need thereof, the method comprising administering a first composition and a second composition, wherein the first composition and / or the second composition comprise an adjuvantation system comprising a mannan. In some embodiments, methods of the disclosure recruit more immune cells relative to a subject treated with a method comprising administering a first composition and a second composition, wherein the first composition and the second composition do not comprise an adjuvantation system comprising a mannan. In some embodiments, the method recruits more immune cells against variants of a virus (e.g., SARS-CoV-2 BA.4 / 5 or SARS-CoV-2 XBB.1.5) relative to a subject treated with a method comprising administering a first composition and a second composition, wherein the first composition and the second composition do not comprise an adjuvantation system comprising a mannan. In some embodiments, the method recruits more immune cells against variants of a viral antigen (e.g., SARS-CoV-2 BA.4 / 5 spike protein or SARS-CoV-2 XBB.1.5 spike protein) relative to a subject treated with a method comprising administering a first composition and a second composition, wherein the first composition and the second composition do not comprise an adjuvantation system comprising a mannan. In some embodiments, the method recruits more immune cells against variants of a viral antigen that is not the same as the first antigen or the second antigen relative to a subject treated with a method comprising administering a first composition and a second composition, wherein the first composition and the second composition do not comprise an adjuvantation system comprising a mannan. For example, in some embodiments, where a subject is treated with a method comprising administering a first composition comprising a nucleic acid encoding for a SARS- CoV-2 WAI spike protein and a second composition comprising a nucleic acid encoding for a

[0132] 12413624.1 SARS-CoV-2 XBB1.5 spike protein, wherein the first composition and / or the second composition comprise an adjuvantation system comprising a mannan, the method recruits more immune cells against variants of a SARS-CoV-2 BA.4 / 5 spike protein relative to a subject treated with a method comprising administering a first composition and a second composition, wherein the first composition and the second composition do not comprise an adjuvantation system comprising a mannan.

[0133] Thus, in some embodiments, the methods described herein enhance the production of cross -reactive antigen- specific antibodies, compared to when the first composition and the second composition do not comprise an adjuvantation system comprising a mannan. In some embodiments, the methods described herein enhance the production of cross-reactive antigenspecific antibodies by at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, or at least 100-fold or more, compared to when the antigen or nucleic acid encoding the antigen is administered alone. In some embodiments, the cross -reactive antigen-specific antibodies target variants of the antigen.

[0134] The methods of administering a composition to a subject, as used herein, can also be referred to as “vaccinating a subject.” This is a process of administering an immunogen (e.g., a composition described herein), to the subject in an amount effective to increase or activate an immune response against the antigen (e.g., a Beta coronavirus antigen), and thus against the pathogen (e.g., Beta coronavirus). In some embodiments, the term “vaccinating a subject” does not require the creation of complete immunity against the virus. In some embodiments, the term “vaccinating a subject” encompasses a clinically favorable enhancement of an immune response toward the viral antigen or pathogen. In some embodiments, vaccinating a subject reduces the risk of developing a viral infection, such as a Beta coronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) infection, and diseases that occur as a result of viral infection, such as those caused by Beta coronavirus infection (e.g., MERS, SARS and / or COVID- 19).

[0135] “Serial vaccination” refers to the administration of two or more compositions (e.g., vaccine compositions) to a subject sequentially over a specific period of time. For example, where a method comprises administering to a subject a first composition comprising a nucleic acid encoding a first antigen and administering to the subject a second composition comprising a nucleic acid encoding a second antigen, the first composition is administered to the subject prior to administration of the second composition. In some embodiments, the second composition is administered between 1 week and 10 weeks after administration of the first composition. In

[0136] 12413624.1 some embodiments, the second composition is administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks after administration of the first composition. In some embodiments, the second composition is administered about 2 weeks after administration of the first composition. In some embodiments, the second composition is administered about 4 weeks after administration of the first composition. In some embodiments, the second composition is administered about 8 weeks after administration of the first composition. In some embodiments, where a third composition is administered to a subject after administration of a first composition and prior to administration of a second composition, the third composition is administered between 1 weeks and 10 weeks after administration of the first composition. In some embodiments, the third composition is administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeks after administration of the first composition. In some embodiments, the third composition is administered about 2 weeks after administration of the first composition. In some embodiments, the third composition is administered about 4 weeks after the first composition. Thus, in some embodiments, where a third composition is administered to a subject after administration of a first composition and prior to administration of a second composition, the third composition is administered about 2 weeks after administration of the first composition and the second composition is administered about 8 weeks after administration of the first composition. In some embodiments, where a third composition is administered to a subject after administration of a first composition and prior to administration of a second composition, the third composition is administered about 4 weeks after administration of the first composition and the second composition is administered about 8 weeks after administration of the first composition.

[0137] A “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)), or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., primate, such as a cynomolgus monkey or a rhesus monkey), commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey). In some embodiments, the non-human animal is a fish, reptile, or amphibian. The non-human animal

[0138] 12413624.1 may be a male or female at any stage of development. The non-human animal may be a transgenic animal or a genetically engineered animal.

[0139] A “subject in need thereof’ refers to a subject (e.g., a human subject or a non-human mammal) in need of treatment of infection (e.g., by a pathogen), such as infection by a Beta coronavirus (e.g., a subject having MERS, SARS or COVID- 19), or in need of reducing the risk of developing an infection (e.g., by a pathogen), such as infection by a Beta coronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2). In some embodiments, methods of the present disclosure treats (i.e., has a therapeutic use for) the infection (e.g., MERS, SARS, or COVID- 19). In some embodiments, methods of the present disclosure reduce the likelihood (e.g., by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) of the subject developing the infection (prophylactic use).

[0140] In some embodiments, the subject is a human subject, e.g., a human neonate, infant, child, adult, or elderly. In some embodiments, the human subject has an undeveloped (e.g., an infant or a neonate), weak (an elderly), or compromised immune system. Immunocompromised subjects include, without limitation, subjects with primary immunodeficiency or acquired immunodeficiency such as those suffering from sepsis, HIV infection, and cancers, including those undergoing chemotherapy and / or radiotherapy. In some embodiments, the human subject has an underlying condition that renders them more susceptible to an infection, such as a Beta coronavirus (e.g., MERS-CoV, SARS-CoV-1, or SARS-CoV-2) infection. In some embodiments, the human subject is immunocompromised, has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease.

[0141] In some embodiments, the subject is a companion animal (i.e., a pet or service animal). The use of the immunogenic composition (e.g., vaccine composition) described herein in a veterinary vaccine is also within the scope of the present disclosure. “A companion animal,” as used herein, refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters. In some embodiments, the subject is a research animal. Non-limiting examples of research animals include: rodents (e.g., ferrets, pigs, rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0142] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have

[0143] 12413624.1 developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of exposure to a pathogen). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. Prophylactic treatment refers to the treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In some embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population.

[0144] An “effective amount” of a composition described herein refers to an amount sufficient to elicit the desired biological response. An effective amount of a composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, an effective amount is a prophylactic treatment. In some embodiments, an effective amount is the amount of a compound described herein in a single dose. In some embodiments, an effective amount is the combined amounts of a compound described herein in multiple doses. When an effective amount of a composition is referred herein, it means the amount is prophylactically and / or therapeutically effective, depending on the subject and / or the disease to be treated. Determining the effective amount or dosage is within the abilities of one skilled in the art.

[0145] The terms “administer,” “administering,” or “administration” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a composition described herein in or on a subject. A composition described herein may be administered systemically (e.g., via intravenous injection) or locally (e.g., via local injection). In some embodiments, a composition described herein is administered orally, intravenously, topically, intranasally, or sublingually. Parenteral administration is also contemplated. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, a composition described herein is administered intramuscularly. In some embodiments, a composition described herein is administered prophylactically.

[0146] 12413624.1 EXAMPLES

[0147] A major limitation of current mRNA-based vaccines is the induction of immune imprinting for the original antigen. This strongly diminishes the possibility to induce protection against new variants of concern. In this Example, the capacity of alum-mannan to overcome antigenic sin was evaluated by using mice immunized intramuscularly with Ancestral Comirnaty (BNT162b2 mRNA, Pfizer®-BioNTech®) on days 0 and 14 that were the rechallenged on day 56 with the Updated XBB.1.5-specific Comirnaty (Pfizer®-BioNTech®) in the presence of absence of alum-mannan (Table 2). Specifically, mice were administered 1 pg of mRNA per mouse with or without alum-mannan (500 pg mannan from C. albicans + 100 pg alum) according to the scheme shown in Table 2. Mouse sera was collected on days 70 and 84 postinitial immunization (FIG. 1).

[0148] Table 2. Vaccination schedule for the experimental groups used in this Example.

[0149] The addition of alum-mannan during the third boost with an updated vaccine (e.g., a modified SARS-CoV-2 vaccine designed to address an emerging variant, SARS-CoV-2 XBB.1.5) was found to strongly potentiates the induction of specific antibodies against the Spike protein of the variants of concern SARS-CoV-2 BA.4 / 5 and SARS-CoV-2 XBB.1.5, compared to a third boost with an “upgraded” vaccine without alum-mannan or a third boost with the Ancestral Comirnaty (FIG. 2). Additionally, boosting with an “upgraded” vaccine with alum- mannan resulted in more diverse T cell responses (FIG. 3). Finally, the peptides of the Spike proteins of the variants of concern SARS-CoV-2 BA.4 / 5 and SARS-CoV-2 XBB.1.5 were also found to be better recognized upon boosting in the presence of the alum-mannan (FIG. 4).

[0150] To further verify that the addition of alum-mannan overcame the antigenic sin, an additional experimental group was evaluated in which mice were intramuscularly administered three doses of the Updated XBB- 1.5. -specific Comirnaty (Pfizer®-BioNTech®) on days 0, 21,

[0151] 12413624.1 and 56. Evaluation of neutralizing antibody titers demonstrated that the mice receiving a dose including alum-mannan had a similar response to a SARS-CoV-2 pseudovirus expressing a SARS-CoV-2 XBB.1.5 as mice receiving three doses of the Updated XBB- 1.5. -specific Comimaty (PfizerO-BioNTech®). Further, only mice receiving a dose including alum-mannan had a significant response to a SARS-CoV-2 pseudovirus expressing a SARS-CoV-2 BA.5, demonstrating expanded protection for multiple variants of concern. Taken together, these data demonstrate that addition of alum-mannan can be used to overcome antigenic sin.

[0152] 12413624.1

Claims

CLAIMSWhat is claimed is:

1. A method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising:(i) administering to the subject a first composition comprising a nucleic acid encoding a first antigen; and(ii) administering to the subject a second composition comprising a nucleic acid encoding a second antigen; wherein the first composition and / or the second composition comprises an adjuvantation system comprising a mannan.

2. The method of claim 1, wherein the first composition comprises the adjuvantation system comprising the mannan.

3. The method of claim 1, wherein the second composition comprises the adjuvantation system comprising a mannan.

4. The method of claim 1, wherein the first composition and the second composition comprise an adjuvantation system comprising a mannan.

5. The method of any one of claims 1-4, wherein the method further comprises administering to the subject a third composition comprising a nucleic acid encoding a third antigen.12413624.

16. The method of claim 5, wherein the third composition is administered to the subject prior to administering the second composition.

7. The method of claim 5 or claim 6, wherein the third composition comprises an adjuvantation system comprising a mannan.

8. The method of any one of claims 1-7, wherein the mannan is a plant mannan or a fungal mannan.

9. The method of any one of claims 1-8, wherein the mannan is a glucomannan, a galactomannan, or a galactoglucomannan.

10. The method of claim 8 or claim 9, wherein the plant mannan is konjac mannan, aloe vera mannan, salep mannan, porang mannan, ivory nut mannan, cassia gum mannan, locust bean gum mannan, guar gum mannan, tar gum mannan, fenugreek gum mannan, or Norway spruce mannan.

11. The method of any one of claims 8-10, wherein the plant mannan is a konjac glucomannan.

12. The method of claim 8 or claim 9, wherein the fungal mannan is a yeast mannan.

13. The method of claim 8 or claim 9, wherein the fungal mannan is a Candida albicans mannan.12413624.

114. The method of any one of claims 1-13, wherein the adjuvantation system further comprises an aluminum salt.

15. The method of claim 14, wherein the aluminum salt is aluminum hydroxide, aluminum phosphate, or aluminum hydroxyphosphate.

16. The method of claim 14 or claim 15, wherein the weight ratio of aluminum salt to mannan in the adjuvantation system is between 1:1 and 1:100.

17. The method of any one of claims 14-16, wherein the weight ratio of aluminum salt to mannan in the adjuvantation system is 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, or 1:100.

18. The method of any one of claims 1-17, wherein the nucleic acid is DNA or RNA.

19. The method of claim 18, wherein the RNA is a messenger RNA (mRNA).

20. The method of any one of claims 1-19, wherein the first antigen is a bacterial antigen, a viral antigen, or a fungal antigen.

21. The method of claim 20, wherein the viral antigen comprises a Beta coronavirus protein or polypeptide.12413624.

122. The method of claim 21, wherein the Beta coronavirus protein or polypeptide comprises a Beta coronavirus spike protein or spike protein receptor binding domain (RBD).

23. The method of claim 22, wherein the Beta coronavirus spike protein is a MERS-CoV spike protein, a SARS-CoV-1 spike protein, or a SARS-CoV-2 spike protein.

24. The method of claim 22, wherein the Beta coronavirus spike protein RBD is a MERS- CoV spike protein RBD, a SARS-CoV-1 spike protein RBD, or a SARS-CoV-2 spike protein RBD.

25. The method of any one of claims 1-24, wherein the second antigen is a variant of the first antigen.

26. The method of any one of claims 1-25, wherein the third antigen and the first antigen are the same.

27. The method of any one of claims 1-26, wherein the ratio of the nucleic acid to the adjuvantation system is between 1:1 and 1:100.

28. The method of any one of claims 1-27, wherein the ratio of the nucleic acid to the adjuvantation system is 1:1, 1:10, or 1:100.

29. The method of any one of claims 1-28, wherein the adjuvantation system and the nucleic acid are admixed.12413624.

130. The method of any one of claims 1-29, wherein the second composition and the third composition are administered between 1 week and 10 weeks after administration of the first composition.

31. The method of claim 30, wherein the second composition is administered about 2 weeks after administration of the first composition.

32. The method of claim 30, wherein the second composition is administered about 8 weeks after administration of the first composition.

33. The method of claim 32, wherein the third composition is administered about 2 weeks after administration of the first composition.

34. The method of any one of claims 1-33, wherein the adjuvantation system increases recruitment of immune cells in the subject, compared to when the nucleic acid is administered alone.

35. The method of claim 34, wherein the immune cells are B cells or T cells.

36. The method of any one of claims 1-35, wherein the subject is a human.

37. The method of any one of claims 1-35, wherein the subject is a human neonate, a human infant, an adult human, or an elderly human.12413624.

138. The method of any one of claim 1-37, wherein the subject is a companion animal or a research animal.

39. The method of any one of claims 1-38, wherein the subject is immune-compromised, has chronic lung disease, asthma, cardiovascular disease, cancer, obesity, diabetes, chronic kidney disease, and / or liver disease.

40. The method of any one of claims 1-39, wherein the administration is intramuscular administration, intradermal administration, oral administration, intravenous administration, topical administration, intranasal administration, or sublingual administration.

41. The method of any one of claims 1-39, wherein the administration is intramuscular administration.

42. The method of any one of claims 1-41, wherein the administration is prophylactic.

43. The method of any one of claims 1-42, wherein first composition and the second composition comprise different mannans.

44. The method of any one of claims 1-42, wherein the first composition and the second composition comprise different amounts of the same mannan.12413624.

145. A method of inducing an immune response against a pathogen in a subject in need thereof, the method comprising:(i) administering to the subject a first composition comprising a nucleic acid encoding a first SARS-CoV-2 spike protein; and(ii) administering to the subject a second composition comprising a nucleic acid encoding a second SARS-CoV-2 spike protein and an adjuvantation system comprising a mannan and an aluminum salt.12413624.1

Images