WO2023240278A2

WO2023240278A2 - Uses of glycolipids as a vaccine adjuvant and methods thereof

Info

Publication number: WO2023240278A2
Application number: PCT/US2023/068255
Authority: WO
Inventors: David D. Ho; Moriya Tsuji; Yaoxing Huang; Manoj S. Nair
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2022-06-10
Filing date: 2023-06-09
Publication date: 2023-12-14
Also published as: WO2023240278A3

Abstract

The subject matter described herein related to a messenger RNA (mRNA)-vaccine adjuvant comprising a glycolipid compound.

Description

USES OF GLYCOLIPIDS AS A VACCINE ADJUVANT AND METHODS THEREOF

[0001] This application claims priority to U.S. Provisional Application 63/351,276, filed on June 10, 2022; and U.S. Provisional Application 63/375,508, filed on September 13, 2022, the contents of each of which is hereby incorporated by reference in its entirety.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0003] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosure of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.

BACKGROUND OF THE INVENTION

[0004] Vaccines reduce the risk developing a disease by stimulating the body’s natural defenses to build protection. Vaccination leads to increased immunity to disease, which saves millions of lives every year. Vaccines available today prevent more than twenty lifethreatening diseases, helping people of all ages live longer, healthier lives. Immunization currently prevents 3.5-5 million deaths every year from various diseases including diphtheria, tetanus, pertussis, influenza and measles.

SUMMARY OF THE INVENTION

[0005] In certain aspects, the subject matter disclosed herein provides an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0006] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273. [0007] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0008] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration.

[0009] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0010] In certain aspects, the subject matter disclosed herein provides a method of stimulating an immune response in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0011] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0012] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0013] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human. [0014] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0015] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0016] In certain aspects, the subject matter disclosed herein provides a method for augmenting an immune response to an antigen in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0017] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0018] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0019] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human.

[0020] In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2.

[0021] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient. [0022] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0023] In certain aspects, the subject matter disclosed herein provides a method for elevating invariant Natural Killer T (zNKT) cells production in a subject, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0024] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0025] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0026] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human. In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0027] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg. [0028] In certain aspects, the subject matter disclosed herein provides a method for stimulating cytokine and/or chemokine production in a subject, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0029] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0030] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0031] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human.

[0032] In some embodiments, the cytokine and/or chemokine production transactivates downstream immune cells. In some embodiments, the downstream immune cells comprise one or more of dendritic cells (DC), natural killer cells (NK), B cells, CD4+T, and CD8+T cells. In some embodiments, the cytokines comprise Thi cytokines. In some embodiments, the cytokines are selected from the groups consisting of interferon-gamma (IFN-y), GM-CSF, TNFoc, interleukin 2, interleukin 12, and interleukin 10.

[0033] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0034] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg. [0035] In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising: (i) a messenger RNA (mRNA)-vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine.

[0036] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof. In some embodiments, the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

[0037] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0038] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0039] In some embodiments, the mRNA-vaccine comprises an mRNA sequence encoding for an antigen. In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intranasally.

[0040] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0041] In certain aspects, the subject matter disclosed herein provides a kit for inoculating a subject in need thereof, the kit comprising (i) a messenger RNA (mRNA)- vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine. [0042] In some embodiments, the vaccine adjuvant further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof. In some embodiments, the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

[0043] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0044] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5. In some embodiments, the mRNA-vaccine comprises a sequence encoding for an antigen to a subject.

[0045] In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2.

[0046] In some embodiments, the vaccine adjuvant and the mRNA vaccine are packaged together. In some embodiments, the vaccine adjuvant and the mRNA vaccine are packaged separately.

[0047] In some embodiments, the adjuvant and the vaccine are for intramuscular administration. In some embodiments, the adjuvant and the vaccine are for intranasal administration.

[0048] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

BRIEF DESCRIPTION OF THE FIGURES

[0049] The patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many of the figures presented herein are black and white representations of images originally created in color.

[0050] FIG. 1 shows a schematic representation of the mRNA vaccine and 7DW8-5 administration in the T-cell analysis of FIGS. 2A-C and FIG. 3.

[0051] FIGS. 2A-C show the relative number of Spike-specific splenic T-cell responses. FIG. 2A shows IFNy secreting spots for CD8+ T-cells. FIG. 2B shows IFNy secreting spots for CD4+ T-cells in pool 1. FIG. 2C shows IFNy secreting spots for CD4+ T-cells in pool 2.

[0052] FIG. 3 shows the relative number of Spike-specific lung-resident T-cell responses.

[0053] FIGS. 4A-C show that intramuscular co-administration of 7DW8-5 enhances T- cell immunogenicity of the Pfizer mRNA vaccine (BNT162b2) in mice. FIG. 4 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration. FIG. 4B shows a Spike-specific splenic T-cell response to administration of the vaccine alone or with 7DW8-5. FIG. 4C shows a Spike-specific lung-resident T-cell response to administration of the vaccine alone or with 7DW8-5. Anti-RBD Ab titers: 1/800 - 1/1600 (mRNA) and 1/400 - 1/800 (mRNA and 7DW8-5).

[0054] FIGS. 5A-C show that intranasal co-administration of 7DW8-5 enhances T-cell immunogenicity of the Pfizer mRNA vaccine (BNT162b2) in mice. FIG. 5 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration.

FIG.5B shows a Spike-specific splenic T-cell response to administration of the vaccine alone or with 7DW8-5. FIG. 5C shows a Spike-specific lung-resident T-cell response to administration of the vaccine alone or with 7DW8-5.

[0055] FIGS. 6A-C show MAIO challenge after priming mice intranasally with Pfizer mRNA vaccine (BNT162b2) and 7DW8-5. FIG. 6 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration prior to the M10 challenge. FIG. 6B shows mouse body weight change with administration of saline, 7DW8-5 alone, or 7DW8-5 with the mRNA vaccine. FIG. 6C shows virus titer on day 3 following the MAIO challenger with administration of saline, 7DW8-5 alone, or 7DW8-5 with the mRNA vaccine.

DETAILED DESCRIPTION OF THE INVENTION

[0056] In certain aspects, the subject matter disclosed herein provides an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0057] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0058] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0059] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration.

[0060] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0061] In certain aspects, the subject matter disclosed herein provides a method of stimulating an immune response in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0062] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273. [0063] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0064] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human.

[0065] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0066] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0067] In certain aspects, the subject matter disclosed herein provides a method for augmenting an immune response to an antigen in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0068] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0069] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0070] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human. [0071] In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2.

[0072] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0073] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0074] In certain aspects, the subject matter disclosed herein provides a method for elevating invariant Natural Killer T (zNKT) cells production in a subject, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0075] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0076] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0077] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human. In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0078] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0079] In certain aspects, the subject matter disclosed herein provides a method for stimulating cytokine and/or chemokine production in a subject, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

[0080] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0081] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0082] In some embodiments, the adjuvant is administered to a subject concurrently with the mRNA-vaccine. In some embodiments, the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine. In some embodiments, the subject is a human.

[0083] In some embodiments, the cytokine and/or chemokine production transactivates downstream immune cells. In some embodiments, the downstream immune cells comprise one or more of dendritic cells (DC), natural killer cells (NK), B cells, CD4+T, and CD8+T cells. In some embodiments, the cytokines comprise Thi cytokines. In some embodiments, the cytokines are selected from the groups consisting of interferon-gamma (IFN-y), GM-CSF, TNFoc, interleukin 2, interleukin 12, and interleukin 10.

[0084] In some embodiments, the adjuvant is for intramuscular administration. In some embodiments, the adjuvant is for intranasal administration. In some embodiments, the adjuvant is administered with a pharmaceutically acceptable excipient.

[0085] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0086] In certain aspects, the subject matter disclosed herein provides a pharmaceutical composition comprising: (i) a messenger RNA (mRNA)-vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine.

[0087] In some embodiments, the composition further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof. In some embodiments, the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

[0088] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0089] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5.

[0090] In some embodiments, the mRNA-vaccine comprises an mRNA sequence encoding for an antigen. In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2. In some embodiments, the composition is administered intramuscularly. In some embodiments, the composition is administered intranasally.

[0091] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 jug, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

[0092] In certain aspects, the subject matter disclosed herein provides a kit for inoculating a subject in need thereof, the kit comprising (i) a messenger RNA (mRNA)- vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine.

[0093] In some embodiments, the vaccine adjuvant further comprises one or more pharmaceutically acceptable excipients. In some embodiments, the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof. In some embodiments, the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

[0094] In some embodiments, the mRNA-vaccine is a COVID-19 vaccine. In some embodiments, the mRNA-vaccine is a SARS-CoV-2 vaccine. In some embodiments, the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein. In some embodiments, the mRNA-vaccine comprises the mRNA BNT162b2. In some embodiments, the mRNA-vaccine comprises the mRNA mRNA-1273.

[0095] In some embodiments, the glycolipid compound is a-galactosylceramide (a- GalCer). In some embodiments, the glycolipid compound is a modification or an analog of a- GalCer. In some embodiments, the glycolipid compound is 7DW8-5. In some embodiments, the mRNA-vaccine comprises a sequence encoding for an antigen to a subject.

[0096] In some embodiments, the antigen comprises at least a portion of a SARS-CoV-2 spike protein. In some embodiments, the antigen is a peptide of Table 1. In some embodiments, the antigen is a peptide of Table 2.

[0097] In some embodiments, the vaccine adjuvant and the mRNA vaccine are packaged together. In some embodiments, the vaccine adjuvant and the mRNA vaccine are packaged separately.

[0098] In some embodiments, the adjuvant and the vaccine are for intramuscular administration. In some embodiments, the adjuvant and the vaccine are for intranasal administration.

[0099] In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 jug, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

Vaccine Adjuvants

[0100] In some embodiments, adjuvants are components used in combination with vaccines. In some embodiments, adjuvants are administered before the vaccine is administered. In some embodiments, adjuvants are administered after the vaccine is administered. In some embodiments, adjuvants are administered concurrently with the vaccine. In some embodiments, adjuvants are administered in the same formulation with the vaccine. In some embodiments, adjuvants are administered in a separate formulation from the vaccine. Adjuvants can help produce a stronger immune response in a subject immunized with a vaccine. In some embodiments, adjuvants can enhance the effects of a vaccine to work. In some embodiments, adjuvants help the subject’s body produce a strong enough immune response. In some embodiments, adjuvants reduce the dose required to be administered to elicit an immune response. In some embodiments, the adjuvant reduces the time required for an immune response to be elicited. In some embodiments, the adjuvant described herein contains a glycolipid. In some embodiments, the adjuvant described herein contains a-galactosylceramide (a-GalCer). In some embodiments, the adjuvant described herein contains an analog of a-GalCer. In some embodiments, the adjuvant described herein contains 7DW8-5.

A pharmaceutically acceptable excipient

[0101] In some embodiments, the adjuvant contains a glycolipid. In some embodiments, the glycolipid is a-galactosylceramide (a-GalCer). In some embodiments, the glycolipid is an analog of a-GalCer. In some embodiments, the glycolipid is 7DW8-5. In some embodiments, the adjuvant further contains a pharmaceutically acceptable excipient. In some embodiments, the excipient is a carrier, antioxidant, binder, buffer, bulking agent, chelating agent, coloring agent, diluent, disintegrant, emulsifying agent, surfactant, solvent, filler, gelling agent, pH buffering agent, preservative, solubilizing agent, stabilizer, or any combination thereof. In some embodiments, the excipient is an oil, oil emulsion, aluminum salt, calcium salt, immune stimulating complex, bacterial and viral derivative, virosome, carbohydrate, cytokine, polymeric microparticle, or any combination thereof. In some embodiments, the excipient is an osmotic pressure keeper, such as water, alcohol, a saline solution (e.g., sodium chloride), or any combination thereof. In some embodiments, the excipient is a preservative, such as an alkyl/aryl alcohol (e.g., benzyl alcohol, chlorbutanol, 2-ethoxyethanol), amino aryl acid ester (e.g., methyl, ethyl, propyl butyl parabens and combinations), alkyl/aryl acid (e.g., benzoic acid, sorbic acid), biguanide (e.g., chlorhexidine), aromatic ether (e.g., phenol, 3-cresol, 2- phenoxyethanol), organic mercurial (e.g., thimerosal, phenylmercurate salt).

ELISPOT Assay

[0102] Enzyme-linked immunosorbent spot (ELISPOT) assays are highly sensitive immunoassays that measure the frequency of antigen-specific T cells present in a sample. They measure the number of cytokine-secreting cells within the blood at a single cell level. ELISPOT assays can also quantify the number of antigen-specific B cells recently activated in vivo. They can also quantify memory B cells which have been activated in response to a specific stimulus ex vivo. There are a number of applications for ELISpot assays pre- clinically and clinically. These include, but are not limited to, monitoring immune responses during preclinical testing and clinical trials, ex vivo stimulation of immune cells to measure frequency of cells specific to the antigen of interest, and vaccine efficacy.

[0103] In some embodiments, to test for the presence of cytokines of interest in a sample, peripheral blood mononuclear cells (PBMCs) or other relevant tissue cells are cultured on a membrane surface coated with a specific capture antibody in the presence or absence of antigenic stimuli. The cytokines secreted by the cultured cells following stimulation can be captured onto the membrane surface. After an appropriate incubation time, cells can be removed, and the secreted cytokines can be detected using a detection antibody. In some embodiments, the binding is captured using enzymatic horseradish peroxidase (HRP). In some embodiments, the binding is captured using alkaline phosphatase (ALP). In some embodiments, the binding is captured using a fluorescent readout. In some embodiments, the binding is captured using any method known in the art which allows the reaction spots to become visible. The plate can then be washed to stop the reaction. In some embodiments, the membrane is allowed to dry before analysis. In some embodiments, the ELISPOT analysis is performed for a single cytokine. In some embodiments, the ELISPOT analysis is performed for combinations of two or more cytokines.

Pfizer mRNA Vaccine (BNT162b2)

[0104] The Pfizer mRNA vaccine (BNT162b2) is a vaccine against the SARS-CoV-2 virus. It is administered to a subject who may come in contact with the virus by an injection in the muscle of the upper arm. As of August 18, 2022, according to the World Health Organization the vaccine is authorized for use for those aged 6 months and older, with an adjustment in the recommended dosage for those aged 6 months to 4 years, and an adjustment for those aged 5-11 years. The BNT162b2 vaccine includes nucleoside-modified RNA (modRNA) sequence, which is encapsulated in a lipid nanoparticle (LNP). The modified RNA sequence encodes the full-length of SARS-CoV-2 spike (S) protein, modified by two proline mutations to ensure antigenically optimal pre-fusion conformation. This mimics the intact virus and elicits virus-neutralizing antibodies. BNT162b2 has shown high efficiency in a wide range of COVID-19-related outcomes in a real -world setting. The Pfizer mRNA BNT162b2is included in Pfizer’s vaccines COMIRNATY® and Bivalent®.

[0105] The Spike protein on the surface of the SARS-CoV-2 virus is involved in receptor recognition, virus attachment, and entry into the host cell. The messenger RNA that encodes Spike protein is made from a DNA template in the laboratory. The lipid nanoparticle, which encapsulates the RNA sequence, can stabilize and protect the RNA and can help it enter the host cell. In some embodiments, an amount of a solution of nanoparticles encapsulating an RNA sequence are injected into a subject. In some embodiments, the nanoparticles fuse with or are taken up by antigen-presenting cells (APCs) in the subject’s body. The messenger RNA can be read by the cell’s translation machinery to produce spike protein. The RNA can then be degraded by the cell. In some embodiments, the produced spike proteins are broken down into shorter polypeptides. These can then be presented on the surface of the APCs by major histocompatibility complex (MHC). The presented Spike proteins or portions thereof can activate circulating T cells, which come in contact with the MHCs of the APCs. The T- cells can be T-helper cells or cytotoxic T-cells. In some embodiments, T-helper cells activate B cells. In some embodiments, B cells produce antigen-specific antibodies which circulate within the subject’s body. Antibodies can prevent the virus from binding to host cells when the subject is infected. Antibodies can also mark the virus for destruction. In some embodiments, cytotoxic T-cells kill virus-infected host cells with which they come in contact. T-cell and B-cell activation represents the vaccine-primed immune response. In some embodiments, T-cells and B-cells become memory cells. In some embodiments, the memory cells can be reactivated when they come in contact with the same spike protein antigen they recognized on the APC.

Moderna mRNA Vaccine (mRNA-1273)

[0106] The Moderna mRNA vaccine (mRNA-1273) is a vaccine against the SARS-CoV- 2 virus. According to the World Health Organization, the vaccine is safe and effective for all individuals aged 6 months and above. Modema’s mRNA-1273 COVID-19 vaccine is an LNP-encapsulated mRNA vaccine expressing the prefusion-stabilized Spike glycoprotein. The vaccine contains a synthetic mRNA (single-stranded, 5 ’-capped) encoding the prefusion- stabilized spike glycoprotein (S) of SARS-CoV-2 virus. The vaccine also contains lipids (SM-102, l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000- DMG), cholesterol, and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), tromethamine, tromethamine hydrochloride, acetic acid, sodium acetate, and sucrose. The vaccine is supplied as a frozen suspension. The Moderna mRNA vaccine (mRNA-1273) (100 pg), is administered intramuscularly as a series of two doses (0.5 ml each), given 28 days apart. [0107] RNA-based vaccines have the advantage of rapid development and reduced side effects. They generally do not integrate with the host cell genome and are able to produce pure viral protein. Because they are transiently expressed, they allow for protein to be made within the cell. Lipid nanoparticle (LNP)-formulated mRNA vaccine technology allows the delivery of precise genetic information together with an adjuvant effect to antigen-presenting cells. The Moderna mRNA mRNA-1273 is included in Modema’s SPIKEVAX® and Bivalent®.

Glycolipids and the 7DW8-5 glycolipid

[0108] Glycolipids, such as a-galactosylceramide (a-GalCer), bind the non-polymorphic MHC class Llike molecule, CD Id and are presented to invariant natural killer T (zNKT) cells. (Padte, N.N., et al. A Glycolipid Adjuvant, 7DW8-5, Enhances CD8+ T Cell Responses Induced by an Adenovirus- Vectored Malaria Vaccine in Non-Human Primates, 2013, 8(10) 1-15) This leads to activation of z'NKT cells to rapidly produce large quantities of Thl and Th2 cytokines, and the subsequent induction of a cascade of immuno-competent cells, including dendritic cells (DCs), natural killer (NK) cells, B cells, and CD4+ and CD8+ T cells. a-GalCer can therefore be utilized as a direct therapy for infectious diseases. a-GalCer has also been used as an adjuvant to enhance the efficacy of various existing or new vaccines, including malaria vaccines. The adjuvant effect of a-GalCer is shown to be mediated by CD Id molecules, iNKT cells, IFN-y, type I interferon, and CD40-CD40L interaction. In some embodiments, 7DW8-5 is the most biologically potent glycolipid among several a-GalCer analogs tested.

[0109] 7DW8-5 is a glycolipid consisting of two lipid tails and a galactose head, which are o-linked. The chemical formula of 7DW8-5 is C41H72FNO9. Its molecular weight is 742.01 kDa. The systematic name of 7DW8-5 is (2S, 3S, 4R)-l-O-(a-D-galactopyranosyl)- N-(l l-(4-fluorophenyl)undecanoyl)-2-amino-l,3,4-octadecanetriol). 7DW8-5 is modified from a-GalCer. This analog differs from a-GalCer in that it possesses a fluorinated benzene ring at the end of CIO length fatty acyl chain.

[0110] 7DW8-5 has been shown to bind an MHC class-I like molecule, CD Id, and activate innate T cells, which are zNKT cells, through their invariant T-cell receptor (5). Upon activation, z'NKT cells can secrete a large number of cytokines that include interferon-y, which is known to have anti-viral activity (5). Activation of z'NKT cells rapidly induces activation and maturation of dendritic cells (DCs), which in turn induce a cascade of various immune competence cells, such as natural killer (NK) cells and CD8+ T cells (2, 3). These NK cells and CD8+ T cells can also produce a large amount of IFN-y. In some embodiments, the subject matter described herein relates to the intranasal administration of the 7DW8-5 glycolipid which activates the “Innate” immune system rather than a pathogen-specific immune response. In some embodiments, 7DW8-5 binds to CD Id in order to activate innate invariant natural killer T (z'NKT) cells. In some embodiments, activation of z'NKT cells leads to maturation of DCs. In some embodiments, activation of z'NKT cells leads to cytokine secretion. In some embodiments, activation of z'NKT cells leads to IFN-y secretion. In some embodiments, IFN-y secretion leads to DCs maturation. In some embodiments, 7DW8-5 administration induces the activation of natural killer cells and CD8+ T cells. In some embodiments, 7DW8-5 can provide a broad protection against all SARS-CoV-2 variants. In some embodiments, 7DW8-5 can provide protection against all respiratory viral infections. In some embodiments, 7DW8-5 administration provides protection against influenza. In some embodiments, the adjuvant described herein contains a glycolipid. In some embodiments, the adjuvant described herein contains a glycolipid and an excipient. In some embodiments, the adjuvant described herein contains a-galactosyl ceramide (a-GalCer). In some embodiments, the adjuvant described herein contains an analog of a-GalCer. In some embodiments, the adjuvant described herein contains 7DW8-5.

[OHl] In some embodiments, the subject matter described herein relates to a method of activation of natural killer T (NKT) cells in a subject in need thereof, the method comprising administering to the subject a pharmaceutical amount of a glycolipid. In some embodiments, the glycolipid is 7DW8-5. In some embodiments, the glycolipid is alpha-GalCer. In some embodiments, the glycolipid is an alpha-GalCer or a 7DW8-5. In some embodiments, the analog has stimulatory activity against iNKT cells. In some embodiments, the analogue is a 4”-modified a-galactosylceramide. In some embodiments, the analogue is a 6”-triazole- substituted a-galactosyl ceramide. In some embodiments, the analogue is an acyl-chain- and galactose-6"-modified analogue of a-GalCer. In some embodiments, the analogue is a c- glycoside analogue of a-galactosylceramide. In some embodiments, the analogue is an a- galactosylceramide with amide-linked phenyl alkane substitutions on the C4” position of the galactose ring. In some embodiments, the analogue is a C-5” and C-6”-modified a-GalCer. In some embodiments, the analogue is KRN7000 (alpha-GalCer). In some embodiments, the analogue is AHI 0-7. In some embodiments, the analogue is AHI 5-1. In some embodiments, the analogue is an a-galactosylsphingamides. In some embodiments, the analogue is a n-acyl variant of a-galactosylceramides. In some embodiments, the analogue is a non-glycosidic analogue. In some embodiments, the analogue is ThrCer 6.

[0112] Additional analogs and compounds, which can be used with the methods described here can be found in Janssens, J. et al., ACS Med. Chem. Lett. 2017, 8, 642-647, Jervis, P.J. et al., Bioorg. Med. Chem. Lett. 2012, 22, 13, 4348-4352, Hsieh, M.H. et al., Chembiochem. 2012, 13, 11, 1689-1697, Schmieg, J. et al., J. Exp. Med. 2003, 198, 11, 1631-1641, Tyznik, A.J. et al., Chem Biol. 2011, 18, 12, 1620-1630, Saavedra-Avila, N. A. et al., ACS Chem Biol. 2020, 15, 12, 3176-3186, Kharkwal, S.S. et al., Cancer Res. 2021, 81, 7, 1788-1801, Guillaume, J. et al., Bioorg Med Chem. 2015, 23, 13, 3175-3182, Chennamadhavuni, D. et al., Cell Chemical Biology 2018, 25, 571-584, Guillaume, J. et al., Scientific Reports 2017, 7, 4276, 1-18, Yu, K.O.A. et al., TWA 2005, 102, 9, 3383-3388, Jukes, J.P. et al., Eur. J. Immunol. 2016. 46, 1224-1234, Veerapen, N. et al., Bioconjug Chem. 2018, 29, 9, 3161-3173, and Hartrampf, N. et al., Chemistry. 2020, 26, 20, 4476-4479, US10,918,714, US9,879,042, US9,782,476, US10,533,034, US10,l 11,951, US8,586,051, and US7,923,013 the contents of each of which is incorporated herein by reference.

[0113] Further embodiments of the adjuvant effects of glycolipids can be found in Padte, N.N., et al. PLOS One. 2013, 8, 10, 1-15, Li, X. et al., PNAS. 2010, 107, 29, 13010-13015, Feng, H. et al., Frontiers in Microbiol. 2019, 10, 2157, 1-9, Feng, H, et al., Viruses. 2022, 14, 1174, 1-9, Pifferi, C., et al. Nat. Rev. Chem. 2021, 5, 2021, 197-216 the contents of each of which is incorporated herein by reference.

[0114] In some embodiments, the adjuvant is co-administered with an mRNA vaccine. In some embodiments, the adjuvant is co-administered with a Pfizer mRNA BNT162b2 vaccine. In some embodiments, the adjuvant is co-administered with a Moderna mRNA-1273 vaccine. In some embodiments, the adjuvant is administered prior to a mRNA vaccine administration. In some embodiments, the adjuvant is administered following a mRNA vaccine administration. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

Adjuvant Formulations

[0115] In some embodiments, adjuvant described herein can be formulated for immediate release or for sustained release formulations. In some embodiments, the adjuvant can be formulated for induction of systemic, or localized mucosal, immunity. Such delivery systems are readily determined by one of ordinary skill in the art. In some embodiments, the adjuvant can be prepared as an injectable composition. In some embodiments, the adjuvant can be prepared as an intranasal composition. In some embodiments, the adjuvant can be a liquid solution or suspension. In some embodiments, the adjuvant contains a glycolipid. In some embodiments, the glycolipid is a-galactosylceramide (a-GalCer). In some embodiments, the glycolipid is an analog of a-GalCer. In some embodiments, the glycolipid is 7DW8-5.

[0116] In some embodiments, the adjuvant can be administered by any suitable mode know in the art. In some embodiments, the adjuvant composition can be administered intradermally, intraperitoneally, intramuscularly, intranasally, orally, subcutaneously, or in any suitable delivery method known in the art. In some embodiments, the adjuvant contains a glycolipid. In some embodiments, the glycolipid is a-galactosylceramide (a-GalCer). In some embodiments, the glycolipid is an analog of a-GalCer. In some embodiments, the glycolipid is 7DW8-5.

[0117] In some embodiments, the adjuvant can also be formulated in a suitable dosage unit form. In some embodiments, the adjuvant composition contains from about 0.1 pg to about 1,000 pg of a glycolipid or a pharmaceutically acceptable salt thereof. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg. In some embodiments, the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg. In some embodiments, the glycolipid is a-galactosylceramide (a-GalCer). In some embodiments, the glycolipid is an analog of a-GalCer. In some embodiments, the glycolipid is 7DW8-5. Effective doses of the adjuvant can vary depending upon many different factors, including method of administration, target site, physiological state of the subject, whether the subject is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. When delivered in multiple doses, the adjuvant may be divided into an appropriate amount per dosage unit form. The administered dosage will depend on the age, weight and general health of the subject as is well known in the therapeutic arts.

EXAMPLES

[0118] The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the statements of the invention which follow thereafter. The Examples described below are provided to illustrate aspects of the present invention and are not included for the purpose of limiting the invention.

EXAMPLE 1 - 7DW8-5 enhances T-cell immunogenicity of Pfizer mRNA vaccine (BNT162b2) in mice

[0119] To test the adjuvant activity of 7DW8-5 on the Pfizer mRNA vaccine (BNT162b2), groups of BALB/c mice (N=5) received an intramuscular (anterior tibialis muscles in both legs) co-administration of a suboptimal dose (1 pg) of the BNT162b2 vaccine and 2 pg of the 7DW8-5 glycolipid, as shown in FIG. 1. Two weeks later, spleens and lungs were harvested, and single cell suspensions were prepared. Then, the relative numbers of Spike protein-specific CD4+ and CD8+ T cells secreting IFN-y among splenocytes and lung mononuclear cells (MNCs) were determined by an ELISPOT assay, using a mouse IFN-y ELISPOT assay kit (Mabtech, Inc). Briefly, 1 x 10⁶ splenocytes and 2 x 10⁵ MNCs were placed in 96-well plates pre-coated with anti-mouse IFN-y mAb (AN18) in the presence or absence of 10 pg/mL of a synthetic peptide corresponding to the CD8+ T-cell epitope (SEQ ID NO: 1 : GYLQPRTF), as well as pools (Pool 1 and Pool 2 shown below) of peptides corresponding to CD4+ T-cell epitopes, both derived from the Spike protein. After incubating the plates for 20 hr at 37 °C in 5% CO2 incubator, the plates were incubated with biotinylated anti-mouse-IFN-y mAb (R4-6A2) for 2 hr at RT, followed by incubation with streptavidin-ALP for 1 hr at RT. Finally, spots were developed by adding BCIP/NBT-plus substrate for 10 min at RT and quantified by stereomicroscopy. [0120] The results show that the 7DW8-5 glycolipid was able to display an adjuvant effect when administered with the Pfizer mRNA vaccine (BNT162b2), as shown in FIGS. 2A-C and FIG. 3. Administration of the 7DW8-5 glycolipid increased the level of Spikespecific splenic CD8+ T-cell responses compared to administration of the vaccine alone, as shown in FIG. 2A. Administration of the 7DW8-5 glycolipid increased by almost 2-fold the level of Spike-specific splenic CD4+ T-cell responses compared to administration of the vaccine alone, as shown in FIGS. 2B and 2C. Administration of the 7DW8-5 glycolipid also increased by almost 2-fold the level of Spike-specific lung-resident CD4+ T-cell responses compared to administration of the vaccine alone, as shown in FIG. 3. Administration of the 7DW8-5 glycolipid increased more than 2-fold the level of Spike-specific lung-resident CD8+ T-cell responses compared to administration of the vaccine alone, as shown in FIG. 3.

S-protein Peptide pool#

Peptide Length Sequence

Table 1

Peptide Length Sequence

Table 2 [0121] In the future, the efficacy of a booster shot/vaccine will be determined. A booster shot/vaccine will be administered to groups of mice 3-4 weeks after priming the mice with a mRNA vaccine administered with or without a dose 7DW8-5 glycolipid The levels of Spikespecific secondary CD4+ and CD8+ T-cell responses will be determined by the ELISPOT assay.

EXAMPLE 2 - the adjuvant effect of the 7DW8-5 glycolipid

[0122] In some embodiments, the 7DW8-5 glycolipid displays an adjuvant effect, increasing a subject’s immune response to an mRNA vaccine. In some embodiments, 7DW8-5 increases activation of helper T-cells compared to the vaccine alone. In some embodiments, 7DW8-5 increases activation of cytotoxic T-cells compared to the vaccine alone. In some embodiments, 7DW8-5 increases activation of B-cells compared to the vaccine alone. In some embodiments, 7DW8-5 increases production of spike-protein specific antibodies compared to the vaccine alone. In some embodiments, the antibodies induced by 7DW8-5 are specific for any one of the spike protein peptides in Table 1 and Table 2.

[0123] For the antibody (humoral) responses against the Spike protein, sera will be collected from groups of mice immunized with an mRNA-vaccine with or without 7DW8-5. The collection will be performed 4 weeks following immunization. Then, ELISA assays will be conducted to determine the level of antibodies specifically recognizing the Receptor Binding Domain (RBD) of the Spike protein. Briefly, the RBD protein will be plated on high-binding 96-well polystyrene plates (Corning) at 1 pg/mL in PBS for overnight at 4 °C. Unbound protein will be washed away with PBST (1 x PBS with 0.5% Tween) followed by blocking with 1% casein (w/v) in PBS (VWR International, Radnor, PA) for 2 hr at room temperature. Serum will then be incubated for 1 hr at room temperature with shaking, followed by washing with PBST. For serum anti-RBD antibody titers, six dilutions of sera in blocking buffer will be plated. Secondary reporter goat anti-mouse IgG HRP (Southern Biotech) will be diluted (1 :2500) in blocking buffer and incubated for 1 h at room temperature with shaking, followed by washing. Plates will be developed by adding l-step Ultra TMB (Fisher Scientific) for 30 seconds, followed by quenching with 2 N H2SO4. Absorbance (450 nm) will be measured by plate reader (Varioskan Flash, Thermo Fisher Scientific). Antibody titers will be calculated by sigmoidal non-linear regression using GraphPad Prism v.8 analysis with log 10 serum dilution plotted against absorbance at 450 nm. EXAMPLE 3 - co-administration of 7DW8-5 and the Pfizer mRNA vaccine

(BNT162b2) in mice

[0124] As shown herein, the immunostimulatory glycolipid 7DW8-5 displays an adjuvant effect to increase the T-cell immunogenicity of an mRNA-based CO VID vaccine when administered intranasally or intramuscularly. The immunogenic effects are observed after a single immunizing dose.

[0125] FIGS. 4A-C show that intramuscular co-administration of 7DW8-5 enhances T- cell immunogenicity of the Pfizer mRNA vaccine (BNT162b2) in mice. FIG. 4 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration. The vaccine was administered with or without 7DW8-5 intramuscularly 12 days prior to immunogenicity analysis or spleen and lung by ELISpot assay. FIG. 4B shows a Spikespecific splenic T-cell response to administration of the vaccine alone or with 7DW8-5. FIG. 4C shows a Spike-specific lung-resident T-cell response to administration of the vaccine alone or with 7DW8-5. Co-administration of 7DW8-5 with the vaccine leads to an increased immunogenicity response compared to administration of the vaccine alone.

[0126] FIGS. 5A-C show that intranasal co-administration of 7DW8-5 enhances T-cell immunogenicity of the Pfizer mRNA vaccine (BNT162b2) in mice. FIG. 5 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration. The vaccine was administered intranasally with or without 7DW8-5 ten days prior to analysis of spleen and lung by ELISpot assay. FIG.5B shows a Spike-specific splenic T-cell response to administration of the vaccine alone or with 7DW8-5. FIG. 5C shows a Spike-specific lungresident T-cell response to administration of the vaccine alone or with 7DW8-5. Intranasal co-administration of the canine and 7DW8-5 significantly increased the immunogenicity response in lungs.

[0127] FIGS. 6A-C show MAIO challenge after priming mice intranasally with Pfizer mRNA vaccine (BNT162b2) and 7DW8-5. FIG. 6 A shows a schematic representation of the BNT162b2 mRNA vaccine and 7DW8-5 administration prior to the MIO challenge. FIG. 6B shows mouse body weight change with administration of saline, 7DW8-5 alone, or 7DW8-5 with the mRNA vaccine. FIG. 6C shows virus titer on day 3 following the MAIO challenger with administration of saline, 7DW8-5 alone, or 7DW8-5 with the mRNA vaccine. Co- administration of the vaccine and 7DW8-5 significantly decreases virus titer in the lungs.

[0128] Whether 7DW8-5 increases the immunogenicity and efficacy of CO VID vaccines with intranasal administration after multiple immunizing doses can be determined using established methods in the art. Whether 7DW8-5 increases the durability of the CO VID vaccines can be determined using established methods in the art.

Claims

CLAIMS What is claimed:

1. An adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

2. The adjuvant of claim 1, wherein the mRNA-vaccine is a COVID-19 vaccine.

3. The adjuvant of claim 1, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

4. The adjuvant of claim 1, wherein the mRNA-vaccine comprises an mRNA encoding a

SARS-CoV-2 Spike protein.

5. The adjuvant of claim 1, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

6. The adjuvant of claim 1, wherein the mRNA-vaccine comprises the mRNA mRNA- 1273.

7. The adjuvant of claim 1, wherein the glycolipid compound is a-galactosylceramide (a-GalCer).

8. The adjuvant of claim 1, wherein the glycolipid compound is a modification or an analog of a-GalCer.

9. The adjuvant of claim 1, wherein the glycolipid compound is 7DW8-5.

10. The adjuvant of claim 1, wherein the adjuvant is administered to a subject concurrently with the mRNA-vaccine.

11. The adjuvant of claim 1, wherein the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine.

12. The adjuvant of claim 1, wherein the adjuvant is for intramuscular administration.

13. The adjuvant of claim 1, wherein the adjuvant is for intranasal administration.

14. The adjuvant of claim 1, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

15. The adjuvant of claim 14, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

16. A method of stimulating an immune response in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

17. The method of claim 16, wherein the mRNA-vaccine is a COVID-19 vaccine.

18. The method of claim 16, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

19. The method of claim 16, wherein the mRNA-vaccine comprises an mRNA encoding a

SARS-CoV-2 Spike protein.

20. The method of claim 16, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

21. The method of claim 16, wherein the mRNA-vaccine comprises the mRNA mRNA- 1273.

22. The method of claim 16, wherein the glycolipid compound is a-galactosylceramide (a-GalCer).

23. The method of claim 16, wherein the glycolipid compound is a modification or an analog of a-GalCer.

24. The method of claim 16, wherein the glycolipid compound is 7DW8-5.

25. The method of claim 16, wherein the adjuvant is administered to a subject concurrently with the mRNA-vaccine.

26. The method of claim 16, wherein the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine.

27. The method of claim 16, wherein the subject is a human.

28. The method of claim 16, wherein the adjuvant is for intramuscular administration.

29. The method of claim 16, wherein the adjuvant is for intranasal administration.

30. The method of claim 16, wherein the adjuvant is administered with a pharmaceutically acceptable excipient.

31. The method of claim 16, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

32. The method of claim 31, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

33. A method for augmenting an immune response to an antigen in a subject, the method comprising administering to the subject a pharmaceutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)-vaccine.

34. The method of claim 33, wherein the mRNA-vaccine is a COVID-19 vaccine.

35. The method of claim 33, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

36. The method of claim 33, wherein the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein.

37. The method of claim 33, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

38. The method of claim 33, wherein the mRNA-vaccine comprises the mRNA mRNA-

39. The method of claim 33, wherein the glycolipid compound is a-galactosylceramide (a-GalCer).

40. The method of claim 33, wherein the glycolipid compound is a modification or an analog of a-GalCer.

41. The method of claim 33, wherein the glycolipid compound is 7DW8-5.

42. The method of claim 33, wherein the adjuvant is administered to a subject concurrently with the mRNA-vaccine.

43. The method of claim 33, wherein the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine.

44. The method of claim 33, wherein the subject is a human.

45. The method of claim 33, wherein the antigen comprises at least a portion of a SARS- CoV-2 spike protein.

46. The method of claim 33, wherein the antigen is a peptide of Table 1.

47. The method of claim 33, wherein the antigen is a peptide of Table 2.

48. The method of claim 33, wherein the adjuvant is administered intramuscularly.

49. The method of claim 33, wherein the adjuvant is administered intranasally.

50. The method of claim 33, wherein the adjuvant is administered with a pharmaceutically acceptable excipient.

51. The method of claim 33, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

52. The method of claim 51, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

53. A method for elevating invariant Natural Killer T (zNKT) cells production in a subject, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)- vaccine.

54. The method of claim 53, wherein the mRNA-vaccine is a COVID-19 vaccine.

55. The method of claim 53, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

56. The method of claim 53, wherein the mRNA-vaccine comprises an mRNA encoding a

SARS-CoV-2 Spike protein.

57. The method of claim 53, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

58. The method of claim 53, wherein the mRNA-vaccine comprises the mRNA mRNA- 1273.

59. The method of claim 53, wherein the glycolipid compound is a-galactosylceramide (a-GalCer).

60. The method of claim 53, wherein the glycolipid compound is a modification or an analog of a-GalCer.

61. The method of claim 53, wherein the glycolipid compound is 7DW8-5.

62. The method of claim 53, wherein the adjuvant is administered to a subject concurrently with the mRNA-vaccine.

63. The method of claim 53, wherein the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine.

64. The method of claim 53, wherein the subject is a human.

65. The method of claim 53, wherein the adjuvant is for intramuscular administration.

66. The method of claim 53, wherein the adjuvant is for intranasal administration.

67. The method of claim 53, wherein the adjuvant is administered with a pharmaceutically acceptable excipient.

68. The method of claim 53, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

69. The method of claim 68, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

70. A method for stimulating cytokine and/or chemokine production in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an adjuvant for a vaccine, the adjuvant comprising a glycolipid compound or a pharmaceutically acceptable salt thereof, wherein the vaccine is a messenger RNA (mRNA)- vaccine.

71. The method of claim 70, wherein the mRNA-vaccine is a COVID-19 vaccine.

72. The method of claim 70, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

73. The method of claim 70, wherein the mRNA-vaccine comprises an mRNA encoding a

SARS-CoV-2 Spike protein.

74. The method of claim 70, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

75. The method of claim 70, wherein the mRNA-vaccine comprises the mRNA mRNA- 1273.

76. The method of claim 70, wherein the glycolipid compound is a-galactosylceramide (a-GalCer).

77. The method of claim 70, wherein the glycolipid compound is a modification or an analog of a-GalCer.

78. The method of claim 70, wherein the glycolipid compound is 7DW8-5.

79. The method of claim 70, wherein the adjuvant is administered to a subject concurrently with the mRNA-vaccine.

80. The method of claim 70, wherein the adjuvant is administered to a subject before administration of the mRNA-vaccine or after administration of the mRNA-vaccine.

81. The method of claim 70, wherein the subj ect is a human.

82. The method of claim 70, wherein the cytokine and/or chemokine production transactivates downstream immune cells.

83. The method of claim 82, wherein the downstream immune cells comprise one or more of dendritic cells (DC), natural killer cells (NK), B cells, CD4+T, and CD8+T cells.

84. The method of claim 82, wherein the cytokines comprise Thi cytokines.

85. The method of claim 82, wherein the cytokines are selected from the group consisting of interferon-gamma (IFN-y), GM-CSF, TNFoc, interleukin 2, interleukin 12, and interleukin 10.

86. The method of claim 70, wherein the adjuvant is for intramuscular administration.

87. The method of claim 70, wherein the adjuvant is for intranasal administration.

88. The method of claim 70, wherein the adjuvant is administered with a pharmaceutically acceptable excipient.

89. The method of claim 70, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

90. The method of claim 89, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

91. A pharmaceutical composition comprising: (i) a messenger RNA (mRNA)-vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine.

92. The composition of claim 91, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

93. The composition of claim 91, wherein the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof.

94. The composition of claim 91, wherein the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

95. The composition of claim 91, wherein the mRNA-vaccine is a COVID-19 vaccine.

96. The composition of claim 91, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

97. The composition of claim 91, wherein the mRNA-vaccine comprises an mRNA encoding a SARS-CoV-2 Spike protein.

98. The composition of claim 91, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

99. The composition of claim 91, wherein the mRNA-vaccine comprises the mRNA mRNA- 1273.

100. The composition of claim 91, wherein the glycolipid compound is a- galactosylceramide (a-GalCer).

101. The composition of claim 91, wherein the glycolipid compound is a modification or an analog of a-GalCer.

102. The composition of claim 91, wherein the glycolipid compound is 7DW8-5.

103. The composition of claim 91, wherein the mRNA-vaccine comprises an mRNA sequence encoding for an antigen.

104. The composition of claim 91, wherein the antigen comprises at least a portion of a SARS-CoV-2 spike protein.

105. The composition of claim 91, wherein the antigen is a peptide of Table 1.

106. The composition of claim 91, wherein the antigen is a peptide of Table 2.

107. The composition of claim 91, wherein the composition is administered intramuscularly.

108. The composition of claim 91, wherein the composition is administered intranasally.

109. The composition of claim 91, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

110. The composition of claim 109, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.

111. A kit for inoculating a subject in need thereof, the kit comprising (i) a messenger RNA (mRNA)-vaccine adjuvant, wherein the vaccine adjuvant comprises a glycolipid compound or a pharmaceutically acceptable salt thereof; and (ii) a mRNA-vaccine.

112. The kit of claim 111, wherein the vaccine adjuvant further comprises one or more pharmaceutically acceptable excipients.

113. The kit of claim 111, wherein the mRNA-vaccine is in an amount sufficient to stimulate an immune response in a subject in need thereof.

114. The kit of claim 111, wherein the mRNA-vaccine adjuvant is in an amount sufficient to stimulate an immune response in a subject in need thereof, when co-administered with the mRNA-vaccine.

115. The kit of claim 111, wherein the mRNA-vaccine is a COVID-19 vaccine.

116. The kit of claim 111, wherein the mRNA-vaccine is a SARS-CoV-2 vaccine.

117. The kit of claim 111, wherein the mRNA-vaccine comprises an mRNA encoding a

SARS-CoV-2 Spike protein.

118. The kit of claim 111, wherein the mRNA-vaccine comprises the mRNA BNT162b2.

119. The kit of claim 111, wherein the mRNA-vaccine comprises the mRNA mRNA-1273.

120. The kit of claim 111, wherein the glycolipid compound is a-galactosylceramide (a- GalCer).

121. The kit of claim 111, wherein the glycolipid compound is a modification or an analog of a-GalCer.

122. The kit of claim 111, wherein the glycolipid compound is 7DW8-5.

123. The kit of claim 111, wherein the mRNA-vaccine comprises a sequence encoding for an antigen to a subject.

124. The kit of claim 123, wherein the antigen comprises at least a portion of a SARS- CoV-2 spike protein.

125. The kit of claim 123, wherein the antigen is a peptide of Table 1.

126. The kit of claim 123, wherein the antigen is a peptide of Table 2.

127. The kit of claim 111, wherein the vaccine adjuvant and the mRNA vaccine are packaged together.

128. The kit of claim 111, wherein the vaccine adjuvant and the mRNA vaccine are packaged separately.

129. The kit of claim 111, wherein the adjuvant and the vaccine are administered intramuscularly.

130. The kit of claim 111, wherein the adjuvant and the vaccine are administered intranasally.

131. The kit of claim 111, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose range from about 1 pg to about 1 mg.

132. The kit of claim 131, wherein the glycolipid compound or a pharmaceutically acceptable salt thereof is administered in a dose of about 1 pg, about 50 pg, about 100 pg, about 150 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 550 pg, about 600 pg, about 650 pg, about 700 pg, about 750 pg, about 800 pg, about 850 pg, about 900 pg, about 950 pg, or about 1 mg.