Glyconjugate adjuvants derived from lipid a

Abstract

The present application describes a new class of vaccine adjuvants, which have the 2,4-nitrodiphenyl (DNP) group or an immunologically active sugar moiety, such as rhamnose (Rha) or α-galactose (α-Gal) epitope covalently linked to monophosphoryl lipid A (MPLA). DNP, Rha, α-Gal, and MPLA are potent immunostimulants / adjuvants that function through different mechanisms. The covalent linkage of DNP, Rha, or α-Gal with MPLA generates synergistic effects for stimulating the immune system, thereby creating more potent adjuvants.

Description

GLYCONJUGATE ADJUVANTS DERIVED FROM LIPID ASTATEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant No. R21 Al 170129, awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0002] Adjuvants derived from monophosphoryl lipid A (MPLA), or lipid A (LA), and either a 2,4-dinitrophenyl group (DNP) or a sugar, such as rhamnose (Rha), o-linked galactose (a-Gal) and o-Gal derivatives, for use in vaccines, intermediate compounds containing a DNP, Rha or o-Gal moiety, and methods for enhancing the immune response in a patient, and methods for preparing adjuvants derived from LA and DNP, Rha or o-Gal, wherein the new adjuvants display a synergistic effect, are described herein.BACKGROUND

[0003] Vaccination is the most economical and the most efficient strategy to control or prevent infectious diseases. Cancer immunotherapies, including treating cancer through activation of the immune system using therapeutic cancer vaccines, have also been proved to be very effective in recent years. For all these applications of vaccines, adjuvants are necessary. Adjuvants are substances that are formulated with vaccines to boost the immune response to vaccines. Without adjuvants, most vaccines would not be potent enough to be clinically useful. However, currently, only a few adjuvants are available, whilst all these adjuvants have limitations. For example, Alum, the most common adjuvant, was discovered in the 1930s, has low efficacy, and its mechanism of action in humans is not clear. Additionally, the few newly approved adjuvants have limited application scopes, for example, AS04 (monophosphoryl lipid A-based) was approved in 2009 for the CERVARIX® vaccine, for use against certain types of cancer-causing human papillomavirus (HPV); MF59 (squalene-based) was approved in 2016 for the FLUAD® vaccine, for use against seasonal influenza; and QS21 (a natural saponin or glycolipid) was approved for the SHI NGRIX® vaccine, for use against shingles. Thus, vaccine adjuvants having greater efficacy when administered to patients in need, enhanced immune response in the patients, improved formulation properties, and / or that are useful across many classes and types of vaccines are needed.SUMMARYIn one aspect, the disclosure provides an MPLA / LA conjugate adjuvant having a structure according to Formula (I) or Formula (II):wherein:R is selected from the group of -(CH2)a-CH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24;Q is absent or selected from -(O-CH2-CH2)n-, — (CH2)m— , -(O-CH2-CH2-CH2)t-, and a combination thereof, wherein n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15; and

[0004] The disclosure further provides an adjuvant, wherein X is - P(=O)(OH)2. The disclosure further provides an adjuvant, wherein X is -S(=O)2(OH). The disclosure further provides an adjuvant, wherein Y is an integer of6-24. The disclosure further provides an adjuvant, wherein L isThe disclosure furtherprovides an adjuvant, wherein L is O The disclosure further provides an adjuvant, wherein Q is -(O-CH2-CH2)n- The disclosure further provides an adjuvant, wherein n is an integer of 1-15, 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6. The disclosure further provides an adjuvant, wherein Q is -(O-CH2-CH2-CH2)t-. The disclosure further provides an adjuvant, wherein t is an integer of 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6. The disclosure further provides an adjuvant, wherein Q is -(CH2)m-. The disclosure further provides an adjuvant, wherein m is an integer of 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18, 2-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3, 4, 5, 6, 10,NO2Hor 12. The disclosure further provides an adjuvant, wherein Z is = The disclosure further provides an adjuvant, whereinThe disclosure further provides an adjuvant, wherein Z isThe disclosure further provides an adjuvant, whereinThe disclosure further provides an adjuvant, whereinThe disclosure furtherThe disclosure further provides an adjuvant, whereinThe disclosure further provides an adjuvant, wherein the MPLA / LA conjugate adjuvant has a structure according to Formula (I). The disclosure further provides an adjuvant, wherein MPLA / LA conjugate adjuvant has a structure according to Formula (II).

[0005] The disclosure further provides an adjuvant selected from the group consisting of:

[0006] In a second aspect, the present application describes a compound having a structure according to Formula (III):wherein: Q is selected from - (CF -CF jn- , — (CH2)m— , - (CF -CF -CF jt- , and a combination thereof, wherein n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15. The disclosure further provides a compound of Formula (III), wherein Q is -(CH2-CH2)n-. The disclosure further provides a compound of Formula (III), wherein n is an integer of 1-15, 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6. The disclosure further provides a compound of Formula (III), wherein Q is -(CH2-CH2-CH2)t-. The disclosure further provides a compound of Formula (III), wherein t is an integer of 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6. The disclosure further provides a compound of Formula (III), wherein Q is — (CH2)m— . The disclosure further provides a compound of Formula (III), wherein m is an integer of 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18, 2-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3, 4, 5, 6, 10, or 12.

[0007] In a third aspect, the disclosure provides methods of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant of the disclosure. The disclosure further provides methods of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant of the disclosure and a vaccine. The disclosure further provides methods of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant of the disclosure and a vaccine, wherein the MPLA / LA conjugate adjuvant and the vaccine are administered concurrently. The disclosure further provides methods of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant of the disclosure and a vaccine, wherein the vaccine and the MPLA / LA conjugate adjuvant are administered stepwise.

[0008] In a fourth aspect, a MPLA / LA conjugate adjuvant according to the present application is used to enhance an immune response.

[0009] In a fifth aspect, a MPLA / LA conjugate adjuvant according to the present application is used in the manufacture of a medicament for enhancing an immune response of a vaccine or an adjuvant.

[0010] In a sixth aspect, the present application describes a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) in its reduced form with a compound of Formula (III):whereinX is selected from-P(=O)(OH)2 and -S(=O)2(OH); and Y is an integer of 6-24; and Q is selected from -(CH2- CH2)n- -(CH2)m- -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0011] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) in its reduced form with a compound of Formula (III):wherein R is selected from the group of - (CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from — P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from — (CH2-CH2)n— , - (CH2)m-, — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0012] The disclosure further describes a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (VI):wherein X is selected from -P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from - (CH2-CH2)n- , -(CH2)m-, -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0013] The disclosure further describes a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising admixing a compound of Formula (V) with a compound of Formula (VI):wherein R is selected from the group of - (CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from — P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from - (CF -CF jn- , - (CH2)m-, — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0014] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II) comprising: admixing a compound of Formula (IV) in its reduced form with a compound of Formula (VII) or (VIII):wherein X is selected from — P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from — (CH2-CH2)n— , - (CH2)m- , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0015] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) in its reduced form with a compound of Formula (VII) or (VIII):wherein R is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)C-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1- 20, and c is an integer of 1-15; X is selected from-P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n- — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

[0016] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (IX):wherein X is selected from-P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2- CH2)n— , -(CH2)m-, - (CH2-CH2-CH2)t- , and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

[0017] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (IX):selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from -P(=O)(OH)2 and -S(=O)2(OH); and Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n-, — (CH2)m— -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

[0018] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) in its reduced form with a compound of Formula (X) or (XI):wherein X is selected from — P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from — (CH2-CH2)n— , - (CH2)m- , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0019] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) in its reduced form with a compound of Formula (X) or (XI):)b-OCH3,-(O-CH2-CH2- CH2)C-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from -P(=O)(OH)2and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n-, -(CH2)m-, -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0020] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (XII):is selected from -P(=O)(OH)2and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n-, - (CH2)m-, -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0021] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (IV) with a compound of Formula (XII):wherein R is selected from -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from - P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n- — (CH2)m— , -(CH2- CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0022] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) in its reduced form with a compound of Formula (XIII), (XIV),wherein X is selected from - P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from — (CH2-CH2)n— , — (CH2)m— -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0023] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) in its reduced form with a compound of Formula (XIII), (XIV),wherein R is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from -P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n- — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0024] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (XIX), (XX), or (XXI):wherein X is selected from— P(=O)(OH)2 and — S(=O)2(OH); Y is an integer of 6-24; and Q is selected from - (CF -CF jn- , — (CH2)m— , -(CH2- CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0025] The disclosure further provides a method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (IV) with a compound of Formula (XIX), (XX), or (XXI):of - (CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15; X is selected from -P(=O)(OH)2 and -S(=O)2(OH); Y is an integer of 6-24; and Q is selected from -(CH2-CH2)n-, — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

[0026] In the methods of the disclosure, the admixing is performed in a solvent selected from the group of water, methanol, dimethylformamide (DMF), dichloromethane, diethyl ether or their mixtures. In the methods of the disclosure, the admixing can be performed for a time of 1 hour to several days at a temperature of 0 °C to 40 °C.

[0027] In the methods of the disclosure, the admixing is performed in a solvent selected from the group of water, methanol, dimethylformamide (DMF), dichloromethane, diethyl ether, t-butyl alcohol, or their mixtures. Cu(ll) salts and sodium ascorbate are used as catalysts. In the methods of the disclosure, the admixing can be performed for a time of 1 hour to several days at a temperature of 0 °C to 40 °C.

[0028] Further aspects and advantages will be apparent to those of ordinary skill in the art from a review of the following detailed description. While the methods disclosed herein are susceptible of embodiments in various forms, the description hereafter includes specific embodiments with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGURE 1 shows the chemical structure of monophosphoryl lipid A (MPLA).

[0030] FIGURE 2 shows the method of action of an MPLA-DNP conjugate adjuvant of the disclosure with the immune system of a patient.

[0031] FIGURE 3 shows representative structure of Lipid A (1), example compounds according to Formula I (2a(P), 2b(a)), compounds according to Formula II (3a(P), 3b(a), 5a, and 5b), linkers (a and b), and intermediates (4a, 4b, and 4c) useful in the synthesis of compounds according to Formula II.

[0032] FIGURE 4 shows the synthesis of rhamnose derivatives 11 a / 11 b.

[0033] FIGURE 5 shows the synthesis of MPLA building blocks 14 and 17.

[0034] FIGURE 6 shows the synthesis 1-N-MPLA-(P)-Rhamnose conjugate 2a and 1-N-MPLA-(o)-Rhamnose conjugate 2b,

[0035] FIGURE 7 shows the synthesis of 6'-N-MPLA-(P)-Rhamnose conjugate 3a, and 6’-N-MPLA-(a)- Rhamnose conjugate 3b.

[0036] FIGURE 8 The synthesis of adjuvants 25a and 25b, wherein Rha is attached at the 1 -O-position of the disaccharide moiety of MPLA / LA via the triazole-amide linker.

[0037] FIGURE 9 shows the synthesis of adjuvants 27a and 27b, wherein Rha is attached at the C-6-position of the disaccharide moiety of MPLA / LA via the triazole-amide linker.

[0038] FIGURE 10 The synthesis of adjuvants 29 and 31, wherein o-Gal is attached at the 1 -O-position of the disaccharide moiety of Lipid A via the amide or triazole-amide linker.

[0039] FIGURE 11 shows the synthesis of adjuvants 5a and 5b, wherein the DNP moiety is attached at the 1- O position of the disaccharide moiety of MPLA / LA via the amide or triazole-amide linker.

[0040] FIGURE 12 shows the structures of the mixture of free MPLA 4b and Rha-lipid B utilized as a negative control.

[0041] FIGURE 13 shows the timeline of immunizations and blood collections as an example to demonstrate the immunological properties of the conjugate adjuvants.

[0042] FIGURE 14A shows the ELISA results (total antibody titers by adjuvant) of sTn-specific antibodies of the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA-NH2 + Rha-Lipid mixture, the control).

[0043] FIGURE 14B shows the ELISA results (IgG antibody titers by adjuvant) of sTn-specific antibodies of the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA- NH2 + Rha-Lipid mixture, the control).

[0044] FIGURE 14C shows the ELISA results (IgM antibody titers by adjuvant) of sTn-specific antibodies of the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA-NH2 + Rha-Lipid mixture, the control).

[0045] FIGURE 15A shows the ELISA results (total antibody titers by adjuvant) of KLH-specific antibodies in the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA-NH2 + Rha-Lipid mixture, the control).

[0046] FIGURE 15B shows the ELISA results (IgG antibody titers by adjuvant) of KLH-specific antibodies in the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA-NH2 + Rha-Lipid mixture, the control).

[0047] FIGURE 15C shows the ELISA results (IgM antibody titers by adjuvant) of KLH-specific antibodies in the day 38 antisera derived from mice immunized with sTn-KLH vaccine plus various adjuvants (4b / B is the MPLA-NH2 + Rha-Lipid mixture, the control).

[0048] FIGURE 16 shows the anti-sTn IgG antibody titers in the day 0, 27, 31 and 38 sera from mice before and after immunization with sTn-KLH using PBS, 4b / B (the MPLA-NH2 + Rha-Lipid mixture, the control), alum, and 3b as the adjuvant.

[0049] FIGURE 17 shows the anti-KLH IgG antibody titers in the day 0, 27, 31 and 38 sera from mice before and after immunization with sTn-KLH using PBS, 4b / B (MPLA-NH2 + Rha-Lipid mixture, the control), alum, and 2b as the adjuvants.DETAILED DESCRIPTION

[0050] Infectious diseases and cancer remain serious threats to human health. Therefore, prevention of infections and immunotherapy of cancer using vaccines, which stimulate the human immune system to fight diseases, are an area of intense research and development. To provoke effective and lasting protective immunities, a vaccine needs to be combined with an adjuvant, which is a substance formulated as part of a vaccine to boost immune responses and enhance the vaccine's effectiveness.

[0051] The disclosure provides a new class of vaccine adjuvants according to Formula (I) and Formula (II), methods for making the adjuvants, and methods for enhancing the immune response in a patient receiving a vaccine containing one or more of the adjuvants:wherein, the moiety Z in both Formulas I and II can be DNP or a specific sugar. R, X, Y, L, and Q are defined herein below.

[0052] Figure 1 illustrates the chemical structure of monophosphoryl lipid A (MPLA), also often referred to as Lipid A (LA). The terms "monophosphoryl lipid A,” "MPLA,” lipid A,” and "LA,” are used interchangeably herein. The compounds of Formulas I and II are a new class of adjuvants having either the DNP or a sugar moiety covalently coupled to LA, through linkers L and Q, to form the "conjugate adjuvants”. The term "lipid” describes a class of organic compounds that are fatty acids or their glycosylated derivatives and are insoluble in water but soluble in organic solvents. Lipids include many natural oils, waxes, and steroids. The term "2,4-dinitrophenyl (DNP)” describes an optionally-substituted moiety of the following structure:The term "sugar” here describes a sugar molecule, such as rhamnose (Rha) and o-linked galactose (o-Gal), wherein it has immunological activities. The disclosure provides sugar moieties such as described in the following group:"linker” describes an optionally-substituted ethyl amide or triazole group L, which is further coupled to an alkyl or alkoxy group containing from 1 to 60 methylene groups Q.

[0053] DNP, Rha, o-Gal, and MPLA (or LA) are potent immunostimulants, but they boost the human innate and adaptive immune systems through different mechanisms and pathways. DNP, Rha, and o-Gal recruit endogenous anti-DNP, -Rha or -o-Gal antibodies, respectively, naturally existing in the human serum to bind Fc receptors on immune cells to show antibody effector functions, while MPLA stimulates the immune system via interactions with toll-like receptor 4 (TLR4). Covalently coupling DNP, Rha or o-Gal and MPLA guarantees their co-localization and concerted actions on and in the same immune cells. These amphiphilic glycoconjugates can be applied in liposomal forms for multivalent presentation of the adjuvants, and the recruited anti-DNP, -Rha or - o-Gal antibodies can also attract more immune cells to the vaccination site to increase vaccine-immune system interaction. These factors can enhance the immunological activities of the resultant conjugates to provide a synergistic effect and form more effective adjuvants with a broader application scope, such as adjuvants used with antiviral, antibacterial, and antifungal vaccines and with therapeutic cancer vaccines.

[0054] Without intending to be bound by theory, it is believed that because monosulforyl lipid A (MSLA) has a structure substantially similar to MPLA, MSLA is expected to demonstrate immunostimulant properties similar to MPLA.

[0055] Further, Rha is known to inhibit tumor growth and this factor can enhance the immunological activities of the resultant conjugates to provide a synergistic effect and form more effective adjuvants with therapeutic cancer vaccines.

[0056] Without intending to be bound by theory, it is believed that due to the structural similarities between Rha and other deoxy sugars, wherein at least one hydroxyl group of a sugar has been replaced with a hydrogen atom such as deoxyribose and fucose, other deoxy sugars are expected to demonstrate immunostimulant properties similar to Rha.

[0057] Vaccine adjuvants are well known in the art, for example, as described in: N. Maeshima and R.C. Fernandez, Recognition of lipid A variants by the TLR4-MD-2 receptor complex. Front. Cell. Infect. Microbiol. 2013, 3, e3; Z. Jiang and R.R. Koganty, Synthetic vaccines: The role of adjuvants in immune targeting. Curr. Med. Chem. 2003, 10, 1423-1439; E. Ribi, J.L. Cantrell, K. Takayama, N. Qureshi, J. Peterson, and H.O. Ribi, Lipid A and immunotherapy. Rev. Infect. Dis. 1984, 6, 567-572; K. Ikeda, K. Miyajima, Y. Maruyama, and K. Achiwa, Synthesis of cancer peptide antigen-lipid A analog conjugates for synthetic vaccines. Chem. Pharm. Bull. 1999, 47, 563-568; K. Miyajima, T. Nekado, K. Ikeda, and K. Achiwa, Synthesis of Tn, sialyl Tn and HIV-1- derived peptide antigen conjugates having a lipid A analog as an immunoadjuvant for synthetic vaccines. Chem. Pharm. Bull. 1998, 46, 1676-1682; Z. Zhou, G. Liao, S.S. Mandal, S. Suryawanshi, and Z. Guo, A fully synthetic self-adjuvanting globo H-based vaccine elicited strong T cell-mediated antitumor immunity. Chem. Sci. 2015, 6, 7112-7121; G. Liao, Z. Zhou, S. Suryawanshi, M.A. Mondal, and Z. Guo, Fully synthetic self-adjuvanting a-2,9- oligosialic acid-based conjugate vaccines against group C meningitis. ACS Cent. Sci. 2016, 2, 210-218; M.J.S. Feigman and M.M. Pires, Synthetic immunobiotics: A future success story in small molecule-based immunotherapy? ACS Infect. Dis. 2018, 4, 664-672; P. Kaewsapsak, O. Esonu, and D.H. Dube, Recruiting the host's immune system to target Helicobacter pylori's surface glycans. ChemBioChem 2013, 14, 721-726; X. Liu, B. Zhang, Y. Wang, H. Haymour, F. Zhang, L. Xu, M. Srinivasarao, and P.S. Low, A universal dual mechanism immunotherapy for the treatment of influenza virus infections. Nat. Commun. 2020, 11, E5597; A. Dubrovska, C. Kim, J. Elliott, W. Shen, T. Kuo, D. Koo, C. Li, T. Tuntland, J. Chang, T. Groessl, X. Wu, V. Gorney, T. Ramirez- Montagut, A.A.C. Spiegel, C.Y. ho, and P.G. Schultz, A chemically induced vaccine strategy for prostate cancer. ACS Chem. Biol. 2011, 6; B. Schrand, E. Clark, A. Levay, A.R. Capote, O. Martinez, R. Brenneman, I. Castro, and E. Gilboa, Hapten-mediated recruitment of polyclonal antibodies to tumors engenders antitumor immunity. Nat. Commun. 2018, 9, e3348; J. Liu, H. Hong, J. Shi, Y. Xie, Z. Lu, Z. Liu, Z. Zhou, Z. Bian, Z. Huang, and Z. Wu, Dinitrophenol-mediated modulation of an anti-PD-L1 VHH for Fc-dependent effector functions and prolonged serum half-life. Eur. J. Pharmaceut. Sci. 2021, 165, e105941; T. Sato, T.N.J. Bullock, L.C. Eisenlohr, M.J. Mastrangelo, and D. Berd, Dinitrophenyl-modified autologous melanoma vaccine induces a T cell response to hapten-modified, melanoma peptides. Clin. Immunol. Immunother. 1997, 85, 265-272; D.K. Sojka, D. Felnerova,and M.B. Mokyr, Anti-metastatic activity of hapten-modified autologous tumor cell vaccine in an animal tumor model. Cancer Immunol. Immunother. 2002, 51 , 200-208; D. Berd, H.C. Maguire Jr, L.M. Schuchter, R. Hamilton, W.W. Hauck, T. Sato, and M.J. Mastrangelo, Autologous hapten-modified melanoma vaccine as postsurgical adjuvant treatment after resection of nodal netastases. J. Clin. Oncol. 1997, 15, 2359-2370; and D. Berd, Autologous, hapten-modified vaccine as a treatment for human cancers. Vaccine 2001 , 19 2565-2570. The above-listed references are incorporated herein by reference in their entirety.

[0058] Figure 2 illustrates the anticipated biological pathway of MPLA in a patient.

[0059] Figure 3 illustrates representative structure of Lipid A (1), compounds according to Formula I (2a(P),2b(a)), compounds according to Formula II (3a(P), 3b(a), 5a, and 5b), linkers within them (a and b), and intermediates (4a, 4b, and 4c) useful in the synthesis of compounds according to Formula II.

[0060] The disclosure provides a MPLA / LA conjugate adjuvant having a structure according to Formula (I) or Formula (II):wherein:R is selected from the group of - (CH2)aC H3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24;Q is absent or selected from -(O-CH2-CH2)n-, — (CH2)m— , -(O-CH2-CH2-CH2)t-, and a combination thereof, wherein n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15; and

[0061] The MPLA / LA conjugate adjuvant of the disclosure can have a structure according to Formula (I). The MPLA / LA conjugate adjuvant of the disclosure can have a structure according to Formula (II).

[0062] In general, R is an alkyl group or derivatives selected from -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O- CH2-CH2-CH2)C-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15. R can be -CHs. R can be -(O-CH2-CH2)-OCH3. R can be ,-(O-CH2-CH2-CH2)-OCH3.

[0063] In general, X is selected from -P(=O)(OH)2and -S(=O)2(OH). X can be -P(=O)(OH)2. Without intending to be bound by theory, it is believed that when X is — P(=O)(OH)2, the MPLA / LA conjugate adjuvant can stimulate the immune system via interactions with toll-like receptor (TLR4) in the same way as MPLA. X can be -S(=O)2(OH). Without intending to be bound by theory, it is believed that when X is — S(=O)2(OH), the MPLA / LA conjugate adjuvant will behave in the same way as a MPLA / LA conjugate adjuvant of the disclosure wherein X is -P(=O)(OH)2due to the structural and electrical similarities.

[0064] In general, Y is an integer between 6-24 (i.e., 6 to 24 carbon atoms or methylene groups) as well as all subgroups (e.g, 6-24, 6-23, 6-22, 6-19, 6-20, 6-18, 6-16, 6-14, 6-12, 6-10, 8-10, 8-18, 10-18, 12-18, 14-18, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, etc.) between and including the end points. Y can be 6-12, for example 8-10, or 6, 8, 10, or 12. Y can be 8. Y can be 10. In general, as Y decreases, for example, below 6, the compound becomes less lipophilic and may be water soluble to affect its immunological activity. Ingeneral, as Y increases, for example, above 24, the compound may not interact with TLR4 effectively to affect its immunological activity. o

[0065] In general, L is an optionally-substituted ethyl or triazole amido moiety. L ean be. L can b

[0066] In general Q is an alkyl or alkoxy having between 1-60 methylene groups and between 0-20 oxygen atoms, but longer linkers or linker with other substituents may also work but synthetically more challenging. Without intending to be bound by theory, it is believed that as the length of Q increases, e.g., above 60 atoms in the chain, the ability of the MPLA / LA moiety and the DNP or sugar moieties to exhibit a concerted action decreases. Q can be -(O-CH2-CH2)n- n can be an integer in a range of 1-15, 1-10, 2-8, or 2-6, for example, 2,3, 4, 5, or 6. Q can be -(O-CH2-CH2-CH2)t-. t can be an integer in a range of 1-10, 2-8, or 2-6, for example, 2, 3,4, 5, or 6. Q can be -(CH2)m-. m can be an integer in a range of 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18, 2-16, 3-14, 3-12, 3-10, 3-8, or 3-6, for example, 3, 4, 5, 6, 10, or 12.

[0067] In general Z is an optionally-substituted DNP moiety or sugar, wherein the sugar has immunological stimulating property, such as Rha and o-Gal. Z can

[0068] The compounds of Formulas I and II can be present as salts. Pharmaceutically acceptable (i.e., nontoxic, physiologically acceptable) salts are preferred. If the compounds of Formulas I and II have, for example, at least one basic center, they can form acid addition salts. These are formed, for example, with strong inorganic acids, such as mineral acids, for example sulfuric acid, phosphoric acid or a hydrohalic acid, with organic carboxylic acids, such as alkane carboxylic acids of 1 to 4 carbon atoms, for example acetic acid, which are unsubstituted or substituted, for example, by halogen as chloroacetic acid, such as saturated or unsaturateddicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or terephthalic acid, such as hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid, such as amino acids, (for example aspartic or glutamic acid or lysine or arginine), or benzoic acid, or with organic sulfonic acids, such as (C1-C4) alkyl or arylsulfonic acids which are unsubstituted or substituted, for example by halogen, for example methyl- or p-toluene-sulfonic acid. Corresponding acid addition salts can also be formed having, if desired, an additionally present basic center. The compounds of Formulas I and II having at least one acid group (for example COCH, -P(=O)(OH)2, -S(=O)2OH) can also form salts with bases. Suitable salts with bases are, for example, metal salts, such as alkali metal or alkaline earth metal salts, for example sodium, potassium or magnesium salts, or salts with ammonia or an organic amine, such as morpholine, thiomorpholine, piperidine, pyrrolidine, a mono, di or tri-lower alkylamine, for example ethyl, tert-butyl, diethyl, diisopropyl, triethyl, tributyl or dimethyl-propylamine, or a mono, di or trihydroxy lower alkylamine, for example mono, di or triethanolamine. Corresponding internal salts may furthermore be formed. Salts which are unsuitable for pharmaceutical use, but which can be employed, for example, for the isolation or purification of free compounds of Formulas I and II or their pharmaceutically acceptable salts, are also included.

[0069] Preferred salts of the compounds of Formulas I and II which contain a basic group include monohydrochloride, hydrogensulfate, methanesulfonate, phosphate, nitrate, or acetate.

[0070] Preferred salts of the compounds of Formulas I and II which contain an acid group include sodium, potassium and magnesium salts and pharmaceutically acceptable organic amines.

[0071] The compounds of Formulas I and II can include modulators, which are also within the scope of the invention. Modulator refers to a chemical compound with the capacity to either enhance (e.g., "agonist” activity) or partially enhance (e.g., "partial agonist” activity) or inhibit (e.g., "antagonist” activity or "inverse agonist” activity) a functional property of biological activity or process (e.g., enzyme activity or receptor binding); such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway, and / or may be manifest only in particular cell types.

[0072] The compounds of Formulas I and II can include a bioactive metabolite, which are also within the scope of the invention. Bioactive metabolite refers to any functional group contained in a compound of Formulas I and II with an open valence for further substitution wherein such substitution can, upon biotransformation, generate a compound of Formulas I and II. Examples of such functional groups of bioactive metabolites include, but are not limited to, —OH, — NH or functional groups wherein the hydrogen can be replaced with a functional group such as — PO3H2for example, which, upon biotransformation generates an —OH or — NH functional group of a compound of Formulas I and II.

[0073] The compounds of Formulas I and II can include prodrug esters, which are also within the scope of the invention. Prodrug esters refers to esters and carbonates formed by reacting one or more hydroxyls of compounds of Formulas I and II with alkyl, alkoxy, or aryl substituted acylating agents employing procedures known to those skilled in the art to generate acetates, pivalates, methylcarbonates, benzoates and the like.Prodrug esters may also include but are not limited to groups such as phosphate esters, phosphonate esters, phosphonamidate esters, sulfate esters, sulfonate esters, and sulfonamidate esters wherein the ester may be further substituted with groups that confer a pharmaceutical advantage such as, but not limited to, favorable aqueous solubility or in vivo exposure to the bioactive component of Formulas I and II.

[0074] The compounds of Formulas I and II can include prodrugs, which are also within the scope of the invention. Prodrugs refers to functionalization of bioactive amine- or hydroxyl-containing compounds of Formulas I and II to form alkyl-, acyl-, sulfonyl-, phosphoryl-, or carbohydrate-substituted derivatives. Such derivatives are formed by reacting compounds of Formulas I and II with alkylating-, acylating-, sulfonylating-, or phosphorylating reagents employing procedures known to those skilled in the art. Alkylation of amines of Formulas I and II may result in— but are not limited to— derivatives that include spacer units to other prodrug moieties such as substituted alkyoxymethyl-, acyloxymethyl-, phosphoryloxymethyl-, or sulfonyloxymethyl-groups. Alkylation of amines of Formulas I and II may result in the generation of quaternary amine salts that act in vivo to provide the bioactive agent (i.e., the compound of Formulas I and II).

[0075] Prodrugs may also consist of a compound of Formulas I and II where a pendant hydroxyl is phosphorylated to generate a phosphate derivative. Such a prodrug may also include a spacer group between the compound of Formulas I and II and the phosphate group, such as a methyleneoxy-group. Methods to generate such a prodrug from a compound of Formulas I and II are known to those skilled in the art and are listed in the references below.

[0076] Any compound that can be converted in vivo to provide the bioactive agent (i.e., the compound of Formulas I and II) is a prodrug within the scope and spirit of the invention.

[0077] Various forms of prodrugs are well known in the art. A comprehensive description of prodrugs and prodrug derivatives are described in: The Practice of Medicinal Chemistry, Camille G. Wermuth et al., Ch 31, (Academic Press, 1996); Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985); A Textbook of Drug Design and Development, P. Krogsgaard-Larson and H. Bundgaard, eds. Ch 5, pgs 113-191 (Harwood Academic Publishers, 1991); Hydrolysis in Drug and Prodrug Metabolism, B. Testa and J. M. Mayer, (Verlag Helvetica Chimica Acta AG, Zurich, Switzerland; Wiley-VCH, Weinheim, Federal Republic of Germany, 2003); Ettmayer, P.; Amidon, G. L; Clement, B.; Testa, B. "Lessons Learned from Marketed and Investigational Prodrugs” J. Med. Chem. 2004, 47 (10), 2393-2404; and Davidsen, S. K. et al. “N-(Acyloxyalkyl)pyridinium Salts as Soluble Prodrugs of a Potent Platelet Activating Factor Antagonist” J. Med. Chem. 1994, 37 (26), 4423-4429. The above-listed references are incorporated herein by reference in their entireties.

[0078] The disclosure also includes molecules which have been isotopical ly enriched at one or more positions within the molecule. The term "isotopically enriched,” as used herein, refers to an adjuvant or compound of the disclosure with the isotopic content for one isotope at a predetermined position within a molecule that is at least 100 times greater than the natural abundance of this isotope. For example, a composition that is isotopically enriched for deuterium includes an adjuvant with at least one hydrogen atom position having at least 100 timesgreater abundance of deuterium than the natural abundance of deuterium. Preferably, an isotopic enrichment for deuterium is at least 1000 times greater than the natural abundance of deuterium. More preferably, an isotopic enrichment for deuterium is at least 4000 times greater (e.g., at least 4750 times greater, e.g., up to 5000 times greater) than the natural abundance of deuterium. Thus, compounds enriched for deuterium fall within the scope of the claims.

[0079] The disclosure provides methods of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant according to Formulas I and II. The methods can further comprise administering at least one vaccine, including anti-virus, -parasite, -bacterium, -fungus, and - cancer vaccines administered individually or in combinations. The vaccine and the MPLA / LA conjugate adjuvant can be administered concurrently. The vaccine and the MPLA / LA conjugate adjuvant can be administered stepwise.

[0080] An administration of a therapeutic agent of the invention includes administration of a therapeutically effective amount of the agent of the invention. The term "therapeutically effective amount” as used herein refers to an amount of a therapeutic agent to treat or prevent a condition treatable by administration of a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic or preventative or ameliorative effect. The effect may include, for example, treatment or prevention of the conditions listed herein. The precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration.

[0081] All stereoisomers of the compounds of the instant invention are contemplated, either in mixture or in pure or substantially pure form. The compounds of the present invention can have asymmetric centers at any of the carbon atoms including any one of the R substituents. Consequently, compounds of Formulas I and II can exist in enantiomeric or diastereomeric forms or in mixtures thereof. The processes for preparation can utilize racemates, enantiomers or diastereomers as starting materials. When diastereomeric or enantiomeric products are prepared, they can be separated by conventional methods for example, chromatographic techniques, chiral HPLC or fractional crystallization.

[0082] The compounds of Formulas I and II of the invention can be prepared as shown in the figures and description herein, as well as relevant published literature procedures that may be used by one skilled in the art. Exemplary reagents and procedures for these reactions appear hereinafter and in the working Examples.

[0083] The disclosure further provides compounds having a structure according to Formulas (III and VI-XXI):wherein Q is selected from -(CH2-CH2)n- — (CH2)m— -(CH2-CH2-CH2)t-, and a combination thereof, wherein n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15. Q can be -(CH2-CH2)n-. n is an integer in a range of 1-15, 1-10, 2-8, or 2-6, for example, 2, 3, 4, 5, or 6. Q can be -(CH2-CH2-CH2)t-. t is an integer in a range of 1-10, 2-8, or 2-6, for example, 2, 3, 4, 5, or 6. Q can be -(CH2)m-. m is an integer in a range of 1-55, 1- 50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18, 2-16, 3-14, 3-12, 3-10, 3-8, or 3-6, for example, 3, 4, 5, 6, 10, or 12.

[0084] The compounds of Formulas I and II can be prepared by admixing azide-containing intermediates IV and V with any of DNP intermediates III and VI, rhamnose intermediates VII, VIII, and IX, or galactose intermediates X, XI, and XII, as well as their derivatives containing sugar moieties,

[0085] In general, the admixing involves one or more reaction steps and is performed in a solvent selected from the group of water, methanol, dimethylformamide, dichloromethane, diethyl ether, or any mixture thereof. In general, the admixing is performed for a time of 1 hour to several days. In general, the admixing is performed at a temperature of 0 °C to 40 °C.EXAMPLES

[0086] The following Examples serve to better illustrate, but not limit, some of the preferred embodiments of the disclosure.

[0087] Synthesis of Rhamnose-MPLA Adjuvants Having Amide Linker:

[0088] The target synthetic adjuvants 2a / 2b and 3a / 3b generated for this study are shown in Figures 6 and 7. To access the synthetic adjuvants 2a / 2b and 3a / 3b, the building blocks 11 a / 11 b and 14 / 17 are required and the coupling of these key intermediates to assemble the target adjuvants was accomplished by amide bond coupling reaction of the intermediates as depicted in Figures 6 and 7. 11 a / 11 b (Figure 4) were synthesized as the configurationally defined (i.e., 0 and DDisomers) L-rhamnose epitopes with 3-O-propanoic acid as a linker. Thus, the benzyl-protected L-rhamnose anomers with free acid functionality could be further utilized in the conjugation process, as the benzyl groups were removed in the final step. The key building block 14 of MPLA / LA has a 2- azidoethyl group at the reducing end (C-1 -O-position) of the disaccharide backbone whereas compound 17 has an azido group at the C-6' position. Advantageously, the azido groups in both MPLA derivatives could be reduced to amine that would be used in the conjugation process with rhamnose acids. Moreover, global deprotection of 14 and 17 would afford free MPLA derivatives that can be explored as independent vaccine adjuvants or for the synthesis of other conjugate adjuvants disclosed herein.

[0089] As depicted in Figure 4, L-rhamnose antigens were prepared from commercially available L-rhamnose monohydrate. L-Rhamnose monohydrate 6 was peraceylated with AC2O and pyridine followed by the introduction of the 4-thiotolyl group at the anomeric position giving the peraceylated thiol donor 7. The required benzyl protections were installed in two steps, all acetate groups were removed in a solution of MeONa / methanol (pH = 9-10) and subsequently protected with benzyl ether using NaH and benzyl bromide, which delivered thiol donor 8 in quantitative yield. The glycosylation reaction between the acceptor methyl 3-hydroxypropanoate 9 and benzyl protected thiol donor 8 was achieved with N-iodosuccinimide (NIS) and trimethylsilyl triflate (TMSOTf) as promoters. The reaction afforded a 1 :1 mixture of the desiredm and D-anomers in excellent yield (93%) and both anomers were purified by column chromatography and confirmed by the C-H bond coupling at the anomeric positions. The D-anomer had a C-H bond coupling of 168.2 Hz while the D-anomer had a C-H bond coupling of 153.8 Hz. The desired 0- and DDpropanoic acid derivatives of rhamnose 11a (83%) and 11b (93%) were individually obtained by the hydrolysis of the methyl ester with aqueous lithium hydroxide, respectively.

[0090] As outlined in Figure 5, intermediates 14 and 17 were synthesized, wherein the MPLA derivative has an azido ethyl group at the reducing end i.e., 1 -O-position or the C-6' position, respectively, of the disaccharide unit. The requisite acceptor and donor were synthesized from the D-glucosamine by following protocols previouslyestablished in our group. A series of transformations of acceptor and donor including glycosylation, and deprotection of phthalimide groups followed by sequential installations of the lipid chains on the disaccharide unit and phosphorylation at the Opposition respectively, provided the protected key building block derivative MPLA 13. The spectral data for compound 13 was matched with literature data. Further, the azido ethyl group at the reducing end (1 -O-position) of the MPLA derivative 14 was ready for the conjugation step with the previously synthesized propanoic acid derivatives of rhamnose 11a / 11 b. Alternatively, compound 16 was converted into 14 with the azido ethyl group at the C-6' position of the MPLA, which was also ready for the conjugation with rhamnose derivatives 11a / 11 b.

[0091] As outlined in Figures 6 and 7, the azide functionality in 14 and 17 was selectively reduced with excess Zn in acidic conditions to yield the crude amines, which were directly subjected to coupling reaction with 11a / 11 b without purification. The coupling reaction was achieved via the use of 1 -ethyl-3-(3-dimethyl aminopropyl)- carbodiimide (EDC) to yield the protected conjugate adjuvant assemblies 18a / 18b and 19a / 19b, respectively. Global deprotection of conjugates 18a, 18b, 19a, and 19b with 10% Pd / C as a catalyst in CH2CI2: MeOH (1 :1) for 2 days under H2 gas atmosphere afforded target adjuvants 2a, 2b, 3a, and 3b, respectively, in good yields. All the final products were purified by column chromatography with a mixture of CHCI3 and CH3OH as eluents. The synthetic targets and the key intermediates were all fully characterized with NMR and MS spectroscopic data.

[0092] Synthesis of Rhamnose-MPLA Adjuvants Having Triazole Linker:

[0093] Illustrated in Figure 8 is the synthesis of Rha-MPLA conjugate adjuvants 25a and 25b with |3-Rha and o-Rha attached to the MPLA reducing end 1-O-postion via a triazole linker. The coupling between azide 14 and alkynes 23a / 23b under the catalysis of CUSO4 and sodium ascorbate afforded directly 24a / 24b. Global deprotection of 24a / 24b as described above afforded the target ants 25a and 25b, respectively.

[0094] Illustrated in Figure 9 is the synthesis of Rha-MPLA conjugate adjuvants 27a and 27b with |3-Rha and o-Rha attached to the MPLA non-reducing end C-6'postion via a triazole linker by the same strategy as described above for 25a / 25b.

[0095] Illustrated in Figure 10 is the synthesis of conjugate adjuvants 29 and 31, wherein an o-Gal residue is attached to the MPLA reducing end 1-O-postion via an amide and the triazole-amide linker, respectively, using key intermediate 14 and activated ester 28 and glycosylated alkyne 30 by similar method for the synthesis of 2a and 25a,

[0096] Illustrated in Figure 11 is the synthesis of adjuvants 5a and 5b, wherein the DNP moiety is attached to the MPLA reducing end 1-O-postion via an amide and the triazole-amide linker, respectively, by the similar strategy. However, in the synthesis of 5b, an extra step was involved to reduce the azide and then reinstall the azide to avoid the reduction of DNP during the global deprotection process.

[0097] (3-Methoxy-3-oxopropyl) 2,3,4-tri-O-benzymethyl-D-L-rhamnopyranoside (10a) and (3-methoxy-3- oxopropyl) 2,3,4-tri-O-benzymethyl-D-L-rhamnopyranoside (10b): A suspension of thiol donor 8 (560 mg, 5.38mmol) and 4 A molecular sieves in dry dichloromethane (10 mL) was stirred at rt for 30 min and then cooled to - 50 °C. N-lodo succinimide (NIS) (1.82 mg, 8.07 mmol) was added into the mixture followed by adding TfOH (47.5 pL, 0.537 mmol) slowly under N2 at -50 °C, and the mixture was stirred while warming to 0 °C for 2 h. After completion of the reaction, the reaction mixture was quenched by TEA and diluted with dichloromethane. The resulting mixture was filtered through Celite, extracted with aqueous sat. NaHCOa solution and concentrated in vacuum. The residue was purified by silica gel column chromatography (petrol ethenethyl acetate = 15: 1) to afford 10a (D-anomer, 1 .2 g, 43%) as pale-yellow oil and 10b (D-anomer, 1 .4 g, 50%) as yellow oil. Compound 10a:1H NMR (600 MHz, CDCI3) 5 7.49 - 7.40 (m, 2H, ArH), 7.35 - 7.25 (m, 13H, ArH), 4.96 (d, J = 11.0 Hz, 1 H, -CH2-Ph), 4.94 (d, J = 12.4 Hz, 1 H, -CH2-Ph), 4.83 (d, J = 12.6 Hz, 1 H, -CH2-Ph), 4.64 (d, J = 10.8 Hz, 1 H, -CH2- Ph), 4.49 (d, J = 11.8 Hz, 1 H, -CH2-Ph), 4.42 (d, J = 11.8 Hz, 1 H, -CH2-Ph), 4.39 (s, 1 H, H-1), 4.15 (dt, J = 9.7, 5.8 Hz, 1 H, -OCH2- ), 3.88 (d, J = 2.9 Hz, 1 H, H-2), 3.81 - 3.74 (m, 1 H, -OCH2-), 3.61 (t, J = 9.3 Hz, 1 H, H-4), 3.44 (dd, J = 9.4, 3.0 Hz, 1 H, H-3), 3.31 (dq, J = 9.3, 6.1 Hz, 1 H, H-5), 2.72- 2.67 (m, 1 H, -OCH2-), 2.64 (dt, J = 10.5, 5.6 Hz, 1 H, -OCH2-), 1.38 (d, J = 6.2 Hz, 1 H, H-6).13C NMR (151 MHz, CDCI3) 5 172.11 , 138.82, 138.64, 138.35, 128.61 , 128.53, 128.51 , 128.47, 128.24, 128.21 , 127.82, 127.69, 127.55, 101.82, 82.23, 80.24, 75.59, 73.97, 73.76, 72.11 , 71.54, 65.35, 51.86, 35.11 , 18.11. HRMS (ESI) m / z: [M + Na]+Calculated for CaiHaeOzNa 543.2353, found 543.2361. Compound 10b:1H NMR (600 MHz, CDCI3) 5 7.40 - 7.27 (m, 15H, ArH), 4.94 (d, J = 10.9 Hz, 1 H, -CH2-Ph), 4.79 (d, J = 1.7 Hz, 1 H, H-1), 4.74 (q, J = 12.4 Hz, 2H, -CH2-Ph), 4.64 (d, J = 10.9 Hz, 1 H, -CH-Ph), 4.62 (brs, 2H, -CH2-Ph), 3.90 (dt, J = 10.0, 6.3 Hz, 1 H, -OCH2-), 3.82 (dd, J = 9.2, 3.1 Hz, 1 H, H-3), 3.76 (dd, J = 3.0, 2.0 Hz, 1 H, H-2), 3.72 -3.58 (m, 6H, H-4, H-5, -OCH3 and -OCH2-), 2.54 (t, J = 6.4 Hz, 2H, - C / 72-CO2Me), 1.34 (d, J = 6.2 Hz, 2H, H-6).13C NMR (151 MHz, CDCI3) 5 171.80, 138.76, 138.73, 138.49, 128.47, 128.46, 128.13, 128.02, 127.76, 127.74, 127.72, 127.62, 98.29, 80.57, 80.19, 75.48, 74.98, 72.92,72.28, 68.33, 62.97, 51.83, 34.74, 18.12. HRMS (ESI) m / z: [M + Na]+Calculated for CaiHaeOzNa 543.2353, found 543.2362.

[0098] (2-Carboxyethyl) 2,3,4-tri-O-benzymethyl-D-L-rhamnopyranoside (11a): To a solution of 10a (200 mg, 0.38 mmol) in THF (4 mL) was added a solution of LIOH'H2O (48.3 mg, 1.15 mmol) in H2O (0.5 mL) at O °C. The mixture was stirred while warming to rt for 12 h until no starting material was detected by TLC. The aqueous layer was acidified to pH 2 with aqueous HCI solution (1 .0 mol / L) and extracted with ethyl acetate (20 mL x 2). The organic layers were washed with brine (20 mL x 2), dried over anhydrous Na2SO4, and concentrated in vacuum. The product was purified by flash chromatography (dichloromethane: MeOH = 10:1) to afford 11a (170 mg, 87%) as colourless oil.1H NMR (400 MHz, CDCI3) 5 7.45 (d, J = 6.5 Hz, 2H), 7.39 - 7.23 (m, 13H, ArH), 5.03 - 4.91 (m, 2H, -CH2-Ph), 4.85 (d, J = 12.5 Hz, 1 H, -CH2-Ph), 4.72 - 4.61 (m, 1 H, -CH2-Ph), 4.57 - 4.42 (m, 2H, -CH2-Ph), 4.40 (d, J = 1.7 Hz, 1 H, H-1), 4.26 - 4.11 (m, 1 H, -OCH2-), 3.91 (s, 1 H, H-2), 3.83 - 3.73 (m, 1 H, - OCH2-), 3.63 (dt, J = 9.3, 4.7 Hz, 1 H, H-4), 3.47 (dd, J = 9.4, 3.0 Hz, 1 H, H-3), 3.33 (td, J = 6.2, 3.4 Hz, 1 H, H-5), 2.84 - 2.60 (m, 1 H, -OCH2-), 1.39 (d, J = 6.0 Hz, 1 H, H-6).13C NMR (101 MHz, CDCI3) 5 177.35, 138.70, 138.54, 138.27, 128.61 , 128.49, 128.46, 128.25, 128.19, 127.83, 127.67, 127.56, 101.71 , 82.17, 80.15, 75.57, 74.01 ,73.75, 72.12, 71.54, 64.94, 35.11 , 18.05. HRMS (ESI) m / z: [M + Na]+Calculated for Cao^NaOz 529.2197, found529.2200.

[0099] (2-Carboxyethyl) 2,3,4-tri-O-benzymethyl-D-L-rhamnopyranoside (11b): Compound 11b (181 mg, 93%) was synthesized from 10b (200 mg, 0.38 mmol) by the same procedure and conditions employed for the synthesis of 11a.1H NMR (400 MHz, CDCI3) 5 7.48 - 7.24 (m, 15H, Ar / - / ), 4.93 (d, J = 10.8 Hz, 1 H, -CH2-Ph ), 4.78 (d, J = 1.7 Hz, 1 H, H-1), 4.72 (q, J = 12.5 Hz, 1 H, -CH2-Ph), 4.63 (d, J = 10.8 Hz, 1 H, -CH2-Ph), 4.60 (q, J = 12 Hz, 2H, -C / 72-Ph), 3.89 (dt, J = 10.1 , 6.2 Hz, 1 H, -OCH2-), 3.82 (dd, J = 9.1 , 3.1 Hz, 1 H, H-3), 3.75 (dd, J = 3.0, 1.9 Hz, 1 H, H-2), 3.73 - 3.57 (m, 3H, -OCH2-, H-4 and H-5), 2.57 (t, J = 6.3 Hz, 2H, -OCH2-), 1.33 (d, J = 6.1 Hz, 3H, H-6).13C NMR (101 MHz, CDCI3) 5 176.26, 138.68, 138.43, 128.49, 128.20, 128.05, 127.80, 127.78, 127.67, 98.34, 80.53, 80.12, 75.51 , 75.00, 72.98, 72.33, 68.41 , 62.68, 34.53, 18.11. HRMS (ESI) m / z: [M + Na]+Calculated for Cst^NaOz 529.2197, found 529.2207.

[0100] Compound 18a (C-Rh-conjugate): To a solution of 14 (36 mg, 0.016 mmol) in AcOH / THF (1 / 3, 2 mL) was added activated Zn (52.4 mg, 0.80 mmol). After the mixture was stirred at rt for 8 h, it was filtered through a Celite pad, and the filtrate was concentrated under reduced pressure. The crude product was stripped with dry toluene (3x3 mL) and then dissolved in 1 mL of dry CH2CI2. To this solution were added D-L-Rhamnose acid 11a (19.2 mg, 0.037 mmol), 1 -ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) (14.5 mg, 0.075 mmol) and DMAP (1 .1 mg, 0.009 mmol) in 2 mL of dry CH2CI2at 0 °C. The mixture was stirred at rt overnight and diluted with CH2CI2. The organic layer was washed with saturated NaHCOs solution, water, and brine and dried over Na2SO4. The solution was concentrated under a vacuum, and the residue was purified by silica gel column chromatography to offer 18a (24.8 mg, 71 %) a colourless liquid.1H NMR (600 MHz, CDCI3) 5 7.42 (d, J= 7.1 Hz, 2H, Ar / - / ), 7.32 - 7.24 (m, 33H, Ar / - / ), 7.23 - 7.20 (m, 8H, Ar / - / ), 7.18 - 7.14 (m, 2H, Ar / - / ), 6.73 (brs, 1 H, - C(O)NH), 6.07 (d, J = 7.8 Hz, 1 H, -C(O)NH), 5.80 (d, J = 8.7 Hz, 1 H, -C(O)NH), 5.48 (dd, J = 9.1 Hz 1 H, H-3’), 5.10 - 5.01 (m, 3H, 2xlipid-H, H-3), 5.00 (d, J = 8.2 Hz, 1 H, H-1’), 4.94 (d, J = 10.8 Hz, 1 H, PhCW2-O), 4.92 - 4.84 (m, 5H, PhCW2-O), 4.82 (d, J = 12.4 Hz, 1 H, PhCH2-O), 4.62 (d, J = 10.8 Hz, 1 H, PhCH2-O), 4.53 - 4.33 (m, 15H, 5xPhCH2-O, H-4’, H-1” and H-1 , H-4), 4.13 - 4.05 (m, 1 H, -OCH2CH2N-), 3.97 (d, J = 10.0 Hz, 1 H, H-3”), 3.92 (d, J = 2.9 Hz, 1 H, H-2”), 3.89 - 3.85 (m, 1 H, H-2), 3.83 - 3.79 (m, 2H, -OCH2CH2CO-), H-5), 3.78 - 3.71 (m, 3H, H-6, -OCH2CH2C(O)-), 3.68 - 3.55 (m, 6H, H-5’, H-5”, H-6’, H-6, H-4” ), 3.54 - 3.49 (m, 1 H, -OCH2CH2N- ), 3.45 (dd, J = 9.5, 2.9 Hz, 2H, -CH2-N-), 3.44 - 3.39 (m, 1 H, H-2’), 3.36 (dt, J = 8.5, 4.2 Hz, 1 H, -CH2-N-), 3.34 - 3.27 (m, 1 H, H-6”), 2.59 - 2.41 (m, 6H, -CO-CH2-), 2.35 (dd, J = 14.6, 6.9 Hz, 1 H, -CO-CH2-), 2.31 - 2.22 (m, 6H, -CO-CH2-), 2.05 (dd, J = 15.1 , 5.6 Hz, 1 H, -CO-CH2-), 1 .61 - 1 .44 (m, 7H, 3x -CH2- ), 1 .36 (d, J = 6.1 Hz, 1 H, H- 6”,), 1.33 - 1.16 (m, 100H, 50xCH2-), 0.89 - 0.86 (m, 18H, 6xCH3).13C NMR (151 MHz, CDCI3) 5 173.70, 173.56, 171.93, 171.56, 171.08, 170.03, 169.98, 138.92, 138.74, 138.69, 138.62, 138.39, 138.33, 137.55, 135.79, 135.77, 135.74, 135.72, 128.68, 128.64, 128.58, 128.50, 128.47, 128.44, 128.42, 128.32, 128.17, 128.10, 128.05, 127.86, 127.77, 127.75, 127.71 , 127.69, 127.68, 127.66, 127.63, 101.69, 101.00, 100.16, 82.22, 80.27, 76.17, 75.74, 75.53, 75.20, 74.88, 74.50, 74.43, 74.21 , 74.19, 74.16, 73.43, 72.38, 72.10, 71.54, 71.47, 71.06, 70.65, 69.74, 69.70, 69.62, 69.58, 68.83, 68.21 , 67.70, 65.89, 55.68, 54.43, 41.93, 41.17, 39.71 , 39.56,38.93, 36.95, 34.65, 34.43, 34.33, 34.28, 32.08, 32.07, 29.89, 29.88, 29.86, 29.84, 29.83, 29.81 , 29.79, 29.76, 29.75, 29.71 , 29.67, 29.64, 29.61 , 29.58, 29.52, 29.50, 29.43, 29.39, 25.50, 25.44, 25.33, 25.20, 22.83, 18.18, 14.26.31P NMR (243 MHz, CDCI3) 5 -2.05. HRMS (ESI) m / z: [M + 2NH4]2+Calculated for Ci62H246N3O28P, 1370.8870 found 1370.8850.

[0101] Compound 18b (C-Rh-conjugate): Compound 18b (25 mg, 73%) was synthesized from 14 (36 mg, 0.016 mmol) and 11b (19.2 mg, 0.037 mmol) by the same procedure and reaction conditions employed for the synthesis of 18a.1H NMR (600 MHz, CDCI3) 5 7.45 - 7.14 (m, 45H, Ar / - / ), 6.50 (brs, 1 H, -C(O)NH), 5.96 (d, J = 7.4 Hz, 1 H, -C(O)NH), 5.78 (d, J = 8.6 Hz, 1 H, , -C(O)NH), 5.65 - 5.48 (dd, J = 9.2 Hz, 1 H, H-3’), 5.17 - 5.02 (m, 3H, lipid-CH2-C / 7(O)-, H-3, H-T), 5.01 - 4.96 (m, 1 H, lipid-CH2-CH(O)-), 4.93 (d, 1 H, J = 10.9 Hz, PhCW2O- ), 4.90 - 4.82 (m, 5H, PhCW2O-, H-1”), 4.75 (q, J = 12.2 Hz, 2H, PhCW2O-), 4.64 (d, J = 10.9 Hz, 1 H, PhCW2O-), 4.62 (brs, 1 H, PhCW2O-), 4.54 - 4.35 (m, 10H, PhCW2O-, 2xlipid-CH2-CH(OBn ), -OCH2CH2CO-, H-4’, H-1 , H- 2”), 4.00 (d, J = 10.9 Hz, 1 H, -OCH2CH2N- ), 3.93 - 3.80 (m, 5H, , -OCH2CH2N-, H-2, H-3” and lipid-H), 3.78 - 3.71 (m, 3H, H-6, H-6’, lipid-H), 3.72 - 3.64 (m, 4H, H-5”, lipid-H), 3.64 - 3.58 (m, 3H, -CH2N-), 3.54 - 3.45 (m, 3H), 3.37 (m, 3H, H-2’,), 2.63 - 2.39 (m, 6H, -CH2-CO-), 2.38 - 2.15 (m, 8H, -CH2-CO-), 2.00 (dd, J = 15.0, 5.6 Hz, 1 H, -CH2-CO-), 1.55 - 1.43 (m, 7H, 3x -CW2-), 1.33 (d, J = 6.2 Hz, 3H, H-6” ), 1.32 - 1.14 (m, 100H, 50x- CH2-), 0.96 - 0.80 (m, 18H, 6xCH3).13C NMR (151 MHz, CDCI3) 5 173.70, 173.58, 172.00, 171.05, 170.05, 138.83, 138.82, 138.72, 138.65, 138.60, 138.29, 137.58, 135.76, 135.72, 128.69, 128.65, 128.60, 128.58, 128.53, 128.51 , 128.45, 128.43, 128.29, 128.19, 128.12, 128.11 , 128.06, 127.97, 127.86, 127.83, 127.78, 127.71 , 127.69, 127.68, 127.59, 101.09, 100.04, 98.02, 80.76, 80.60, 80.26, 79.70, 76.10, 75.83, 75.74, 75.50, 75.42, 75.20, 75.12, 74.75, 74.55, 74.43, 74.39, 74.20, 73.43, 72.93, 72.13, 71.43, 71.13, 71.10, 70.61 , 69.76,69.72, 69.63, 69.59, 68.88, 68.31 , 68.24, 68.17, 67.78, 63.92, 63.58, 61.42, 56.04, 54.51, 42.00, 41.86, 41.23,39.66, 39.38, 38.86, 37.35, 36.54, 34.64, 34.62, 34.45, 34.34, 34.29, 32.08, 32.07, 29.91, 29.88, 29.84, 29.83,29.81 , 29.79, 29.76, 29.75, 29.70, 29.63, 29.59, 29.53, 29.52, 29.50, 29.44, 29.39, 25.53, 25.42, 25.35, 25.32,25.19, 22.83, 18.21 , 14.26.31P NMR (243 MHz, CDCI3) 5 -2.06. HRMS (ESI) m / z: [M + 2NH4]2+Calculated for Ci62H246N3O28P, 1370.8870 found 1370.8843.

[0102] Compound 2a (C-Rh-conjugate): To a solution of 18a (24 mg, 8.87 pmol) in CH2CI2(3 mL) and MeOH (1 mL) was added Pd / C (10%, 5.2 mg) under a N2atmosphere. The reaction mixture was placed under a H2atmosphere with stirring at rt for 2 days. The mixture was filtered, and the filtrate was concentrated. The residue was purified by column chromatography with 3:1 CHCI3-CH3OH as eluents to give 2a as a white solid (11.3 mg, 67%).1H NMR (600 MHz, CDCI3:MeOD:D2O, 2: 1 :0.2) 5 5.17 (t, J = 9.9 Hz, 1 H, H-3’), 5.11 - 5.04 (m, 2H, 2xlipid-H), 4.95 (t, J = 9.6 Hz, 1 H, H-3), 4.55 (d, J = 8.5 Hz, 1 H, H-1’), 4.52 (s, 1 H, H-1”), 4.47 (d, J = 8.4 Hz, 1 H, H-1), 4.25 - 4.17 (m, 1 H, H-4’), 4.14 - 4.00 (m, 3H), 3.98 - 3.89 (m, 4H), 3.84 - 3.71 (m, 5H), 3.55 (dd, J = 12.3, 6.6 Hz, 2H), 3.51 - 3.44 (m, 3H), 3.41 (d, J = 9.9 Hz, 3H), 3.39 - 3.33 (m, 3H), 3.26 (ddd, J = 15.0, 7.9, 4.8 Hz, 3H, -CH2N-), 3.21 - 3.15 (m, 1 H, -CH2N-), 2.57 - 2.40 (m, 7H, -CH2-CO-), 2.40 - 2.21 (m, 7H, -CH2-CO-), 1.63 - 1.08 (m, 115H, 56xlipid-CH2-, H-6”), 0.85 (t, J = 6.9 Hz, 18H, 6x-CH3).13C NMR (151 MHz, CDCI3:MeOD:D2O, 2:1 :0.2) 5 174.90, 174.70, 173.39, 173.28, 173.02, 172.11 , 171.87, 102.21 , 101.91 , 100.53,77.35, 76.24, 76.00, 75.33, 74.22, 74.08, 73.05, 71.96, 71.81, 71.65, 71.49, 70.52, 69.13, 68.92, 68.84, 68.57,66.07, 65.36, 63.99, 61.77, 60.76, 54.25, 53.72, 42.78, 42.43, 41.67, 41.49, 41.38, 40.21, 39.74, 39.36, 38.73,37.87, 37.09, 37.01, 36.99, 35.13, 34.73, 34.54, 32.54, 30.95, 30.29, 29.98, 29.86, 29.79, 26.24, 26.17, 26.04,25.91, 25.75, 25.71, 23.26, 17.81, 14.43.31P NMR (243 MHz, CDCI3:MeOD:D2O, 1 : 1 :0.2) 5 -0.34. HRMS (ESI) m / z: [M - H]- Calculated for C99H183N3O28P 1893.2720, found 1893.2785.

[0103] Compound 2b (C-Rh-conjugate): Compound 2b (10.6 mg, 63%) was synthesized from 18b (24 mg, 8.87 pmol) by the same procedure and reaction conditions employed for the synthesis of 2a.1H NMR (600 MHz, CDCI3:MeOD:D2O, 2:1:0.2)55.20 (t, J= 10.1 Hz, 1H, H-3’), 5.13-5.05 (m, 3H, 2xlipid-H), 4.99 (dd, J= 10.4, 8.9 Hz, 1H, H-3), 4.72 (s, 1H, H-1”), 4.59 (d, J = 8.4 Hz, 1H, H-1’), 4.50 (d, J= 10.8 Hz, 1H, H-1), 4.22 (q, J= 9.3 Hz, 1H, H-4’), 4.09 (d, J = 10.3 Hz, 1H), 4.04 (dq, J = 8.8, 4.5 Hz, 1H), 3.97 (dq, J= 10.7, 3.8 Hz, 1H), 3.94- 3.85 (m, 3H), 3.85 - 3.83 (m, 1 H), 3.82 - 3.74 (m, 4H), 3.71 - 3.64 (m, 2H), 3.62 - 3.56 (m, 2H), 3.55 - 3.47 (m, 2H), 3.47- 3.34 (m, 4H, -CH2N-), 3.25-3.20 (m, 1H, -CH2N-), 2.58-2.21 (m, 14H, -CH2-CO-), 1.71 -1.05 (m, 115H, 56xlipid-CH2-, H-6”), 0.86 (t, J = 6.9 Hz, 18H, 6x-CH3).13CNMR(151 MHz, CDCI3:MeOD:D2O, 1:1:0.2)5 174.93, 174.75, 174.57, 174.44, 173.31, 173.05, 172.15, 102.21, 101.70, 100.63, 77.70, 76.13, 75.92, 75.33, 74.21, 74.00, 73.22, 71.81, 71.62, 71.22, 69.24, 69.13, 68.99, 68.94, 68.78, 64.07, 60.75, 54.50, 54.34, 42.76,42.38, 41.72, 41.61, 40.14, 39.96, 37.85, 36.98, 35.19, 35.14, 34.54, 32.55, 30.33, 30.30, 30.17, 30.06, 29.99,29.87, 29.80, 26.27, 26.21, 26.05, 25.94, 25.78, 25.72, 23.27, 17.83, 14.47.31P NMR (243 MHz, CDCI3: MeOD:D2O, 2: 1:0.2) 5 -1.47. HRMS (ESI) m / z: [M - Hp Calculated for C99H183N3O28P 1893.2720, found 1893.2684.

[0104] Compound 19a (C-Rh-conjugate): Compound 19a (17.5 mg, 71%) was synthesized from 17 (21 mg, 0.0098 mmol) and 11a (11.8 mg, 0.023 mmol) by the same procedure and reaction conditions employed for the synthesis of 18a.1H NMR (600 MHz, CDCI3) 57.43 (d, J = 6.9 Hz, 2H, Ar / - / ), 7.34 - 7.12 (m, 38H, Ar / - / ), 6.64 (brs, 1H, -C(O)NH-), 5.99 (brs, 1H, -C(O)NH-), 5.54 (d, J= 7.7 Hz, 1H, -C(O)NH-), 5.40 (t, J = 9.3 Hz 1H, H-3’), 5.19 (dd, J= 9.2, 9.4 Hz, 1H, H-3), 5.08 (dt, J= 12.5, 6.6 Hz, 1H, lipid-H), 5.01 -4.85 (m, 7H, lipid-H, PhCH2O-, H-1’), 4.81 (d, J = 12.5 Hz, 1H, PhCH2O-), 4.61 (d, J = 10.9 Hz, 1H, PhCH2O-), 4.52 (d, J= 11.1 Hz, 1H, PhC / 72O-), 4.50 (d, J= 11.6 Hz, 1H, PhCH2O-), 4.48-4.34 (m, 7H, PhCH2O-, H-1”, H-1), 4.24 (q, J= 9.2 Hz, 1 H, H-4), 4.07 (dt, J = 9.7, 5.9 Hz, 1 H, -OCH2CH2CO-), 3.96 - 3.88 (m, 3H, H-2”, H-6,), 3.87 - 3.76 (m, 4H, - OCH2CH2CO-, H-6’, H-4’, H-4”), 3.75 - 3.68 (m, 2H, H-6), 3.60 - 3.54 (m, 2H, H-5’, H-5”), 3.52 - 3.44 (m, 1 H, H- 5, H-2), 3.44-3.41 (m, 4H, -OCH3), 3.38-3.31 (m, 2H, H-6’), 3.29 (ddd, J= 12.3, 7.7, 4.6 Hz, 1H, H-2’), 2.56 (dd, J = 16.0, 7.2 Hz, 1H, -CH2-CO-), 2.54-2.21 (m, 11 H, -CH2-CO-), 2.18 (dd, J= 15.2, 7.0 Hz, 1H, -CH2-CO-), 2.00 (dd, J = 15.2, 5.4 Hz, 1H, -CH2-CO-), 1.67-1.38 (m, 13H, lipid-CH2), 1.36 (d, J = 6.1 Hz, 3H, H-6”), 1.35- 1.06 (m, 98H, lipid-C / 72-), 0.91 -0.85 (m, 18H, 6x-CH3).13CNMR(151 MHz, CDCI3) 5173.84, 173.60, 171.78, 171.10, 170.98, 169.93, 169.80, 138.95, 138.70, 138.68, 138.63, 138.42, 137.69, 128.81, 128.78, 128.75, 128.73, 128.62, 128.57, 128.47, 128.44, 128.42, 128.29, 128.27, 128.23, 128.19, 128.09, 127.88, 127.83, 127.82, 127.77, 127.66, 127.63, 127.57, 101.75, 100.46, 82.22, 80.27, 76.33, 75.74, 75.57, 75.52, 75.28, 75.25, 74.86, 74.49, 74.12, 73.00, 72.05, 71.95, 71.94, 71.43, 71.41, 71.19, 71.12, 70.63, 70.04, 70.01, 69.97, 69.00, 65.78, 56.49, 56.06, 54.07, 41.93, 41.36, 39.85, 39.72, 38.76, 37.11, 37.09, 34.63, 34.39, 34.33, 34.29, 34.06,32.07, 29.87, 29.83, 29.81, 29.79, 29.78, 29.76, 29.74, 29.70, 29.67, 29.63, 29.60, 29.56, 29.52, 29.50, 29.42, 29.39, 25.48, 25.41, 25.33, 25.30, 25.16, 22.84, 18.18, 14.26.31P NMR (243 MHz, CDCI3) 5 -1.34. HRMS (ESI) m / z: [M +2NH4]2+Calculated for C154H238N5O28P 1310.8582, found 1310.8596.

[0105] Compound 19b (C-Rh-conjugate): Compound 19b (17.9 mg, 72%) was synthesized from 17 (21 mg, 0.0098 mmol) and 11a (11.8 mg, 0.023 mmol) by the same procedure and reaction conditions employed for the synthesis of 18a.1H NMR (600 MHz, CDCI3) 57.53 - 7.07 (m, 40H, ArH), 6.55 (s, 1 H, -C(O)-NH-), 5.93 (d, J = 5.8 Hz, 1H, -C(O)-NH-), 5.54 (d, J= 7.7 Hz, 1H, -C(O)-NH-), 5.40 (t, J= 9.2 Hz, 1H, H-3’), 5.19 (t, J = 9.2 Hz, 1H, H-3), 5.10 - 5.03 (m, 1H, lipid-H), 5.01 -4.95 (m, 2H, lipid-H, H-T), 4.94-4.84 (m, 4H, PhCW2O-) 4.81 (d, J = 1.31 Hz, 1H, H-1”), 4.71 (d, J = 12.3 Hz, 2H, PhCW2O- ), 4.63 (d, J = 10.9 Hz, 1H, PhCW2O-), 4.59 (brs, 2H, PhC / 72O-), 4.53 (d, J = 11.1 Hz, 1 H, PhCW2O- ), 4.50 (d, J = 11.6 Hz, 1 H, PhCW2O-), 4.45 (d, J = 11.7 Hz, 1 H, PhC / 72O-), 4.43 (d, J= 11.1 Hz, 1H, PhCW2O-), 4.42 (d, J = 8.2 Hz, 1H, H-1), 4.41 (d, J= 11.1 Hz, 1H, PhCW2O- ), 4.37 (d, J= 11.3 Hz, 1H, PhCW2O-), 4.24 (q, J = 9.3 Hz, 1H, H-4’), 4.03-3.77 (m, 7H, H-6’, H-2, - OCH2CH2CO-, H-4”), 3.77 - 3.63 (m, 4H, -OCH2CH2CO-, H-5”, H-6), 3.63 - 3.54 (m, 2H), 3.54 - 3.45 (m, 2H, H- 5’), 3.42 (s, 3H, -OCH3), 3.39-3.32 (m, 1H, H-2’), 3.28 -3.20 (m, 1H, H-6’), 2.56 (dd, J= 15.9, 7.1 Hz, 1H, -CW2- CO-), 2.46 (dd, J= 15.9, 5.1 Hz, 1H, -CH2-CO-), 2.40-2.20 (m, 10H, -CH2-CO-), 2.17 (dd, J= 15.3, 7.0 Hz, 1H, -CH2-CO-), 1.99 (dd, J= 15.3, 5.3 Hz, 1H, -CH2-CO-), 1.61 -1.11 (m, 112H, 54xlipid-CH2-, H-6”), 0.96-0.80 (m, 18H, 6X-CH3).13C NMR (151 MHz, CDCI3) 5173.84, 173.57, 171.78, 170.96, 170.68, 169.95, 169.81, 140.22, 139.95, 139.93, 138.83, 138.66, 138.62, 137.68, 135.50, 128.81, 128.74, 128.63, 128.57, 128.46, 128.31, 128.23, 128.15, 128.10, 127.96, 127.83, 127.70, 101.76, 100.35, 98.18, 80.64, 80.32, 76.38, 76.00, 75.74, 75.57, 75.46, 75.37, 75.25, 75.14, 74.91, 74.49, 72.88, 72.16, 71.42, 71.17, 70.04, 68.87, 68.28, 64.70,63.99, 63.55, 56.51, 56.07, 54.14, 42.42, 41.91, 41.35, 39.78, 39.73, 38.74, 36.76, 34.63, 34.40, 34.33, 34.05,32.08, 29.81, 29.63, 29.52, 29.42, 25.48, 25.40, 25.33, 25.15, 22.84, 18.21, 14.26.31P NMR (243 MHz, CDCI3) 5-1.23. HRMS (ESI) m / z: [M + 2NH4]2+Calculated for C154H238N5O28P 1310.8582, found 1310.8584.

[0106] Compound 3a (C-Rh-conjugate): Compound 3a (6.1 mg, 60%) was synthesized from 19a (14 mg, 5.41 pmol) by the same procedure and reaction conditions employed for the synthesis of 2a.1H NMR (600 MHz, CDCl3:MeOD:D2O, 2:1 :0.2) 55.18-5.05 (m, 3H, 2xlipid-H, H-3’), 4.97 (dd, J = 12.5, 7.0 Hz, 1H, H-3), 4.39 (d, J = 8.3 Hz, 1H, H-1), 4.15-4.05 (m, 1H, H-4’), 4.04-3.98 (m, 3H), 3.98-3.92 (m, 2H), 3.91 -3.85 (m, 2H), 3.82 - 3.76 (m, 4H), 3.74 - 3.68 (m, 2H), 3.57 - 3.51 (m, 1 H), 3.51 - 3.44 (m, 2H), 3.44 - 3.41 (m, 1 H), 3.40 (s, 3H, - OCH3), 3.26-3.21 (m, 1H), 3.16 -3.11 (m, 1H), 2.51 -2.22 (m, 14H, -CH2-CO-), 1.63-1.11 (m, 112H, lipid- CW2-), 0.85 (t, J= 7.0 Hz, 18H, 6x-CH3).31P NMR (243 MHz, CDCl3:MeOD:D2O, 2: 1 :0.2) 5 -0.56. HRMS (ESI) m / z: [M +NH4]+Calculated for CgsH eN^zP 1883.3070, found 1883.3163.

[0107] Compound 3b (C-Rh-conjugate): Compound 3b (4.7 mg, 51%) was synthesized from 19b (11 mg, 4.25 pmol) by the same procedure and reaction conditions employed for the synthesis of 2a.1H NMR (600 MHz, CDCl3:MeOD:D2O, 2: 1:0.2) 55.15 (t, J=9.9 Hz, 1H, H-3’), 5.14-5.04 (m, 2H, 2xlipid-H), 4.95 (dd, J= 10.6, 8.9 Hz, 1H, H-2), 4.74 (s, 1H, H-1”), 4.58 (d, J= 8.4 Hz, 1H, H-1’), 4.39 (d, J = 8.4 Hz, 1H, H-1), 4.12-4.04 (m, 1 H,-H4'), 4.05 - 3.98 (m, 2H), 3.97 - 3.90 (m, 2H), 3.85 - 3.76 (m, 3H), 3.74 - 3.62 (m, 4H), 3.60 - 3.48 (m, 3H), 3.47 - 3.43 (m, 1 H), 3.40 (s, 3H, -OCH3), 3.14 (q, J = 7.4 Hz, 1 H), 2.51 - 2.22 (m, 14H, -CH2-CO-), 1.68 - 1.11 (m, 112H, lipid-C / 72-), 0.85 (t, J = 7.0 Hz, 18H, 6x-CH3).31P NMR (243 MHz, CDCI3:MeOD:D2O, 2: 1 :0.2) 5 -0.57. HRMS (ESI) m / z: [M + NH4]+Calculated for C98H186N4O27P 1883.3070, found 1883.3154.

[0108] Preparation of the mixture of MPLA and Rha-lipid. An efficient and general synthesis of MPLA derivative 14 is illustrated in Figure 5, and its global deprotected as described above gave free MPLA-NH24b (Figure 12) without the Rha moiety. Compound 4b without the Rha moiety was mixed with a simple lipid conjugate of Rha-Lipid (compound B, Figure 12) in 1 :1 ratio, and this mixture 4b / B was utilized as a negative control to study whether covalent linkage between MPLA and Rha would result in enhanced adjuvant activity for the conjugate adjuvants. Rha-Lipid was employed because it was easily incorporated together with MPLA-NH2in liposomal formulations.

[0109] Immunological studies of the designed conjugate adjuvants. The immunological properties of MPLA-Rha conjugates 2a, 2b, 3a, and 3b as vaccine adjuvants were evaluated in vivo using female C57BL / 6 J mice. In these studies, phosphate-buffered saline (PBS), in addition to the MPLA / Rha mixture 4b / B, was used as another negative control, while the positive control was Alum, which is currently the most widely accepted vaccine adjuvant. Alum was utilized as emulsions following the manufacturer's instruction, whereas the experimental adjuvants and the negative control 4b / B were employed in the form of liposomes. The liposomes were prepared with 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. Briefly, the mixture of an experimental adjuvant or 4b / B, DSPC, and cholesterol in a 10:65:50 molar ratio was dissolved in CH2CI2and MeOH (1 :1, v / v, 2 mL) in a round-bottomed flask. The solvents were removed in vacuum to form a thin lipid film on the flask wall, which was hydrated by the addition of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (20 mM, pH 7.5). The mixture was shaken at 40 °C for 1 h and then sonicated for 1 min. to yield the desired liposomes. The liposomes generated by this method without extrusion through a size-limiting film were heterogeneous, but they were sufficient for being used as adjuvants.

[0110] The sTn-KLH conjugate, which was prepared according to Q. Wang, S.A. Ekanayaka, J. Wu, J. Zhang, and Z. Guo (Synthetic and immunological studies of 5'-N-phenylacetyl sTn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins. Bioconjugate Chem. 2008, 19, 2060-2068) herein incorporated by reference in the entirety, was employed as the model vaccine to test the experimental adjuvants. Glycoconjugate vaccine sTn-KLH, rather than a protein vaccine, was used in this evaluation because the antibody responses to both the carrier protein (KLH) as a protein antigen and the carbohydrate antigen sTn were able to be examined under the influence of the tested adjuvants. The vaccine preparations were obtained by dissolving sTn-KLH in a PBS buffer followed by adding the solution into the Alum emulsion or liposomes 2a, 2b, 3a, 3b and 4b / B prior to immunization.

[0111] The animal model used was female C57BL / 6J mice. This strain of mice has been used for the investigation of vaccines due to the similarity of these mice's immune system to that of a human. Each groupcontained six mice for statistical reasons. This and subsequent animal use protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Florida. The animal immunization protocol and schedule were as follows. Each of the above vaccine preparations (0.1 mL) containing about 8 pig of the sTn antigen (5-8% sTn antigen loading) was subcutaneously injected into to each mouse. The vaccine was injected a total of 4 times, with one injection on days 1, 15, 22, and 29, respectively (Figure 13). Blood samples were collected from each mouse on day 0 (pre-inoculation, the negative control) and on day 27, 31, and 38 postimmunizations (Figure 13). The blood samples were utilized to prepare antisera and detect the antibody responses in each animal.

[0112] Blood samples were processed to obtain antisera following the conventional protocol. Further analysis of the antibody titers in each antiserum was conducted using enzyme-linked immunosorbent assay (ELISA). To detect KLH- and sTn-specific antibodies, KLH protein and sTn-HSA conjugate were employed as the capture reagents. The secondary antibodies used to detect antibodies were goat anti-mouse antibodies coupled with alkaline phosphatase (AP). The total antibody and the IgG and IgM antibodies were analyzed, respectively. Antibody titers were derived from the curves obtained with optical density (OD) values and serum dilution numbers and were defined as the dilution number corresponding to an OD value of 0.1.

[0113] The results concerning anti-sTn antibodies in the day 38 antisera derived from mice immunized with sTn-KLH in combination with various adjuvants are depicted in Figure 14 and Table 1. Overall, these results clearly indicated that the adjuvants, including the analogous MPLA-Rha conjugates, possess very different immunological properties. For example, 3b helped provoke significantly higher levels of total antibody titers against sTn antigen than all other adjuvants (1,938 vs <100 for all others, Figure 14A and Table 1). However, this result may not reflect the whole landscape, as anti-kappa antibodies detect all antibodies that have different affinities. Thus, further examination of different isotypes of antibodies against sTn was performed.

[0114] Table i . The titers of anti-sTn total, IgG, and IgM antibodies (group mean value ± standard deviation) of the day 38 antisera derived from mice immunized with sTn-KLH in combination with various adjuvants.PBS Alum 4b / BAdjuvants 2a 2b 3a 3b(control) (control) (control)Total 1209 8 53 26 1,938 antibody ± 11 ± 7 ± 3 ± 19 ± 23 ± 336IgG03,587 1,377 6,689 11,014 3,010 24,075 antibody ± 1,281 ± 1,102 ± 2,814 ± 2,991 ± 1,402 ± 3,411IgM 56 74 616 9,880 4,946 2,568 3,315 antibody ± 50 ± 71 ± 370 ± 728 ± 1,316 ± 969 ± 513

[0115] The results of IgG antibody analysis (Figure 14B and Table 1) showed that again 3b provided the most significant augmentation of anti-sTn IgG antibodies. However, mice in other MPLA-Rha conjugates 2a, 2b, and 3a also produced significant levels of IgG antibodies, and the antibody titers in the 2a and 2b groups were slightlyhigher than that of the positive control (Alum) group, and the 3a and Alum groups were comparable. Interestingly, 2a helped raise the highest level of IgM antibody (Figure 14C and Table 1), although the IgM antibody levels of all the MPLA-Rha conjugate groups were higher than that of the Alum group. Additionally, both IgG and IgM antibody levels of the MPLA-Rha conjugate groups were significantly higher than that of the MPLA+Rha mixture group and the PBS group.

[0116] Similar as above, the results about anti-KLH antibodies in the day 38 antisera from mice immunized with sTn-KLH in combination with various adjuvants (as depicted in Figure 15A, Figure 15B, Figure 15C, and Table 2) also indicated distinct immunological properties of various adjuvants. However, the trends or patterns are different. In the current case, 2a and 2b, rather than 3b, were the most efficient to boost the production of total anti-KLH antibodies in mice (Figure 15A and Table 2). Moreover, it was shown that all MPLA-Rha conjugates helped induce significant levels of both IgG and IgM antibodies. Most interestingly, 2b provoked higher levels of IgG antibodies than 2a (Figure 15B and Table 2), whilst 2a provoked higher levels of IgM antibodies than 2b (Figure 15C and Table 2), suggesting the great influence of the structures of conjugates on their immunological properties.

[0117] Table 2. The titers of anti-KLH total, IgG, and IgM antibodies (group mean value ± standard deviation) of the day 38 antisera derived from mice immunized with sTn-KLH in combination with various adjuvants.PBS Alum 4b / BAdjuvants2a 2b 3a 3b(control) (control) (control)Total 47 1390 2,409 2,287 190 167 antibody ± 36 ± 1±266 ± 362 ± 520 ± 181 ± 131IgG 496 725 1,140 6,913 14,843 5,592 2,495 antibody ± 313 ± 441 ± 704 ± 990 ± 2,639 ± 666 ±1,195IgM 1,240 460 2,144 17,684 3,283 796 3,645 antibody ± 1,221 ± 459 ± 930 ± 1,661 ± 702 ± 283 ± 1,178

[0118] The dynamics of immune responses induced by the vaccine in the presence of different adjuvants, using the antisera collected at different time points, such as at day 27, 31, and 38, after 3 and 4 times of immunization and more than a week after the final immunization. For sTn-specific immune responses, we are especially focused on conjugate 3b since it boosted the production of the highest IgG antibodies and thus a promising adjuvant for carbohydrate-based conjugate vaccines. The results of anti-sTn IgG antibodies in Day 0, 27, 31, and 38 antisera for different adjuvants including 3b and negative and positive controls (PBS, 4b / B, and Alum) are depicted in Figure 16 and Table 3. In all groups except for the PBS group, anti-sTn antibody titers were increased with the number of immunizations, suggesting further enhancement of the immune response to sTn after each booster immunization. This is expected and desirable. Even more than a week after the final booster immunization, a high level of antibodies was still observed. More importantly, not only the antibody levels in all antisera of 3b were higher than that in the antisera of positive control (Alum) and MPLA / Rha mixture 4b / Bgroups, but also a more significant enhancement of the immune response was observed with the 3b group, verifying that this adjuvant is superior to other adjuvants.

[0119] Table 3. Observed titers of anti-sTn IgG antibodies in day 27, 31, and 38 antisera from mice immunized with adjuvants PBS, 4b / B, Alum, and 3b.Adjuvants PBS 4b / B Alum 3bDay 27 sera 0 305 ± 247 1136 ± 431 8,492 ± 709Day 31 sera 0 968 ± 772 2582 ± 930 13,427 ± 1,614Day 38 sera 0 1,377 ± 1,102 3,587 ± 1,281 24,075 ± 3,411

[0120] Similar results, as depicted in Figure 17 and Table 4, were also observed with day 0, 27, 31, and 38 sera using KLH protein to coat ELISA plates for the detection of KLH-specific IgG antibodies as described above. However, in this study, we were focused on conjugate 2b, as it boosted the production of the highest levels of IgG antibodies against KLH (Figure 15B) and thus seemed to be an excellent adjuvant for protein vaccines. Again, MPLA-Rha conjugate 2b was superior to Alum and MPLA / Rha mixture 4b / B.

[0121] Table 4. Observed titers of anti-KLH IgG antibodies in day 27, 31, and 38 antisera from mice immunized with adjuvants PBS, 4b / B, Alum, and 2b.Adjuvants PBS 4b / B Alum 2bDay 27 sera 44 ± 42 534 ± 359 360 ± 233 6,498 ±1,134Day 31 sera 298 ± 192 710 ± 446 454 ± 282 9,135 ± 1,736Day 38 sera 496 ± 314 1,140 ± 704 725 ± 441 1,4843 ± 2,639

[0122] Discussion about immunological results. Previous studies indicate that the linkage site to MPLA and the glycosidic linkage form, e.g., o-Gal form vs its anomer, can have a big impact on the immunological properties of the conjugates. Thus, the disclosure provides MPLA-Rha conjugates 2a, 2b, 3a, and 3b that have different linkage sites and forms from known conjugates. The results of the immunological evaluations demonstrated that, overall, these conjugates are much stronger adjuvants than the positive control Alum and assist in eliciting stronger IgG and IgM antibody responses to both carbohydrate and protein antigens. The conjugates of the disclosure are also much stronger adjuvants than the MPLA and Rha mixture, suggesting the synergistic effect of the two epitopes in covalently conjugated forms. The production of IgG antibodies in these experiments further indicates antibody isotype switch and maturation and the involvement of cellular immunities, which is desired for both antibacterial and antitumor vaccines.

[0123] However, the MPLA-Rha conjugates have exhibited significantly different efficacies in boosting immune responses against the vaccine. For the carbohydrate antigen sTn, results in Figure 14 and Table 1 show that the strongest IgG antibody response against sTn was elicited in the presence of conjugate 3b, which was followedby conjugate 2b; both conjugates have Rha o-linked to MPLA. Therefore, it seems that o-linked Rha may be more effective than [3-linked Rha to boost cellular immunities against sTn. On the other hand, higher IgM antibody responses were observed with conjugates 2a and 2b, especially the former, suggesting that they may be more effective to boost innate immunities.

[0124] For protein antigen KLH, the results in Figure 15 and Table 2 show that conjugate 2b boosted the strongest IgG antibody response, which was followed by conjugates 2a, and both conjugates have Rha a- and |3- linked, respectively, to the same 6'-position of MPLA. Thus, the 6'-linked MPLA-Rha conjugates seem to be more effective than 1 -linked conjugates for boost cellular immunities, which result is consistent with our previous observations for MPLA-based conjugate vaccines. Again, the most effective conjugate adjuvant is 2b with o- linked Rha. Furthermore, like the observations with sTn antigen, the highest IgM antibody response against KLH was obtained with conjugate 2a. These results further suggest that 2a is more effective in boosting innate immunities.

[0125] Analysis of anti-sTn and anti-KLH IgG antibody titers in the day 0, 27, 31, and 38 sera (Figures 16 and 17; Tables 3 and 4) from mice immunized with sTn-KLH using MPLA-Rha conjugates 3b and 2b as adjuvants, respectively, disclose a steady increase of IgG antibody responses upon repeated immunization, a pattern consistent with the generation of memorable cellular immunity that is desired and important for both preventative and therapeutic vaccines.

[0126] Overall, these results have proved the efficacy of the new adjuvants for the model vaccine and that they are better adjuvants than the most used adjuvant Alum. The result that adjuvants of different structures and linkage forms have different properties suggest that they can be most applicable to different vaccines, thereby providing multiple choices for medical applications.

[0127] Materials and Methods of Immunological Studies: Alum, DSPC, and cholesterol were purchased from Sigma-Aldrich. AP-linked goat anti-mouse kappa, IgM, and IgG antibodies (the secondary antibodies) were purchased from Southern Biotechnology. MPLA-Rha conjugates 2a, 2b, 3a, and 3b, as well as free MPLA-NH2 and Rha-lipid used to prepare their mixture 4b / B, model vaccine sTn-KLH and coating antigen conjugate sTn- HSA were synthesized. Female C57BL / 6 mice of 6-8 weeks of age used for immunological studies were purchased from The Jackson Laboratory. After the animals arrived at the animal facility at the University of Florida, they were examined and waited in the cage for 1-2 days to set in the environment before the start of experiments.

[0128] General Procedure for the Preparation of the Liposomes of Conjugate Adjuvants: The mixture of an MPLA-Rha conjugate (0.477 pmol) or the mixture of MPLA-NH2 and Rha-Lipid (0.477 pmol each), DSPC (2.45 mg, 3.1 pmol), and cholesterol (0.922 mg, 2.385 pmol) (10:65:50 molar ratio) was dissolved in a mixture of CH2CI2 and MeOH (1 :1, v / v, 2 mL) in a 10 mL round-bottomed flask. Then, the solvents were removed in vacuum with a rotary evaporator to generate a thin lipid film on the flask wall, which was followed by adding 2.0 mL of HEPES buffer (20 mM, pH 7.5) containing NaCI (150 mM) for lipid hydration and shaking the mixture under anargon atmosphere at 40 °C for 1 h. The milky suspension was finally sonicated for 1 min to obtain the desired liposomes. Then, the liposomes were sealed and preserved at 4 °C for immunizations.

[0129] Immunization of Mouse: The vaccine formulations were prepared by integrating 8 pig of the carbohydrate antigen sTn (calculated based on the carbohydrate loading of conjugate vaccine) dissolved in 50 piL PBS and 50 piL of an adjuvant (each prepared liposomes of conjugate adjuvants 2a, 2b, 3a, and 3b or 4b / B, Alum, or PBS) per dose. Prior to injection, each vaccine was mixed vigorously by vortex mixer. For immunological studies, each experimental group is composed of five female C57BL / 6 mice. The mice were immunized on day 1 by subcutaneous (s.c.) injection of 0.1 mL of a vaccine formulation using a 26G 1 / 2 (0.45 mm 13 mm) needle. Following the initial immunization, mice were boosted 3 times on day 15, 22, and 29 through s.c. injection of the same vaccine / adjuvant formulation by the same immunization protocol. Blood samples of each mouse were collected through the saphenous vein prior to initial immunization on day 1 and after booster immunizations on day 27, 31, and 38, respectively. The blood samples were clotted to prepare antisera by standard protocols, which were stored at -80 °C before use. The animal use protocol for this study (#201609560) was approved by the IACUC of the University of Florida.

[0130] ELISA Protocols: A solution of the sTn-HSA conjugate or KLH (2 pg / mL, 100 pL) dissolved in the coating buffer (0.1 M bicarbonate, pH 9.6) was added to each well of ELISA plates, and the plates were incubated at 37 °C for 1 h. After the plates were washed with PBS buffer containing 0.05% Tween 20 (PBST) 3 times, the blocking solution (1% BSA in PBST, 100 pL) was added to each well to block the plates. The plates were washed with PBST 3 times, and then a pooled Day 0 serum or an individual mouse antiserum with serial half-log dilutions from 1 :300 to 1 :218700 in PBS was added to the coated ELISA plates (100 pL / well), which was followed by incubation at 37 °C for 2 h. The plates were washed with PBS and then incubated with a 1:1000 diluted solution of AP-linked goat anti-mouse kappa, IgM, or IgG antibody (100 pL / well), respectively, at rt for 1 h. Finally, the plates were washed with PBS and developed with a solution of p-nitrophenylphosphate (PNPP) in PBS (1.67 mg / mL, 100 pL) at rt for 30 min, followed by colorimetric readout using a BioTek plate reader at 405 nm wavelength. For antibody titer analysis, obtained optical density (OD) values were plotted against antiserum dilution values, and a best-fit line was obtained. The equation of this line was applied to calculate the dilution value at which an OD of 0.1 was achieved, and the antibody titer was calculated at the inverse of the dilution value.

[0131] Statistical analysis: All data were analyzed by one-way analysis of variance (for data of more than 2 groups) or independent t-test (for data between 2 groups), using GraphPad software. P < 0.05 was considered statistically significant.These pharmacological, immunological, and statistical methods are well known in the art, for example, as described in: Q. Wang, S.A. Ekanayaka, J. Wu, J. Zhang, and Z. Guo, Synthetic and immunological studies of 5'- N-phenylacetyl sTn to develop carbohydrate-based cancer vaccines and to explore the impacts of linkage between carbohydrate antigens and carrier proteins. Bioconjugate Chem. 2008, 19, 2060-2068. L. Wang, S.Feng, S. Wang, H. Li, Z. Guo, and G. Gu, Synthesis and Immunological Comparison of Differently Linked Lipoarabinomannan Oligosaccharide-Monophosphoryl Lipid A Conjugates as Antituberculosis Vaccines. J. Org. Chem. 2017, 82, 12085-12096. J. Palma, B. Tokarz-Deptula, J. Deptula, and W. Deptula, Natural antibodies - facts known and unknown. Cent. Eur .J. Immunol. 2018, 43, 466-475. U. Galili, Conversion of tumors into autologous vaccines by intratumoral injection of o-Gal glycolipids that induce anti-Gal / o-Gal epitope interaction. Clin. Dev. Immunol. 2011, 2011, 134020. The above-listed references are incorporated herein by reference in their entirety.

[0132] It will be understood that the specific dose level and frequency of dosage for any particular subject can be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the species, age, body weight, general health, sex and diet of the subject, the mode and time of administration, rate of excretion, drug combination, and severity of the particular condition.It should be understood that while this invention has been described herein in terms of specific embodiments set forth in detail, such embodiments are presented by way of illustration of the general principles of the invention, and the invention is not necessarily limited thereto. Certain modifications and variations in any given material, process step or chemical Formula will be readily apparent to those skilled in the art without departing from the true spirit and scope of the present invention, and all such modifications and variations should be considered within the scope of the claims that follow.

Claims

WHAT IS CLAIMED IS:

1. A MPLA / LA conjugate adjuvant having a structure according to Formula (I) or Formula (II):wherein:R is selected from -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24;Q is absent or selected from -(O-CH2-CH2)n- — (CH2)m— , -(O-CH2-CH2-CH2)t-, and a combination thereof, wherein n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15; and2. The adjuvant of claim 1 , wherein X is -P(=O)(OH)2.

3. The adjuvant of claim 1 , wherein X is -S(=O)2(OH).

4. The adjuvant of any one of claims 1-3, wherein Y is an integer of 6-24.O5. The adjuvant of any one of the preceding claims, wherein L is6. The adjuvant of any one of claims 1 to 4, wherein L is O7. The adjuvant of any one of the preceding claims, wherein Q is -(O-CH2-CH2)n-.

8. The adjuvant of claim 7, wherein n is an integer of 1-15, 1-10, 2-8, 2-6, 2, 3, 4,5, or 6.

9. The adjuvant of any one of claims 1 to 6, wherein Q is -(O-CH2-CH2-CH2)t-.

10. The adjuvant of claim 9, wherein t is an integer of 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6.11 . The adjuvant of any one of claims 1 to 6, wherein Q is - (CH2)m- ■12. The adjuvant of claim 11 , wherein m is an integer of 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18,-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3, 4, 5, 6, 10, or 12.

13. The adjuvant of any one of the preceding claims, wherein Z is14. The adjuvant of any one of claims 1 to 12, wherein15. The adjuvant of any one of claims 1 to 12, wherein Z isThe adjuvant of any one of claims 1 to 12, wherein17. The adjuvant of any one of claims 1 to 12, wherein Z is OH18. The adjuvant of any one of claims 1 to 12, whereinThe adjuvant of any one of claims 1 to 12, wherein20. The adjuvant of any one of the preceding claims, wherein the MPLA / LA conjugate adjuvant has a structure according to Formula (I).21 . The adjuvant of any one of claims 1 to 19, wherein MPLA / LA conjugate adjuvant has a structure according to Formula (II).

22. The adjuvant of claim 1 , selected from the group consisting of:

23. A compound having a structure according to Formula (III) or one of Formula (VI l)-(XXI):wherein:Q is selected from — (CH2-CH2)n— , — (CH2)m— -(CH2-CH2-CH2)t-, and a combination thereof, wherein n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

24. The compound of claim 23, wherein Q is -(CH2-CH2)n-25. The compound of claim 24, wherein n is an integer of 1-15, 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6.

26. The compound of claim 23, wherein Q is -(CH2-CH2-CH2)t-.

27. The compound of claim 26, wherein t is an integer of 1-10, 2-8, 2-6, 2, 3, 4, 5, or 6.

28. The compound of claim 23, wherein Q is -(CH2)m-29. The compound of claim 28, wherein m is an integer of 1-55, 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 2-18, 2-16, 3-14, 3-12, 3-10, 3-8, 3-6, 3, 4, 5, 6, 10, or 12.

30. A method of enhancing an immune response in a subject, comprising: administering to the subject a MPLA / LA conjugate adjuvant according to claims 1-21 .31 . The method of claim 30, further comprising administering a vaccine.

32. The method of claim 31 , wherein the vaccine and the MPLA / LA conjugate adjuvant are administered concurrently.

33. The method of claim 31 , wherein the vaccine and the MPLA / LA conjugate adjuvant are administered stepwise.

34. Use of a MPLA / LA conjugate adjuvant according to any one of claims 1-22 for enhancing an immune response.

35. Use of a MPLA / LA conjugate adjuvant according to any one of claims 1-22 in the manufacture of a medicament for enhancing an immune response.

36. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (III) or (VI):whereinX is selected from-P(=O)(OH)2 and — S(=O)2(OH); andY is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m— , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

37. A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (III) or (VI):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m— , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

38. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II) comprising: admixing a compound of Formula (IV) with a compound of Formula (VI I), (VIII) or (IX):X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m— , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

39. A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (VII), (VIII) or (IX):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from-P(=O)(OH)2 and -S(=O)2(OH); andY is an integer of 6-24; andQ is selected from -(CH2-CH2)n-, — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

40. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (X), (XI) or (XII):whereinX is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from -(CH2-CH2)n-, — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.41 . A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (X), (XI) or (XII):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from-P(=O)(OH)2 and -S(=O)2(OH); andY is an integer of 6-24; andQ is selected from -(CH2-CH2)n-, — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1 -20, m is an integer of 1-60, and t is an integer of 1-15.

42. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (XIII), (XIV) or (XIX):whereinX is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m— , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

43. A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (XIII), (XIV) or (XIX):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m— , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

44. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (XV), (XVI) or (XX):whereinX is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from -(CH2-CH2)n- - (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

45. A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (XV), (XVI) or (XX):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from -(CH2-CH2)n- — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

46. A method of preparing a MPLA / LA conjugate adjuvant of Formula (II), comprising: admixing a compound of Formula (IV) with a compound of Formula (XVII), (XVIII) or (XXI):whereinX is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from — (CH2-CH2)n— , - (CH2)m- , — (CH2-CH2-CH2)t— , and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

47. A method of preparing a MPLA / LA conjugate adjuvant of Formula (I), comprising: admixing a compound of Formula (V) with a compound of Formula (XVII), (XVIII) or (XXI):whereinR is selected from the group of -(CH2)aCH3, -(O-CH2-CH2)b-OCH3,-(O-CH2-CH2-CH2)c-OCH3, and a combination thereof, wherein a is an integer of 0-30, b is an integer of 1-20, and c is an integer of 1-15;X is selected from -P(=O)(OH)2and -S(=O)2(OH);Y is an integer of 6-24; andQ is selected from -(CH2-CH2)n- — (CH2)m— , -(CH2-CH2-CH2)t-, and a combination thereof, n is an integer of 1-20, m is an integer of 1-60, and t is an integer of 1-15.

48. The method of any one of claims 36 to 41 , wherein the admixing is performed in a solvent selected from the group of water, methanol, dimethylformamide (DMF), dichloromethane, diethyl ether or their mixtures.

49. The method of any one of claims 36 to 41 , wherein the admixing is performed for a time of 1 hour to several days at a temperature of 0 °C to 40 °C.

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