Adjuvant Compositions Comprising STING Agonists

Abstract

The present application relates to adjuvant compositions comprising: (i) a STING agonist of formula (I) or a pharma- ceutical acceptable salt thereof; and (ii) aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, immunogenic compositions comprising the adjuvant compositions, their use in methods of immunizing a subject, and related aspects thereof. TIFF2024540888000063.tif88142

Description

[Technical field]

[0001] The present application relates to the field of adjuvant compositions, immunogenic compositions comprising adjuvant compositions, their use in methods of immunizing a subject, and related aspects. [Background technology]

[0002] Vaccine adjuvants are included in the formulation to enhance humoral and cellular immune responses, especially in the case of poorly immunogenic subunit vaccines or in patients with difficult immune conditions (e.g., young children, the elderly, and immune comprised patients). Similar to natural infection with a pathogen, adjuvants rely on activation of the innate immune system to promote long-term adaptive immunity. Because simultaneous activation of multiple innate immune pathways is a feature of natural infection, adjuvants may combine multiple immune stimulants to promote adaptive immune responses to vaccination. Adjuvant system 01 (AS01) stimulates antigen-specific CD4 + It is of particular interest due to its ability to promote T cells and antigen-specific antibodies. The AS01 adjuvant system relies on the synergistic activity of two immune stimulants, 3-O-deacylated-4'-monophosphoryl lipid A (3D-MPL) and QS-21, and is formulated as a liposome-based formulation (Garcon and Van Mechelen, 2011; Didierlaurent et al., 2017). QS-21 is a purified plant extract and is therefore complex to synthesize and relies on natural sources. It is therefore desirable to develop an adjuvant system that induces a similar or even better immune response and / or shows a similar or improved reactogenicity profile, but is easier to prepare.

[0003] Recently developed STING (STimulator of Interferon Genes) agonists have been proposed as vaccine adjuvants. Specifically, International Publication No. 2017 / 175147 (PCT / IB2017 / 051945) discloses a series of dimeric amide benzimidazole (diABZI)-based compounds and their use as vaccine adjuvants. However, there remains a need to develop STING agonist formulations that can provide adjuvant compositions with improved immunogenicity and / or reduced reactogenicity. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2017 / 175147 (PCT / IB2017 / 051945) [Non-patent literature]

[0005] [Non-Patent Document 1] Garcon and Van Mechelen, 2011 [Non-Patent Document 2] Didierlaurent et al., 2017 Summary of the Invention

[0006] In a first aspect, the present invention provides a method for producing a composition comprising the steps of: (i) a STING agonist of formula (I) or a pharma- ceutical acceptable salt thereof [ka] [In the formula, X is -halo(C1-C5)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl; R 1 and R 9are independently H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I 2) optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkyloxy; In this case, the optionally substituted group includes hydroxyl, -OP(O)(OH), -OP(O)(R I )2, C1-C4 alkoxyl, -N(R A )2, -CO2(R B ), optionally substituted phenyl, and optionally substituted 5- to 6-membered heterocycloalkyl, wherein the optionally substituted phenyl or the optionally substituted 5- to 6-membered heterocycloalkyl is selected from the group consisting of halogen, hydroxy, -OP(O)(OH), -OP(O)(R I )2, amino, (C1-C6 alkyl)amino-, (C1-C6 alkyl)(C1-C6 alkyl)amino-, halo(C1-C6 alkyl), hydroxy-(C1-C4 alkyl)-, -(C1-C4 alkyl)-OP(O)(OH)2, -(C1-C4 alkyl)-OP(O)(R I )2, halo(C1-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)-OP(O)(OH)2, -(C2-C4 alkoxy)-OP(O)(R I )2, -(C1-C6 alkyl)-NH2, -C1-C4 alkyl-(C1-C4 alkoxyl) and C1-C4 alkoxy-(C1-C4 alkoxy)-, wherein R A and R B are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 2 and R 7are each independently hydrogen, -CON(R C )2, -COOH, or CO2(R D ) or R 2 and R 7 One of the -CON(R C )(R D ) and the other is H, -COOH, or CO2(R E ), where R C and R D are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 3 and R 7 are each independently H, halo(C1-C6 alkyl), halo(C1-C6 alkoxy)-, hydroxy, -OP(O)(OH), -OP(O)(R I )2, -NR C R D , -COR C , -CO2R C , -N(R D )COR C , -N(R D )SO2R D , -N(R g )SO2(C1-C2 alkyl)-N(R h )(R f ), -N(R g )CO(C1-C2 alkyl)-N(R h )(R f ) and; R e , R f , R g , and R h are each independently H or C1-C4 alkyl; R 4 , R 5 , R 11 and R 12 are each independently H or C1-C4 alkyl; R 6 and R10 are each C1-C4 alkyl; and R I each occurrence is independently C1-C6 alkyloxy-; and (ii) Aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, or a combination thereof. The present invention provides an adjuvant composition comprising:

[0007] In a second aspect, the present invention provides an immunogenic composition comprising the adjuvant composition of the invention and an antigen. In a third aspect, the present invention provides an adjuvant composition of the invention for use in therapy.

[0008] In a further aspect, the invention provides the adjuvant composition of the invention for use in a method of immunizing a subject comprising administering to the subject the adjuvant composition of the invention and an antigen.

[0009] In a fourth aspect, the present invention provides a method of immunising a host comprising administering to the host an adjuvant composition as defined herein and an antigen.

[0010] In a further aspect, the invention provides a method of adjuvanting an immune response in a subject comprising administering to the subject an adjuvant composition of the invention and an antigen. In a further aspect, the invention provides the use of the adjuvant composition in the manufacture of a medicament for adjuvanting an immune response in a subject. In a further aspect, the present invention provides a kit comprising (i) a first container comprising an adjuvant composition of the invention, and (ii) a second container comprising an antigen. The invention will be further explained with reference to the accompanying non-limiting drawings in which: [Brief description of the drawings]

[0011] [Figure 1]FIG. 13 depicts the adsorption of STING agonists to Al(OH)3 (for Al3+) at different ratios of STING agonist to Al(OH)3 (as indicated) tested. [Diagram 2] FIG. 1 depicts HSV-2 gI-specific IgG antibody responses induced in mice by different vaccine formulations containing antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different doses of STING agonist and different ratios of Al(OH)3 (as indicated). IgG titers were measured 2 weeks after immunization I (Post I) and 2 weeks after immunization II (Post II). Antigen HSV-2 gE-gI alone was used as a control. Vaccine formulations containing HSV-2 gE-gI adjuvanted with AS01 were used as a comparison. Individual geometric means and 95% confidence intervals (Ci) are provided. [Diagram 3] FIG. 1 depicts HSV-2 gE-specific IgG antibody responses induced in mice by different vaccine formulations containing antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. IgG titers were measured 2 weeks after immunization I (Post I) and 2 weeks after immunization II (Post II). Antigen HSV-2 gE-gI alone was used as a control. Vaccine formulations containing HSV-2 gE-gI adjuvanted with AS01 were used as a comparison. Individual geometric means and 95% CI are provided. [Figure 4]FIG. 1 represents HSV-1 gE-gI specific IgG antibody responses induced in mice by different vaccine formulations containing antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. IgG titers were measured 2 weeks after immunization II (Post II). Antigen HSV-2 gE-gI alone was used as control. Vaccine formulations containing HSV-2 gE-gI adjuvanted with AS01 were used as comparator. Individual geometric means and 95% CI are provided. [Diagram 5] FIG. 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II in mice after ex vivo stimulation with HSV-2 gE peptide pools. The administered vaccines contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. [Figure 6]FIG. 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II after ex vivo stimulation with HSV-2 gI peptide pools or HSV-1 gE or gI pools in mice. The administered vaccines contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. [Figure 7] FIG. 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II in mice after ex vivo stimulation with HSV-1 gI peptide pools. The administered vaccine contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. [Figure 8]FIG. 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II in mice after ex vivo stimulation with HSV-1 gE peptide pools. The administered vaccine contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. [Figure 9] Figure 1 depicts HSV-2 gE-specific CD4+ T cell polyfunctionality analysis after in vitro stimulation. The percentage of HSV-2 gE-specific CD4+ T cells expressing one, two, three or four cytokines is depicted. Pie charts represent the mean percentage of total HSV-2-specific CD4+ T cells expressing a single marker and CD4+ T cells positive for any combination of INF-g, IL-2, TNF-a, IL-13 and IL-17 markers. Vaccines used in this analysis were as follows (as indicated): HSV-2 gE-gI antigen adjuvanted with either (i) AS01, (ii) 0.74 μg soluble STING agonist, or (iii) 0.74 μg STING agonist and 5.55 μg AL(OH)3. [Figure 10]Figure 13 depicts HSV-2 gI-specific CD4+ T cell polyfunctionality analysis after in vitro stimulation. The percentage of HSV-2 gI-specific CD4+ T cells expressing one, two, three or four cytokines is depicted. Pie charts represent the average percentage of total HSV-2-specific CD4+ T cells expressing a single marker and CD4+ T cells positive for any combination of INF-g, IL-2, TNF-a, IL-13 and IL-17 markers. The vaccines used in this analysis were as follows (as indicated): HSV-2 gE-gI antigen adjuvanted with either (i) AS01, (ii) 0.74μg soluble STING agonist, or (iii) 0.74μg STING agonist and 5.55μg AL(OH)3. [Figure 11] Figure 1 depicts an analysis of cytokine (IL-6, IP10 and IFN-γ, as indicated) measurements in serum of mice 3, 6, 24 and 48 hours after immunization with either VZV gE antigen alone, VZV gE adjuvanted with a soluble STING agonist, or VZV gE adjuvanted with a STING agonist and Al(OH)3. A vaccine formulation containing VZV gE adjuvanted with AS01 was used as a comparison. [Figure 12] Figure 1 depicts an analysis of cytokine (IFN-a, IFN-b, and IFN-γ, as indicated) measurements in serum of mice 3, 6, 24, and 48 hours after immunization with either VZV gE antigen alone, VZV gE adjuvanted with a soluble STING agonist, or VZV gE adjuvanted with a STING agonist and Al(OH)3. A vaccine formulation containing VZV gE adjuvanted with AS01 was used as a comparison. [Figure 13]FIG. 1 represents HSV-2 gE-specific IgG antibody responses induced in mice by different vaccine formulations containing different doses of soluble STING agonist or different doses of STING agonist and antigen HSV-2 gE-gI adjuvanted with either Al(OH)3, AS01 or alum in different ratios (as indicated). IgG titers were measured 2 weeks after immunization I (Post I) and 2 weeks after immunization II (Post II). Antigen HSV-2 gE-gI alone was used as a control. Vaccine formulations containing HSV-2 gE-gI adjuvanted with AS01 were used as a comparison. Individual geometric means and 95% confidence intervals (Ci) are provided. [Figure 14] FIG. 1 depicts HSV-2 gI-specific IgG antibody responses induced in mice by different vaccine formulations containing antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different doses of STING agonist and different ratios of Al(OH)3 (as indicated). IgG titers were measured 2 weeks after immunization I (Post I) and 2 weeks after immunization II (Post II). Antigen HSV-2 gE-gI alone was used as a control. Vaccine formulations containing HSV-2 gE-gI adjuvanted with AS01 were used as a comparison. Individual geometric means and 95% confidence intervals (Ci) are provided. [Figure 15]FIG. 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II in mice after ex vivo stimulation with HSV-1 gE peptide pools. The administered vaccine contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. [Figure 16] Figure 1 represents the frequency of vaccine-specific CD4+ T cells expressing IL-2 and / or INF-g and / or TNF-a and / or IL-13 and / or IL-17 from splenocytes collected 2 weeks after immunization II in mice after ex vivo stimulation with HSV-1 gI peptide pools. The administered vaccine contained the antigen HSV-2 gE-gI adjuvanted with either different doses of soluble STING agonist or different ratios (as indicated) of STING agonist to Al(OH)3. The antigen HSV-2 gE-gI alone was used as a control. A vaccine formulation containing HSV-2 gE-gI adjuvanted with AS01 was used as a comparison. Individual geometric means and 95% CI are provided. Brief description of sequences SEQ ID NO: 1: SARS-CoV-2 S protein. SEQ ID NO: 2: SARS-CoV-2 S protein ectodomain. SEQ ID NO:3: SARS-CoV-2 S protein receptor binding domain. SEQ ID NO:4: Prefusion stabilized SARS-CoV-2 S protein ectodomain. SEQ ID NO:5: Polypeptide sequence of HSV-2 gE P317R. SEQ ID NO:6: Polypeptide sequence of HSV-2 gI. SEQ ID NO:7: Polypeptide sequence of VZV gE. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] As noted above, in one aspect, (i) a STING agonist of formula (I) or a pharma- ceutical acceptable salt thereof [ka] [In the formula, X is -halo(C1-C5)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl; R 1 and R 9 are independently H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I 2) optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkyloxy; In this case, the optionally substituted group includes hydroxyl, -OP(O)(OH), -OP(O)(R I )2, C1-C4 alkoxyl, -N(R A )2, -CO2(R B ), optionally substituted phenyl, and optionally substituted 5- to 6-membered heterocycloalkyl, wherein the optionally substituted phenyl or the optionally substituted 5- to 6-membered heterocycloalkyl is selected from the group consisting of halogen, hydroxy, -OP(O)(OH), -OP(O)(R I )2, amino, (C1-C6 alkyl)amino-, (C1-C6 alkyl)(C1-C6 alkyl)amino-, halo(C1-C6 alkyl), hydroxy-(C1-C4 alkyl)-, -(C1-C4 alkyl)-OP(O)(OH)2, -(C1-C4 alkyl)-OP(O)(R I )2, halo(C1-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)-OP(O)(OH)2, -(C2-C4 alkoxy)-OP(O)(R I)2, -(C1-C6 alkyl)-NH2, -C1-C4 alkyl-(C1-C4 alkoxyl) and C1-C4 alkoxy-(C1-C4 alkoxy)-, wherein R A and R B are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 2 and R 7 are each independently hydrogen, -CON(R C )2, -COOH, or CO2(R D ) or R 2 and R 7 One of the -CON(R C )(R D ) and the other is H, -COOH, or CO2(R E ), where R C and R D are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 3 and R 8 are each independently H, halo(C1-C6 alkyl), halo(C1-C6 alkoxy)-, hydroxy, -OP(O)(OH), -OP(O)(R I )2, -NR C R D , -COR C , -CO2R C , -N(R D )COR C , -N(R D )SO2R C , -N(R g )SO2(C1-C2 alkyl)-N(R h )(R f), -N(R g )CO(C1-C2 alkyl)-N(R h )(R f ) and; R e , R f , R g , and R h are each independently H or C1-C4 alkyl; R 4 , R 5 , R 11 and R 12 are each independently H or C1-C4 alkyl; R 6 and R 10 are each C1-C4 alkyl; and R I each occurrence is independently C1-C6 alkyloxy-; and (ii) Aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, or a combination thereof. There is provided an adjuvant composition comprising:

[0013] definition As used herein, the term "STING agonist" (also referred to herein as "STINGa") refers to a compound of formula (I) that is capable of binding to and activating STING receptor and STING signal transduction. For example, upon contact with STING receptor, STING agonist causes one or more of the following: (1) stimulate or activate STING receptor, (2) upregulate IRF3 and NFkB signal transduction pathways, and / or (3) induce IFN-b and other cytokines. STING agonist activity can be measured in vitro by various assays known in the art, such as, but not limited to, measuring cell signal transduction, cell proliferation, immune cell activation markers, cytokine production. STING agonist activity can also be measured in vivo by various assays that measure surrogate endpoints, such as, but not limited to, measuring T cell proliferation or innate immune-related cytokine production, particularly type I interferon.

[0014] The alternative definitions for the various groups and substituents of formula (I) provided throughout the specification are intended to describe each compound species disclosed herein individually, as well as groups of one or more compound species. The scope of STING agonists as used herein includes any combination of these group and substituent definitions.

[0015] It will be understood by those skilled in the art that the compounds may exist in other tautomeric or isomeric forms, including zwitterionic forms. All tautomeric (including zwitterionic) and isomeric forms of the compounds of formula (I) and described herein are intended to be encompassed within the scope of this disclosure.

[0016] For example, it will be understood by one of ordinary skill in the art that the compounds for use herein may exist in tautomeric forms, including but not limited to, Formula (A), Formula (B) and / or Formula (C), or zwitterionic forms, including but not limited to, Formula (D) or Formula (E).

[0017] [ka]

[0018] Chemical names provided for intermediate compounds and / or compounds for use described herein may refer to any one of the tautomeric representations of such compounds (in some instances, such alternative names are provided accompanying the experiment). It should be understood that any reference to a compound (intermediate compound or compound of the present disclosure) shown by name or whose structure is depicted is intended to encompass all tautomeric forms of such compounds, including zwitterionic forms, and any mixtures thereof.

[0019] As used herein, the term "alkyl" refers to a saturated straight or branched chain hydrocarbon group having the specified number of carbon atoms. The term "C1-C4 alkyl" refers to a straight or branched chain alkyl moiety containing from 1 to 4 carbon atoms. Exemplary alkyls include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, pentyl, and hexyl.

[0020] When a substituent term such as "alkyl" is used in combination with another substituent term, such as in "hydroxy(C1-C4 alkyl)", the linking substituent term (e.g., alkyl) is intended to encompass a divalent moiety where the point of attachment is through the linking substituent. Examples of "hydroxy(C1-C4 alkyl)" groups include, but are not limited to, hydroxymethyl, hydroxyethyl, and hydroxyisopropyl.

[0021] As used herein, the term "halo(alkyl)" refers to a saturated straight or branched chain hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms. For example, the term "halo(C1-C5 alkyl)" refers to a group having one or more halogen atoms, which can be the same or different, on one or more carbon atoms of the alkyl portion containing 1 to 5 carbon atoms. Examples of "halo(C1-C5 alkyl)" groups include, but are not limited to, -CF3 (trifluoromethyl), -CCl3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl.

[0022] "Alkenyl" means a straight or branched chain hydrocarbon group having the specified number of carbon atoms and at least one, and at most three, carbon-carbon double bonds. Examples include ethenyl and propenyl.

[0023] "Alkoxy-" or "(alkyl)oxy-" refers to an "alkyl-oxy-" group that includes an alkyl moiety having a specified number of carbon atoms linked through an oxygen linking atom. For example, the term "C1-C4 alkoxy-" represents a saturated straight or branched chain hydrocarbon moiety having at least one and up to four carbon atoms linked through an oxygen linking atom. Exemplary "C1-C4 alkoxy-" or "(C1-C4 alkyl)oxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy. Exemplary "C1-C6 alkoxy-" or "C1-C6 alkyloxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy.

[0024] As used herein, the term "halo(alkoxy)-" refers to a saturated straight or branched chain hydrocarbon group having a specified number (n) of carbon atoms and one or more (up to 2n+1) halogen atoms linked via an oxygen linking atom. For example, the term "halo(C1-C4 alkoxy)-" refers to a "haloalkyl-oxy-" group that includes a "halo(C1-C4 alkyl)" moiety linked via an oxygen linking atom. Exemplary "halo(C1-C4 alkoxy)-" groups include, but are not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy), and -OCH(CF3)2 (hexafluoroisopropoxy).

[0025] Heterocyclic groups or moieties are cyclic groups or moieties having atoms of at least two different elements as ring members which can be saturated, partially unsaturated (non-aromatic) or fully unsaturated (aromatic).

[0026] "Heterocycloalkyl" means a non-aromatic monocyclic or bicyclic group containing 3 to 10 ring atoms and containing one or more (typically 1 or 2) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The point of attachment of the heterocycloalkyl group can be any suitable carbon or nitrogen atom.

[0027] The term "5- to 6-membered heterocycloalkyl" refers to a saturated monocyclic group containing 5 or 6 ring atoms, including 1 or 2 heteroatoms independently selected from oxygen, sulfur, and nitrogen. Illustrative examples of 5- to 6-membered heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.

[0028] The terms "halogen" and "halo" refer to a halogen radical, for example, a fluoro, chloro, bromo, or iodo substituent. "Oxo" refers, for example, to a double bonded oxygen moiety which, when attached directly to a carbon atom, forms a carbonyl moiety (C=O). "Hydroxy" or "hydroxyl" is intended to mean the group --OH. As used herein, the term "cyano" refers to a nitrile group, -C≡N.

[0029] As used herein, the term "optionally substituted" means that a group (such as an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group) or ring or moiety can be unsubstituted or that the group, ring, or moiety meets the definitions of substituents (A, R, etc.) provided herein. 3 etc. In the case where groups may be selected from a number of alternative groups, the selected groups may be the same or different.

[0030] The term "independently" means that when two or more substituents are selected from a number of possible substituents, the substituents can be the same or different.

[0031] The term "pharmaceutical acceptable" means compounds, materials, compositions, and dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without undue toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit / risk ratio. As used herein, the term "vaccine" is optionally interchangeable with the term "immunogenic composition."

[0032] As used herein, the term "reactogenicity" refers to a subset of adverse events associated with inflammatory responses to vaccination. Adverse events can be classified as both local (e.g., pain, swelling, erythma, and induration) and systemic (e.g., fever, nausea / vomiting, diarrhea, headache, fatigue, and muscle pain). Improving a vaccine by reducing its reactogenicity can improve the accessibility of the vaccine to certain populations, for example, by reducing pain in adolescents and fever in young children. Thus, reduced reactogenicity can improve vaccine uptake, which leads to greater population coverage and thus reduces morbidity / mortality. Furthermore, excessive inflammation can also negatively impact the quality of the immune response induced by a vaccine or immunogenic composition. Therefore, it is an objective of the present disclosure to reduce the reactogenicity of a vaccine. As used herein, the term "about" is used to indicate a variation of ±10%.

[0033] Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with the reference amino acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0034] Sequence identity can be determined by standard methods commonly used to compare the similarity at the amino acid positions of two polypeptides. Using computer programs such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their individual amino acids (over the entire length of one or both sequences, or over a predetermined portion of one or both sequences). The program provides default opening and gap penalties, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, percent identity can then be calculated as follows: the total number of identical matches is multiplied by 100, and then divided by the sum of the length of the longer sequence in the matched span and the number of gaps introduced into the shorter sequence to align the two sequences.

[0035] STING agonists In one embodiment, the STING agonist is of formula (II) or a pharma- ceutical acceptable salt thereof: [ka] [where X, R 1 , R 5 , R 6 , R 9 , R 10 and R 11 is as defined in relation to formula (I)].

[0036] In one embodiment, R in formula (I) and (II) 1 and R 9are each independently H, halogen, an optionally substituted (C1-C6 alkyl), or an optionally substituted (C1-C6 alkyl)oxy-, and the C1-C6 alkyl of the optionally substituted (C1-C6 alkyl) or optionally substituted (C1-C6 alkyl)oxy- is hydroxyl, -OP(O)(OH), -OP(O)(R I )2, -N(R e )(R f ), C1-C4 alkoxyl, phenyl, and optionally substituted 5- to 6-membered heterocycloalkyl containing at least one nitrogen or oxygen ring member; e is independently selected from H, (C1-C4 alkyl), -(C1-C4 alkyl)-NH2, or -(C1-C4 alkyl) C1-C4 alkoxy; f is independently H or (C1-C4 alkyl).

[0037] In one embodiment, the compound of formula (I) or (II) contains at least one phosphate group. In one embodiment, R 1 and R 9 One of the groups is -OP(O)(OH)2, -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I In one embodiment, R in formula (I) or (II) is a C1-C6 alkyl, or a C1-C6 alkyloxy group substituted with 1 and R 9 One of the groups is -OP(O)(OH)2, -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I In one embodiment, one is -OP(O)(OH), -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I)2, and the other is H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I )2, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkyloxy, where optionally substituted means hydroxyl, -OP(O)(OH)2, -OP(O)(R I )2, C1-C4 alkoxyl, -N(R A )2, -CO2(R B In one embodiment, R in formula (I) or (II) is substituted with 1 to 4 substituents each independently selected from the group consisting of 1 and R 9 One of them is -OP(O)(OH)2, -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I )2, and the other is H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I )2, optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkyloxy, where optionally substituted means hydroxyl, -OP(O)(OH)2, -OP(O)(R I )2, -N(R A )2, -CO2(R B In one embodiment, R in formula (I) or (II) is substituted with 1 to 4 substituents each independently selected from the group consisting of 1 and R 9 One of them is -OP(O)(OH)2, -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I )2, and the other is H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I )2, C1-C6 alkyl or C1-C6 alkyloxy.

[0038] In one embodiment, R in formula (I) or (II) 3 is H. In one embodiment, R in formula (I) or (II) 8 is H. In one embodiment, R in formula (I) or (II) 3 and R 8 is H.

[0039] In one embodiment, R in formula (I) or (II) 4 is H. In one embodiment, R in formula (I) or (II) 12 is H. In one embodiment, R in formula (I) or (II) 4 and R 12 is H.

[0040] In one embodiment, R in formula (I) or (II) 6 In one embodiment, R in formula (I) or (II) is ethyl. 10 In one embodiment, R in formula (I) or (II) is ethyl. 6 and R 10 is ethyl.

[0041] In one embodiment, R in formula (I) and (II) 1 and R 9 At least one of is selected from the following groups: [ka] [wherein a is a number from 2 to 6];

[0042] [ka] [wherein b is a number from 1 to 6];

[0043] [ka] [wherein c is a number from 1 to 6];

[0044] [ka] [In the formula, d is a number from 1 to 6, and R J and R K is C1-C3 alkyl; or

[0045] [ka] [In the formula, e is a number from 1 to 6, and Q is O and N(R X ), wherein R X is C1-C6 alkyl.

[0046] In one embodiment, the STING agonist is (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide;

[0047] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0048] (Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0049] (E)-4-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)butanoic acid;

[0050] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazole-5-carboxamide;

[0051] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0052] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0053] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxamide;

[0054] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0055] (Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0056] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide;

[0057] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0058] (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate;

[0059] 3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate;

[0060] 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate; or Pharmaceutically acceptable salts thereof is selected from the group consisting of:

[0061] In one embodiment, the STING agonist is 4-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)butanoic acid, as shown below, or a pharma- ceutically acceptable salt thereof.

[0062] [ka]

[0063] Depending on its isomeric / tautomeric form, this compound may also be called (E)-4-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)butanoic acid or 4-(((Z) It may also be written as -6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)butanoic acid.

[0064] In one embodiment, the STING agonist is (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide, as represented by the structure below.

[0065] [ka]

[0066] Depending on its isomeric / tautomeric form, this compound may be designated as (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazole-5-carboxamide.

[0067] In one embodiment, the STING agonist is (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide, as shown by the structure below.

[0068] [ka]

[0069] In one embodiment, the STING agonist is (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide (as shown below), or a pharma- ceutically acceptable salt thereof.

[0070] [ka]

[0071] Depending on its isomeric / tautomeric form, this compound may also be called (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxamide or (Z)-1-( It may also be written as (E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide.

[0072] In one embodiment, the STING agonist is (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide (as shown by the structure below), or a pharma- ceutically acceptable salt thereof.

[0073] [ka]

[0074] Depending on its isomeric / tautomeric form, this compound may also be called (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide or (E)-1-( It may also be written as (E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide.

[0075] In one embodiment, the STING agonist is (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide, as represented by the formula:

[0076] [ka]

[0077] Depending on its isomeric / tautomeric form, this compound may also be called (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-hydroxypropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide or (Z)-1-( It may also be written as (E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide.

[0078] In one embodiment, the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate, as represented by the formula:

[0079] [ka]

[0080] Depending on its isomeric / tautomeric form, this compound may also be called (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate or 3-(((E) It may also be written as -6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate. In one embodiment, the STING agonist of Formula (I) or (II) is in free base or free acid form.

[0081] As used herein, the STING agonists of Formula (I) or Formula (II) as defined herein may be in any form, i.e., any tautomeric form, any isomeric form, any salt or non-salt form (e.g., as a free acid or base form, or as a salt, e.g., a pharma- ceutically acceptable salt thereof) and in any physical form (e.g., including non-solid forms, such as liquid or semi-solid forms, and solid forms, such as amorphous or crystalline forms, specific polymorphic forms, solvate forms, including hydrate forms, such as monohydrate, dihydrate and hemihydrate, mixtures of various forms).

[0082] Thus, the compounds of formula (I) or formula (II) as defined herein in any salt or non-salt form and in any physical form thereof, as well as mixtures of various forms, are included for use herein.While such are included for use within the scope of this disclosure, it will be understood that the compounds of formula (I) or (II) as defined herein in any salt or non-salt form and in any physical form thereof may have different levels of activity, different bioavailability and different handling properties for formulation purposes.

[0083] It will be readily appreciated that the STING agonist of formula (I), or a pharma- ceutically acceptable salt thereof, intended for use in an immunogenic composition is preferably provided in a substantially pure form, e.g., at least 60% pure, more suitably at least 75% pure, preferably at least 85%, especially at least 98% pure (percentages being by weight on a weight basis). Impure preparations of the compounds can be used to prepare the purer forms used in the immunogenic compositions.

[0084] STING agonists for use herein can contain one or more asymmetric centers (also referred to as chiral centers), such as a chiral carbon or a chiral -SO- moiety. The STING agonists, which can contain one or more chiral centers, can exist as racemic mixtures, diastereomeric mixtures, enantiomerically enriched mixtures, diastereomerically enriched mixtures, or as enantiomerically or diastereomerically pure individual stereoisomers.

[0085] The stereochemistry of chiral centers present in the compounds used herein is generally represented in the compound name and / or in the chemical structures depicted.When the stereochemistry of chiral centers present in the compounds of the present disclosure or in any chemical structures depicted herein is not specified, the structure is intended to encompass any stereoisomers and all mixtures thereof.Thus, all STING agonists and salts thereof of formula (I) or (II) are encompassed for use herein, whether as individual isomers isolated, such as for being substantially free of other isomers (i.e., pure), or as mixtures (i.e., racemates and racemic mixtures).Individual isomers isolated, such as for being substantially free of other isomers (i.e., pure), can be isolated such that there is less than 10%, particularly less than about 1%, for example less than about 0.1% of other isomers.

[0086] Individual stereoisomers of STING agonists can be resolved (or mixtures of stereoisomers can be enriched) using methods known to those skilled in the art. For example, such resolution can be carried out (1) by the formation of diastereoisomeric salts, complexes or other derivatives; (2) by selective reaction with stereoisomer-specific reagents, for example, by enzymatic oxidation or reduction; or (3) by gas-liquid or liquid chromatography in a chiral environment, for example, on a chiral support such as silica containing a bound asymmetric ligand or in the presence of a chiral solvent. It will be understood that when the desired stereoisomer is converted to another chemical entity by one of the above separation procedures, a further step is required to liberate the desired form. Alternatively, specific stereoisomers can be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation.

[0087] The definition of STING agonist used herein also includes various deuterated forms of the compound. Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art will know how to synthesize the deuterated forms of the compounds used herein. For example, α-deuterated α-amino acids are commercially available or can be prepared by conventional techniques (see, for example, Elemes, Y. and Ragnarsson, UJ Chem. Soc., Perkin Trans. 1, 1996, 6, 537-40). The use of such compounds can allow the preparation of compounds in which the hydrogen atom of the chiral center is replaced by a deuterium atom. Other commercially available deuterated starting materials can be utilized in the preparation of deuterated analogs of compounds (see, for example, methyl-d3-amine available from Aldrich Chemical Co., Milwaukee, WI) or can be synthesized using conventional techniques utilizing deuterated reagents (e.g., by reduction with lithium aluminum deuteride or sodium borodeuteride, or by metal-halogen exchange followed by quenching with DO or methanol-d3).

[0088] In one embodiment, the STING agonist of Formula (I) or (II) is used in the form of a pharma- ceutically acceptable salt. Suitable pharma- ceutically acceptable salts of the STING agonist of formula (I) or (II) include acid addition salts or base addition salts.For a review of suitable pharma-ceutically acceptable salts, see Berge et al., J. Pharm. Sci., 66:1-19, (1977) and PH Stahl and CG Wermuth, Eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim / Zurich:Wiley-VCH / VHCA (2002).

[0089] Salts of STING agonists of formula (I) or (II) containing a basic amine or other basic functional group can be prepared by any suitable method known in the art, such as treatment of the free base with a suitable inorganic or organic acid. Examples of pharma- ceutically acceptable salts so formed include acetate, adipate, ascorbate, aspartate, benzenesulfonate, benzoate, camphorate, camphorsulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), carbonate, bicarbonate, cinnamate, citrate, cyclamate, dodecyl sulfate (estrate), ethanoate, ... Salts include 1,2-dimethylformamide (dimethylformamide), ... salts, etc.), hydroiodide, isobutyrate, lactate, lactobionate, laurate, maleate, malate, malonate, mandelate, methanesulfonate (mesylate), naphthalene-1,5-disulfonate (napadisilate), naphthalene-sulfonate (napsylate), nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, phosphate (diphosphate, etc.), propionate (propriate onate), pyroglutamate, salicylate, sebacate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undecylenate, 1-hydroxy-2-naphthoate, 2,2-dichloroacetate, 2-hydroxyethanesulfonate (isethionate), 2-oxoglutarate, 4-acetamidobenzoate, and 4-aminosalicylate.

[0090] Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such pharma-ceutically acceptable salts can be prepared using a base that provides a pharma-ceutically acceptable cation, including alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, N,N'-dibenzylethylenediamine, 2-hydroxyethylamine, bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine, collidine, choline, quinine, quinoline, and salts formed from basic amino acids such as lysine and arginine.

[0091] The present disclosure includes within its scope all possible stoichiometric and non-stoichiometric forms of salts of STING agonists of formula (I) or (II) (e.g., hydrobromide, dihydrobromide, fumarate, hemifumarate, etc.).

[0092] When the disclosed compound or its salt is designated by name or depicted by structure, it should be understood that the compound or salt, including its solvate (particularly hydrate), can exist in crystalline form, non-crystalline form, or mixtures thereof. The compound or salt, or its solvate (particularly hydrate), may also exhibit polymorphism (i.e., the ability to appear in different crystalline forms). These different crystalline forms are typically known as "polymorphs". It should be understood that the present disclosure includes all polymorphic forms of any compound designated by name or depicted by structure herein, including any polymorphic forms of any compound herein, for example, any salt and / or solvate (particularly hydrate) thereof.

[0093] Polymorphs have the same chemical composition but differ in packing, geometric arrangement, and other descriptive properties of the crystalline solid state. Thus, polymorphs may have different physical properties such as shape, density, hardness, deformability, stability, and dissolution properties. Polymorphs typically exhibit different melting points, IR spectra, and X-ray powder diffraction patterns that can be used for identification. It will be understood that different polymorphs can be produced, for example, by changing or adjusting the conditions used in the crystallization / recrystallization of a compound. Polymorphic forms can be characterized and differentiated using a number of conventional analytical techniques, including, but not limited to, X-ray powder diffraction (XRPD) patterns, infrared (IR) spectra, Raman spectra, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and solid-state nuclear magnetic resonance (SSNMR).

[0094] Those skilled in the art will understand that pharma- ceutical acceptable solvates (particularly hydrates) of STING agonists, including pharma- ceutical acceptable solvates of pharma- ceutical acceptable salts of STING agonists, may be formed when solvent molecules are incorporated into the crystal lattice during crystallization. Solvates may include non-aqueous solvents such as ethanol, or may include water as the solvent incorporated into the crystal lattice. Solvates in which water is the solvent incorporated into the crystal lattice are typically referred to as "hydrates". STING agonists, as defined herein, include within their scope all possible stoichiometric and non-stoichiometric salt and / or hydrate forms.

[0095] Salts and solvates (e.g., hydrates and hydrates of salts) of STING agonists as used in this disclosure include all that are suitable for pharmaceutical use where the counterion or associated solvent is pharma- ceutical acceptable. Salts having pharma-ceutical unacceptable counterions are also within the scope, e.g., for use as intermediates in the preparation of other compounds.

[0096] Typically, a pharma- ceutically acceptable salt can be readily prepared by using a desired acid or base, as appropriate, The resulting salt can crystallize or precipitate from solution or can be formed by trituration and can be recovered by filtration or by evaporation of the solvent.

[0097] The present disclosure includes all prodrugs of STING agonists of formula (I) that are capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof upon administration to a recipient. Such derivatives are recognizable to those of skill in the art without undue experimentation. Nonetheless, to the extent that they teach such derivatives, see Burger's Medicinal Chemistry and Drug Discovery, 5th ed., incorporated herein by reference. th Reference is made to the teachings in the International Standards Board for Business Conduct, Vol 1: Principles and Practice.

[0098] It should be further understood that the present disclosure includes within its scope all tautomeric or isomeric forms of any free base form of the compounds, and all possible stoichiometric and non-stoichiometric salt forms.

[0099] Aluminum Compounds The adjuvant composition comprises aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, or a combination thereof.

[0100] Suitable forms of aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate for adjuvant use are well known to those of skill in the art. In one embodiment, the adjuvant composition comprises aluminum phosphate, aluminum hydroxide, or a combination thereof. Suitable forms of aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate include, but are not limited to, Rehydragel.TM HS, Alhydrogel TM 85. Rehydragel TM PM, Rehydragel TM AB, Rehydragel TM HPA, Rehydragel TM LV, Alhydrogel TM or a combination thereof.

[0101] In one embodiment, the adjuvant composition comprises aluminum hydroxide. In one embodiment, the adjuvant composition comprises aluminum hydroxide and is free (i.e., substantially free) of aluminum oxyhydroxide, or aluminum hydroxyphosphate. In one embodiment, the adjuvant composition comprises aluminum phosphate. In one embodiment, the adjuvant composition comprises aluminum oxyhydroxide. In one embodiment, the adjuvant composition comprises aluminum hydroxyphosphate. In one embodiment, the adjuvant composition comprises aluminum hydroxide. In one embodiment, the adjuvant composition comprises aluminum hydroxide, and

[0102] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-hydroxypropoxy)-1H-benzo[d]imidazole-5-carboxamide;

[0103] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0104] (Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0105] (E)-4-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)butanoic acid;

[0106] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazole-5-carboxamide;

[0107] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0108] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0109] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxamide;

[0110] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0111] (Z)-1-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0112] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazole-5-carboxamide;

[0113] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0114] (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate;

[0115] 3-(((Z)-6-carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate;

[0116] 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate; or Pharmaceutically acceptable salts thereof The STING agonist of formula (I) is selected from the group consisting of:

[0117] In one embodiment, the adjuvant composition comprises aluminum hydroxide and a STING agonist that is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate (Compound 1), or a pharma- ceutically acceptable salt thereof.

[0118] In one embodiment, the aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate (e.g., aluminum hydroxide) has a protein (BSA) / mL Al concentration of 2.5-3.5, 2.6-3.4, 2.7-3.3, or 2.9-3.2, 2.5-3.7, 2.6-3.6, 2.7-3.5, or 2.8-3.4. 3+ In one embodiment, the aluminum hydroxide has a protein adsorption capacity of 2.9 to 3.2 mg BSA / mg Al. 3+The protein adsorption capacity of the aluminum compound can be measured by any means known to those skilled in the art. The protein adsorption capacity of the aluminum compound can be measured using the method (utilizing BSA) as described in Example 1 of WO 12 / 136823, or a modification thereof. The aluminum hydroxide described herein (i.e., having the protein adsorption capacity described herein) can have a crystal size of 2.8-5.7 nm as measured by X-ray diffraction, e.g., 2.9-5.6 nm, 2.8-3.5 nm, 2.9-3.4 nm, or 3.4-5.6 nm, or 3.3-5.7 nm as measured by X-ray diffraction. X-ray diffraction is well known to those skilled in the art. In certain embodiments, the crystal size is measured using the method described in Example 1 of WO 12 / 136823, or a modification thereof.

[0119] In embodiments in which the adjuvant composition further comprises an antigen, the antigen can be adsorbed to the aluminum compound prior to mixing with the STING agonist or the adjuvant composition.

[0120] In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto the same aluminium hydroxide.In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto different aluminium hydroxides.

[0121] In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto the same aluminum phosphate. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto different aluminum phosphates.

[0122] In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto the same aluminum oxyhydroxide. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto different aluminum oxyhydroxides.

[0123] In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto the same aluminum hydroxyphosphate.In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed onto different aluminum hydroxyphosphates.

[0124] The term "adsorbed antigen" is taken to mean, for example, more than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% adsorbed. The amount of adsorbed antigen can be measured using HPLC (e.g., by centrifuging the composition and performing HPLC on the supernatant). In one embodiment, the antigen is adsorbed onto aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate in an amount of more than 20% (e.g., more than 50%). In one embodiment, the antigen is adsorbed onto aluminum hydroxide in an amount of more than 50%. In some embodiments, the antigen is adsorbed onto aluminum hydroxide in an amount of more than 60%. In some embodiments, the antigen is adsorbed onto aluminum hydroxide in an amount of more than 70%. In some embodiments, the antigen is adsorbed onto aluminum hydroxide in an amount of more than 80%. In one embodiment, the antigen is adsorbed onto aluminum hydroxide in an amount of more than 90%.

[0125] "Adsorbed STING agonist" is taken to mean, for example, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% adsorbed. The amount of adsorbed STING agonist can be measured using HPLC (e.g., by centrifuging the composition and performing HPLC on the supernatant).

[0126] In one embodiment, the adjuvant composition comprises a ratio of STING agonist to aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate of 1:2.5-250. In one embodiment, the adjuvant composition contains a STING agonist as defined herein and aluminum hydroxide in an amount of 1:1-250. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:2.5-100.

[0127] In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:1 to 10. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:1. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:5.

[0128] In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:10-100. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:10. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:25. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:50. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:75. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:50-100. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:50-125. In one embodiment, the ratio of the STING agonist to aluminum hydroxide is 1:100. Alternatively, the ratio of STING agonist to aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate is 1:0.1 to 1:250, or 1:0.1 to 1:100, or 1:0.1 to 1:50, or 1:0.1 to 1:25, or 1:0.1 to 1:10, or 1:0.1 to 1:5.

[0129] In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:100-200. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:150. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:175. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:200. In one embodiment, the ratio of STING agonist to aluminum hydroxide is 1:250. Alternatively, the ratio of STING agonist to aluminum hydroxide is 1:0.1-1:250, or 1:0.1-1:100, or 1:0.1-1:50, or 1:0.1-1:25, or 1:0.1-1:10, or 1:0.1-1:5.

[0130] In one embodiment, the adjuvant composition comprises aluminum hydroxide, and the ratio of antigen to aluminum hydroxide is 1:1 to 1:50. In one embodiment, the adjuvant composition comprises aluminum hydroxide, and the ratio of antigen to aluminum hydroxide is 1:1 to 1:25. In one embodiment, the adjuvant composition comprises aluminum hydroxide, and the ratio of antigen to aluminum hydroxide is 1:1 to 1:10. In one embodiment, the adjuvant composition comprises aluminum hydroxide, and the ratio of antigen to aluminum hydroxide is 1:1 to 1:5. In one embodiment, the ratio of antigen to aluminum hydroxide is 1:3.125.

[0131] formulation The adjuvant or immunogenic composition may be administered via a variety of suitable routes, including parenteral routes such as intramuscular or subcutaneous administration. In one embodiment, the adjuvant composition is suitable for intramuscular administration. In one embodiment, the adjuvant composition is suitable for subcutaneous administration. In one embodiment, the adjuvant composition is suitable for intranasal administration.

[0132] In one embodiment, the immunogenic composition or vaccine of the present invention is administered by intramuscular delivery route. Intramuscular administration can be to the thigh or upper arm. Injection is typically via a needle (e.g., a hypodermic needle). A typical intramuscular dose is 0.5 mL.

[0133] The adjuvant composition can be administered together with the antigen. In embodiments in which the adjuvant composition is administered in combination with the antigen, the antigen can be administered separately from the adjuvant composition or can be administered in the same composition as the adjuvant composition. The antigen can also be administered via a different route than the adjuvant composition when administered separately and simultaneously. In one embodiment, the adjuvant composition is in aqueous form. In one embodiment, the adjuvant composition is in a non-aqueous form.

[0134] The pH of the adjuvant composition can be adjusted in view of the ingredients and the necessary suitability for administration to a subject. In one embodiment, the pH of the adjuvant composition is at least 4, at least 5, at least 5.5, at least 5.8, at least 6. The pH of the adjuvant composition can be less than 9, less than 8, less than 7.5, or less than 7.

[0135] In one embodiment, the pH of the adjuvant composition is 4-9, 5-8, for example 5.5-8. In a further embodiment, a buffering agent is added to the formulation. For parenteral administration, it is well known that the solution should have a pharma- ceutically acceptable osmolality to avoid cell deformation or lysis. Pharmaceutically acceptable osmolality would generally mean that the solution has an osmolality that is approximately isotonic or slightly hypertonic. Suitably, the adjuvant composition has an osmolality in the range of 250-750 mOsm / kg, for example, the osmolality may be in the range of 250-550 mOsm / kg, for example, in the range of 280-500 mOsm / kg.

[0136] Osmolality can be measured according to techniques known in the art, such as using a commercially available osmometer, for example, Advanced Model 2020 available from Advanced Instruments, Inc. (USA). The desired osmolality can be achieved by including salts or through the use of non-ionic tonicity agents. In one embodiment, suitable non-ionic tonicity agents are polyols, sugars (especially sucrose, fructose, dextrose or glucose) or amino acids such as glycine. In one embodiment, the polyol is a sugar alcohol, particularly C 3-6 Sugar alcohol. Exemplary sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, sorbitol, mannitol, dulcitol and iditol. In a specific embodiment of this embodiment, the preferred non-ionic tonicity agent is sorbitol. In a particular embodiment, the non-ionic tonicity agent in the formulation is sucrose and / or sorbitol. The parenteral composition is preferably sterile.

[0137] In one embodiment, the adjuvant composition is in the form of a parenteral dosage form suitable for administration in the form of sterile or sterilizable injectable solutions, suspensions, dry and / or lyophilized products (reconstitutable powders) and emulsions that are immediately dissolved or suspended in a pharma- ceutically acceptable vehicle for injection.Vehicles used in such dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles, such as, but are not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles, such as, but are not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles, such as, but are not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0138] antigen The adjuvant composition can be administered in combination with the antigen, which can be formulated separately or in the same formulation (i.e., the antigen can be administered as part of the same formulation or as a separate formulation).

[0139] The term "antigen" refers to any molecule capable of eliciting an immune response in a human or animal body. The immune response is a protective immune response, e.g., partially or completely reducing the severity of one or more symptoms and / or the time that one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge, and / or slowing the progression of an associated disease (e.g., prolonging survival).

[0140] The antigen may be a whole organism, a protein / polypeptide, a polysaccharide, a peptide, a protein-polysaccharide conjugate, or a hapten capable of eliciting an immune response in a human or animal body, and each of these types of antigens, or any combination of two or more thereof, is specifically considered as a possible antigen in specific embodiments of the adjuvant composition. In this sense, the terms "protein" and "polypeptide" are synonymous and interchangeable.

[0141] The immune response may be against a pathogen, such as, for example, a virus, a bacterium, a parasite, or a fungus. Thus, in one embodiment, the antigen is derived from a human pathogen. In one embodiment, the antigen is derived from a human pathogen selected from the group consisting of a bacterium, a virus, a fungus, a parasitic microorganism, and a multicellular parasite. In one embodiment, the antigen is derived from a combination of two or more bacteria, viruses, fungi, parasitic microorganisms, and multicellular parasites.

[0142] Alternatively, the antigen may be derived from a tumor cell (ie, the antigen may be a tumor-associated antigen) and the adjuvant composition may be useful for immunotherapeutic treatment of cancer.

[0143] In this sense, an "antigen derived from an organism" specifically includes the organism as a whole (such as a whole organism, e.g., a whole virus or bacterium), or one or more molecules derived only from the organism. The antigen can be a naturally occurring whole organism, and one or more molecules derived from the organism, e.g., one or more polypeptides, can be isolated and purified from such a naturally occurring whole organism.

[0144] Alternatively, antigens can be artificially generated, for example, by recombinant technology or by chemical synthesis. Such recombinant antigens can be in wild-type form, i.e., their nucleotide or amino acid sequences are identical to the sequences of the corresponding antigens derived from naturally occurring whole organisms. Alternatively, said recombinant antigens can advantageously contain one or more mutations, i.e., their nucleotide or amino acid sequences contain one or more mutations when compared to the sequences of the corresponding wild-type antigens. Whole organisms can be live attenuated or killed / inactivated. Inactivation processes using physical and / or chemical means are known to those skilled in the art. Such recombinant / modified / designed antigens are considered to fall within the definition of "derived from" an organism within the context of this disclosure.

[0145] In some embodiments, the antigen comprises at least one B or T cell epitope, and the antigen comprises B and T cell epitopes. The immune response generated can be an antigen-specific B cell response that generates neutralizing antibodies. The immune response generated can be an antigen-specific T cell response that can be a systemic and / or local response. An antigen-specific T cell response is a CD4+ T cell response that expresses multiple cytokines, e.g., IFNγ, TNFα, and / or IL2. + Responses involving T cells, such as CD4 + Alternatively, or in addition, the antigen-specific T cell response may include CD8 T cells expressing multiple cytokines, e.g., IFNγ, TNFα, and / or IL2. + Responses involving T cells, such as CD8 + Including T cell responses.

[0146] Suitably the encoded antigen comprises 3000 or less residues, particularly 2000 or less residues, especially 1500 or less residues. The encoded antigen may comprise 1000 or less residues, 800 or less residues, 600 or less residues, 400 or less residues or 200 or less residues. Suitably the antigen comprises 50 or more residues, particularly 100 or more residues, especially 150 or more residues. Suitably, the antigen comprises from 50 to 3000 residues, particularly from 100 to 1500 residues, especially from 200 to 1000 residues.

[0147] Viral antigens The antigen used in or with the adjuvant composition can be derived from a virus.Thus, in certain embodiments, the antigen is derived from a virus.In particular, the antigen can be a whole virus.The whole virus can be live attenuated or killed / inactivated.Alternatively, the antigen can be a polypeptide derived from a virus.

[0148] Suitable viruses include those from the Orthomyxoviridae family, such as influenza virus, Paramyxoviridae, such as respiratory syncytial virus (RSV), mumps virus or measles, Togaviridae, such as rubella virus, Papovaviridae, such as human papillomavirus (HPV), Herpesviridae, such as herpes simplex virus (HSV), human cytomegalovirus (HCMV), Epstein-Barr virus (EBV) or varicella zoster virus (ZVZ), Picornaviridae, such as rabies virus (HBV), ... The antigen is derived from the family of viruses, such as Enterovirus, Rhinovirus, Poliovirus, Flaviviridae, such as Dengue virus or Hepatitis C virus (HCV), Hepadnaviridae, such as Hepatitis B virus (HBV), Retroviridae, such as Human Immunodeficiency Virus (HIV), Reoviridae, such as Rotavirus, Rhabdoviridae, such as Rabies virus, or Filoviridae, such as Ebola virus. In one embodiment, the antigen used in or with the adjuvant composition is derived from a virus selected from the group consisting of influenza virus, RSV, HPV, measles virus, rubella virus, mumps virus, HCMV, VZV, dengue virus, poliovirus, HIV, HBV, Ebola virus, and rotavirus, or any combination of two or more thereof.

[0149] In a particular embodiment, the antigen is derived from HCMV. Preferably, the HCMV antigen is glycoprotein gB, which may lack the transmembrane domain (as disclosed in EP 0802979), optionally combined with one or more of the HCMV proteins pp65, IE1, pUL131, gL, gH, pUL128, and pUL130. Preferably, the HCMV antigen is a combination of gB, gL, gH, pUL131, pUL128, and pUL130. Alternatively, the HCMV antigen is a combination of gL, gH, pUL131, pUL128, and pUL130.

[0150] In one embodiment, the antigen is a Varicella Zoster Virus (VZV) antigen. In one embodiment, the VZV antigen is a gE antigen. Suitably, the VZV antigen is glycoprotein gE, which may be deleted from its transmembrane domain, as disclosed in EP 0 405 867 B1. In one embodiment, the VZV antigen is a protein from Varicella Zoster Virus, one of gpI, gpII or gpIII, as defined on pages 5-7 of EP 0 405 867 B1, deleted from 4-20 percent of the total amino acid residues of the full-length glycoprotein at the carboxy terminus.

[0151] In a further embodiment, the antigen is derived from RSV. Preferably, the RSV antigen is a polypeptide selected from the group consisting of fusion protein (F), junction protein (G), matrix protein (M2) and nucleoprotein (N). Conformationally constrained F polypeptides are particularly suitable as RSV polypeptide antigens to be included in or administered with adjuvant compositions. Conformationally constrained F polypeptides have been previously described in both pre-fusion (PreF) and post-fusion (PostF) conformations. Exemplary F protein antigens conformationally constrained in pre-fusion conformation have been described in the art and are disclosed in detail in, for example, WO 09 / 079796, WO 10 / 149745, WO 11 / 008974 and WO 12 / 158613. Similarly, conformationally constrained F protein antigens in post-fusion conformation are also known in the art and can be used in or administered with adjuvant compositions.Examples of conformationally constrained F protein polypeptides in post-fusion are disclosed in detail in, for example, WO 11 / 008974 and Swanson et al. (PNAS, 2011, Vol. 108: 9619-9624).In certain embodiments, the adjuvant composition comprises an antigen polypeptide from RSV selected from the group consisting of F protein, preF protein, N protein and M2 protein.

[0152] In a further embodiment, the antigen is derived from HBV. Suitably, the antigen is Hepatitis B surface antigen (HBS). In one embodiment, the antigen is derived from an RNA virus, hi one embodiment, the antigen is derived from a coronavirus, in particular SARS-CoV-2.

[0153] Multiple antigens may be encoded. As a result, in some embodiments, the antigen is derived from at least one coronavirus, such as SARS-CoV-2. In some embodiments, the antigen is derived from more than one coronavirus (such as two, three, four or five), such as SARS-CoV-2 (such as multiple SARS-CoV-2 variant antigens).

[0154] SARS-CoV-2 utilizes the densely glycosylated spike (S) protein for entry into host cells. In coronaviruses, the S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes substantial structural rearrangements to fuse the viral membrane with the host cell membrane (Li F. Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu Rev Virol. 2016 Sep 29;3(1):237-261;Bosch BJ, van der Zee R, de Haan CA, Rottier PJ. The coronavirus spike protein is a class I viral fusion protein: structural and functional characterization of the fusion core complex. J Virol. 2003 Aug;77(16):8801-11.).

[0155] Useful coronavirus proteins are fragments or variants of native coronavirus proteins that are capable of raising neutralizing antibodies and / or T cell responses (such as CD4 or CD8 T cell responses) against the coronavirus, preferably protective immune responses.

[0156] Useful SARS-CoV-2 S proteins include, for example consist of, fragments or variants of native SARS-CoV-2 S protein capable of eliciting a neutralizing antibody and / or T cell response (such as a CD4 or CD8 T cell response), preferably a protective immune response, against SARS-CoV-2.

[0157] The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, a full-length S protein (such as SEQ ID NO: 1). Alternatively, the encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 1. The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 95% identity to the amino acid sequence shown in SEQ ID NO: 1, particularly at least 98% identity to the amino acid sequence shown in SEQ ID NO: 1, particularly at least 99% identity to the amino acid sequence shown in SEQ ID NO: 1, e.g. 100% identity to the amino acid sequence shown in SEQ ID NO: 1.

[0158] The encoded SARS-CoV-2 S protein can comprise or consist of one or more domains of the full-length SARS-CoV-2 S protein, such as the ectodomain (SEQ ID NO:2) or the receptor binding domain (RBD, SEQ ID NO:3), or a variant thereof.

[0159] The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 2. The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 2, particularly at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 2, particularly at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2, e.g. 100% identity to the amino acid sequence set forth in SEQ ID NO: 2.

[0160] The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 3. The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 3, particularly at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 3, particularly at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 3, e.g. 100% identity to the amino acid sequence set forth in SEQ ID NO: 3.

[0161] Advantageously, the encoded SARS-CoV-2 S protein is stabilized pre-fusion to facilitate proper presentation to the immune system. For example, Wrapp and colleagues (Wrapp et al., Science 367, 1260-1263 (2020)) have generated recombinant pre-fusion S ectodomains using a stabilization strategy that has proven effective for other betacoronavirus S proteins (Pallesen et al., Proc Natl Acad Sci US A. 2017 Aug 29;114(35):E7348-E7357; Kirchdoerfer, RN et al. Sci Rep 8, 15701 (2018)). For this purpose, starting from the SARS-CoV-2 polynucleotide sequence (GenBank accession number MN908947.3), a gene encoding residues 1 to 1208 of the SARS-CoV-2 S protein (UniProt accession number P0DTC2 version 1, dated 22 April 2020) containing proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site (residues 682 to 685), a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrep tag as well as an 8xHis tag was synthesized and cloned into the mammalian expression vector pαH.

[0162] Residues 1-1208 of the SARS-CoV-2 S protein, including proline substitutions at residues 986 and 987 and a "GSAS" substitution at the furin cleavage site, are provided in SEQ ID NO:4 and are an example of a prefusion stabilized ectodomain of the SARS-CoV-2 S protein.

[0163] The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 4. The encoded SARS-CoV-2 S protein can comprise, e.g. consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 4, particularly at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 4, particularly at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, e.g. 100% identity to the amino acid sequence set forth in SEQ ID NO: 4. Suitably, the SARS-CoV-2 S protein is a pre-fusion stabilized protein.

[0164] In one embodiment, the SARS-CoV-2 S protein is a stabilized recombinant pre-fusion S ectodomain as disclosed by Wrapp et al., Science 367, 1260-1263 (2020).

[0165] The SARS-CoV-2 S protein (such as a prefusion stabilized SARS-CoV-2 S protein) may desirably be in a trimer form and, as a result, may include a trimerization motif, such as a T4 fibritin trimerization motif, more preferably a C-terminal T4 fibritin trimerization motif. Alternative trimerization motifs include collagen-derived domains referred to as "trimerization tags," such as those disclosed in Liu et al., 2017, or molecular clamps, such as those disclosed in WO 2018 / 176103.

[0166] The encoded SARS-CoV-2 S protein is desirably 1800 residues or less in length, particularly 1500 residues or less in length, especially 1400 residues or less in length, for example 1300 residues or less in length.

[0167] The encoded SARS-CoV-2 S protein is desirably at least 150 residues long, particularly at least 200 residues long, in particular at least 400 residues long, for example at least 600 residues long. In one embodiment, the antigen is a human cytomegalovirus (CMV) antigen. In one embodiment, the antigen is a Zika virus antigen. In one embodiment, the antigen is a human parainfluenza virus (PIV) antigen, such as a human PIV type 3 antigen. In one embodiment, the antigen is a human metapneumovirus (hMPV) antigen. In one embodiment, the antigen is a respiratory syncytial virus (RSV) antigen. In one embodiment, the antigen is an influenza virus antigen, such as hemagglutinin or neuraminidase. In one embodiment, the antigen is an Epstein-Barr Virus (EBV) antigen.

[0168] In one embodiment, the antigen is a herpes simplex virus (HSV) antigen, such as gE and / or gI antigen. Suitable antigens are disclosed in WO2021 / 013798. In one embodiment, the antigen comprises (i) HSV2 gE antigen or HSV1 gE antigen, such as as defined in FIG. 1 of WO2021 / 013798, and / or (ii) HSV2 gI or HSV2 gI antigen, such as as defined in FIG. 2 of WO2021 / 013798. In a particular embodiment, the antigen comprises a HSV2 gE and gI heterodimer or an immunogenic fragment thereof.

[0169] bacterial antigen The antigens used in or with the adjuvant composition can be derived from bacteria. Thus, in certain embodiments, the antigens are derived from bacteria. In one embodiment, the antigens are derived from bacteria selected from the group consisting of B. pertussis, S. pneumoniae, and N. meningitidis, or any combination of two or more thereof.

[0170] The antigen may be a whole bacterium and may be killed / inactivated or live attenuated. A particular whole bacterial antigen for use in the compositions of the invention is Bordetella pertussis. In one embodiment, the Bordetella pertussis antigen is a whole bacterium (Pw antigen), optionally in combination with tetanus toxoid (T) and / or diphtheria toxoid (D). In some embodiments, the adjuvant composition comprises or is administered in combination with Pw, tetanus toxoid and diphtheria toxoid (DTPw). The Pw antigen may be inactivated by several known methods, including mercury-free methods. Such methods include heat, formaldehyde, glutaraldehyde, acetone-I, or acetone-IO inactivation (see, e.g., Gupta et al., 1987, j. Biol. Stand. 15:87; Gupta et al., 1986, Vaccine, 4:185). Methods for preparing inactivated Pw antigens suitable for use in formulations are disclosed in WO 93 / 24148. In one embodiment of the Pw antigen-containing adjuvant composition of the present disclosure, the Pw component of the formulation produces reduced reactogenicity. The reactogenicity of Pw vaccines is primarily caused by lipooligosaccharide ("LOS"), an endotoxin derived from the bacterial outer membrane. The lipid A portion of LOS is primarily responsible for reactogenicity. To produce a Pw antigen-containing vaccine with relatively low reactogenicity (compared to "conventional" Pw vaccines, such as those produced by the inactivation procedures described above), endotoxins can be genetically or chemically detoxified and / or extracted from the outer membrane. In one embodiment, the Bordetella pertussis antigen used in or with the adjuvant composition comprises a "low reactogenic" Pw antigen from which the LOS has been genetically or chemically detoxified and / or extracted. For example, the Pw antigen can be subjected to treatment with a mixture of an organic solvent, such as butanol, and water, as described in WO 06 / 002502 and Dias et al. (Human Vaccines & Immunotherapeutics, 2012, 9(2):339-348).

[0171] In an alternative embodiment, "low reactogenicity" is achieved by deriving the Pw antigen from a strain of Bordetella pertussis that has been genetically engineered to produce a less toxic LOS. WO 06 / 065139 discloses genetic 3-O-deacylation and detoxification of Bordetella pertussis LOS resulting in a strain that contains at least partially 3-O-deacylated LOS. The Bordetella pertussis antigen used in or with the adjuvant composition may thus be a Pw antigen derived from a strain of Bordetella pertussis that has been genetically engineered to express a lipid A modifying enzyme such as de-O-acylase. In particular, such strains may express the 3-O-deacylase PagL as described in WO 06 / 065139, and in Geurtsen et al. (Infection and Immunity, 2006, 74(10):5574-5585) and Geurtsen et al. (Microbes and Infection, 2007, 9:1096-1103). Alternatively or additionally, the strain from which the Pw antigen is derived may naturally or as a result of genetic engineering lack the ability to modify its lipid A phosphate group with glucosamine and express LOS species that have a lipid A diglucosamine backbone substituted at the C-3' position by C10-OH or C12-OH and / or lack a terminal heptose. Such a strain, 18-323, is disclosed in Marr et al. (The Journal of Infectious Diseases, 2010, 202(12): 1897-1906).

[0172] Further specific bacterial antigens for use in or with the adjuvant composition may be derived from Streptococcus pneumoniae. At least one streptococcal protein and / or at least one streptococcal capsular saccharide, optionally conjugated to a carrier protein, may suitably be included as an antigen in the adjuvant composition or administered with such a formulation. Suitable protein and saccharide antigens from pneumococcus are described in WO 14 / 060385. In some embodiments, the at least one pneumococcal protein is selected from the group consisting of polyhistidine triad family (PhtX), choline binding protein family (CbpX), CbpX truncations, LytX (autolysate) family, LytX truncations, CbpX truncation-LytX truncation chimeric proteins, PcpA (Pneumococcal choline binding protein A), PspA (Pneumococcal surface protein A), PsaA (Pneumococcal surface adhesion protein A), Sp128 (Pneumococcal 128), Sp101 (Pneumococcal 101), Sp130 (Pneumococcal 130), SP125 (Pneumococcal 125) and SP133 (Pneumococcal 133).

[0173] In one embodiment the adjuvant composition comprises or is administered with one or more (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) pneumococcal capsular saccharides, optionally conjugated to a carrier protein. In a particular embodiment the one or more pneumococcal capsular saccharides included in or administered with the adjuvant composition, optionally conjugated to a carrier protein, comprise a saccharide from a serotype selected from the following serotypes: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F or 33F.

[0174] The term "saccharide" can refer to polysaccharides or oligosaccharides, including both. Polysaccharides can be isolated from bacteria and sized to some extent by known methods (see, for example, EP 0497524 and EP 0497525) and optionally by microfluidization. Polysaccharides can be sized to reduce viscosity in polysaccharide samples and / or to improve filterability for the conjugated product. The term "conjugate" refers to capsular saccharides covalently bound to a carrier protein. The carrier protein can be any peptide or protein. Suitable carrier proteins are described in WO 14 / 060385. Carrier proteins include tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin, diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin, e.g., CRM176, CRM228, CRM 45; CRM 9, CRM 45, CRM102, CRM103 and CRM107 (CRM stands for cross-reactive substance), pneumococcal pneumolysin, OMPC (outer membrane protein C), heat shock proteins, pertussis proteins, cytokines, lymphokines, growth factors or hormones, multiple human CD4 antigens from various pathogen-derived antigens, +The engineered protein comprising a T cell epitope may be, for example, N19 protein, pneumococcal surface protein PspA, iron uptake protein, C. difficile toxin A or B, H. influenzae protein, Pneumococcal PhtA (polyhistidine triad protein A), Pneumococcal PhtD (polyhistidine triad protein D), Pneumococcal PhtB (polyhistidine triad protein B), or PhtE (polyhistidine triad protein E). In one embodiment, the at least one pneumococcal capsular saccharide conjugate is conjugated to a carrier protein selected from the group consisting of tetanus toxoid (TT), fragment C of TT, diphtheria toxoid, CRM197 (cross-reactive substance 197), detoxified pneumolysin, protein D (from H. influenzae), PhtD, PhtDE and N19. The saccharide may be linked to the carrier protein by any known method. A further particular bacterial antigen for use in the present disclosure is derived from Neisseria meningitidis. In some embodiments the antigen is a meningococcal capsular saccharide from a serogroup selected from the group consisting of serogroup A (MenA), serogroup C (MenC), serogroup Y (MenY) and serogroup W-135 (MenW), or any combination of two or more thereof, optionally conjugated to a carrier protein. In practice these saccharides may suitably be conjugated to any of the carrier proteins described above in relation to the streptococcal saccharides. In some embodiments the antigen is meningococcal serogroup A capsular saccharide (MenA), meningococcal serogroup C capsular saccharide (MenC), meningococcal serogroup Y capsular saccharide (MenY) and meningococcal serogroup W-135 capsular saccharide (MenW), optionally conjugated to carrier protein CRM197 or carrier protein TT.

[0175] Further specific bacterial antigens from Neisseria meningitidis for use herein are derived from Neisseria meningitidis serogroup B ("MenB"). Antigens suitable for generating an anti-MenB response include polypeptides, lipooligosaccharides and / or membrane vesicles. The adjuvant composition may comprise or be administered with one or more serogroup B Neisseria meningitidis polypeptide antigens. In some embodiments, the antigen is a Neisseria meningitidis serogroup B polypeptide selected from the group consisting of NadA protein (also known as protein "961"), NHBA protein (also known as protein "287"), fHBP protein (also known as protein "741"), GNA1030 protein (also known as protein "953"), and GNA2091 protein (also known as protein "936"), optionally in combination with a Neisseria meningitidis serogroup B derived OMV, or any combination of two or more thereof. These antigens will usefully be present as purified, e.g., recombinant, polypeptides. Suitable forms of these antigens are described in WO 04 / 032958. The five antigens can be present in the formulation as five separate proteins, or preferably at least two of the antigens are expressed as a single polypeptide chain (a "hybrid" protein), such that the five antigens form fewer than five polypeptides, for example as described in WO 04 / 032958. In some embodiments, the adjuvant composition comprises or is administered with at least NadA, NHBA, fHBP, GNA1030 and GNA2091 proteins. In certain embodiments, the adjuvant composition comprises or is administered with SEQ ID NO:2 and SEQ ID NO:6, as disclosed in WO 04 / 032958. In further embodiments, the adjuvant composition comprises or is administered with N. meningitidis serogroup B derived OMV, as described below.

[0176] Further specific bacterial antigens are outer membrane vesicles (OMVs). These include any protein-liposome vesicles obtained by disruption or budding of the outer membrane to form vesicles containing the protein components of the outer membrane therefrom. Gram-negative bacteria such as Neisseria secrete OMVs during active growth. The main immunogenic components of OMVs are outer membrane proteins (OMPs) and membrane-bound lipopolysaccharide (LPS). OMVs can be prepared from any Gram-negative bacteria, including pathogenic Neisserial bacteria such as Neisseria gonorrhoea and Neisseria meningitidis. The OMV approach is particularly useful for Neisseria meningitidis serogroup B, as its polysaccharide capsule is hardly immunogenic. Thus, in some embodiments, the adjuvant composition comprises or is administered with OMVs from Neisseria meningitidis serogroup B strains, optionally in combination with any of the above serogroup B meningitidis polypeptide antigens. OMVs are artificially prepared from bacteria and can be prepared using detergent treatment (using deoxycholate) or by non-detergent means, for example as described in WO 12 / 020326.

[0177] parasite antigen The antigen used in or administered with the adjuvant composition can be derived from a parasite. In some embodiments, the antigen can be derived from a parasite that causes malaria. Thus, in some embodiments, the antigen administered in or with the adjuvant composition is derived from a parasite that causes malaria, such as, for example, Plasmodium falciparum or Plasmodium vivax. Suitably, the Plasmodium falciparum derived antigen is RTS,S. As disclosed in WO 93 / 10152, RTS,S is a hybrid protein consisting of the C-terminal portion of the circumsporozoite (CS) protein of Plasmodium falciparum linked to the surface (S) antigen of the Hepatitis B virus via four amino acids of the preS2 portion of the Hepatitis B surface antigen.

[0178] Tumor-associated antigens The antigen in the immunogenic composition or administered primarily with the adjuvant composition may be a tumor-associated antigen. Suitably, the antigen may be a tumor rejection antigen, such as for prostate cancer, breast cancer, colorectal cancer, lung cancer, pancreatic cancer, rectal cancer or melanoma. Exemplary non-limiting antigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens, such as those disclosed in WO 99 / 40188.

[0179] Additional antigens The compositions of the invention can include multiple antigenic components, for example to generate a broad immune response against one pathogen or to generate a response against multiple pathogens. As a result, more than one antigen can be present. Polysaccharides, such as polysaccharide conjugates, can also be present.

[0180] Dosage The therapeutically effective amount will vary depending, among other things, on the applicable disease, the severity of the disease, the age and relative health of the subject, the potency of the compound administered, the mode of administration, and the treatment desired. In certain embodiments, with regard to the daily dosage of the STING agonist of formula (I), it has been shown that satisfactory results are obtained systemically at a daily dose of about 0.03-2.5 mg / kg body weight.

[0181] The amount of conjugated antigen in each immunogenic composition or vaccine dose is selected as an amount that induces an immunoprotective response without significant adverse side effects in a typical vaccine. Such amounts will vary depending on which specific immunogen is utilized and how it is presented. The content of each protein antigen will typically be in the range of 1-200 μg, preferably 1-100 μg, preferably 5-50 μg. The content of each saccharide antigen will typically be in the range of 0.1-50 μg, preferably 0.1-10 μg, preferably 1-5 μg.

[0182] Doses in volumes suitable for human use are generally 0.25-1.5 mL, although lower volumes of 0.05 mL-0.2 mL can be used for administration to the skin. In one embodiment, the human dose is 0.5 mL. In a further embodiment, the human dose is greater than 0.5 mL, for example 0.6, 0.7, 0.8, 0.9 or 1 mL. In a further embodiment, the human dose is 1 mL-1.5 mL. In another embodiment, the human dose can be less than 0.5 mL, such as 0.25-0.5 mL, particularly when the immunogenic composition is for a pediatric population.

[0183] In certain embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 0.5-250 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 1.0-100 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 1.0 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 10.0 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 25.0 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 50.0 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 75.0 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist of formula (I) in an amount of 100.0 μg per dose.

[0184] In certain embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 50-500 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 100-400 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 50 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 75 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 100 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 125 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 150 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 175 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 200 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 225 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 250 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 275 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 300 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 325 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 350 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 375 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 400 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 425 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 450 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 475 μg per dose. In some embodiments, the adjuvant composition comprises aluminum hydroxide in an amount of 500 μg per dose.

[0185] In one embodiment, the adjuvant composition comprises a STING agonist of formula (I) (e.g., 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate, compound 1) in an amount of 0.5-250 μg per dose and aluminum hydroxide in an amount of 50-500 μg per dose. In some embodiments, the adjuvant composition comprises a STING agonist compound 1 in an amount of 1.0-100 μg per dose and aluminum hydroxide in an amount of 50-500 μg per dose (e.g., 100-400 μg per dose).

[0186] In one embodiment, the adjuvant composition comprises STING agonist compound 1 in an amount of 1.0 μg per dose and aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises STING agonist compound 1 in an amount of 25 μg per dose and aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises STING agonist compound 1 in an amount of 50 μg per dose and aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises STING agonist compound 1 in an amount of 75 μg per dose and aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises STING agonist compound 1 in an amount of 100 μg per dose and aluminum hydroxide in an amount of 375 μg per dose.

[0187] In one embodiment, the adjuvant composition comprises a STING agonist (e.g., compound 1) in an amount of 1-50 μg per dose, and aluminum hydroxide in an amount of 100-400 μg per dose.

[0188] In one embodiment, the adjuvant composition comprises a STING agonist in an amount of 3.1 μg per dose and comprises aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises a STING agonist in an amount of 6.3 μg per dose and comprises aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises a STING agonist in an amount of 12.5 μg per dose and comprises aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises a STING agonist in an amount of 25 μg per dose and comprises aluminum hydroxide in an amount of 375 μg per dose. In one embodiment, the adjuvant composition comprises a STING agonist in an amount of 50 μg per dose and comprises aluminum hydroxide in an amount of 375 μg per dose.

[0189] In one embodiment, the adjuvant composition is included in an immunogenic composition comprising an antigen. In one embodiment, the antigen is an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6.

[0190] In one embodiment, the immunogenic composition comprises a STING agonist in an amount of 3.1 μg per dose, aluminum hydroxide in an amount of 375 μg per dose, and an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6 in an amount of 80 μg per dose.

[0191] In one embodiment, the immunogenic composition comprises a STING agonist in an amount of 6.3 μg per dose, aluminum hydroxide in an amount of 375 μg per dose, and an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6 in an amount of 80 μg per dose.

[0192] In one embodiment, the immunogenic composition comprises a STING agonist in an amount of 12.5 μg per dose, aluminum hydroxide in an amount of 375 μg per dose, and an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6 in an amount of 80 μg per dose.

[0193] In one embodiment, the immunogenic composition comprises a STING agonist in an amount of 25 μg per dose, aluminum hydroxide in an amount of 375 μg per dose, and an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6 in an amount of 80 μg per dose.

[0194] In one embodiment, the immunogenic composition comprises a STING agonist in an amount of 50 μg per dose, aluminum hydroxide in an amount of 375 μg per dose, and an HSV2 gE-gl heterodimer of HSV2 gE (ectodomain) having the amino acid sequence set forth in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence set forth in SEQ ID NO:6 in an amount of 80 μg per dose.

[0195] How to use The adjuvant compositions described herein can be used in therapy (e.g., as a medicament). In one embodiment, the adjuvant composition is for use in a method of immunizing a host comprising administering to the host the adjuvant composition and the vaccine composition.

[0196] The adjuvant compositions described herein can be used in combination with a vaccine to improve the immunogenicity of the vaccine, or can be used as a vaccine when an antigen is included in the adjuvant composition.

[0197] In one aspect, there is provided a method of adjuvanting (i.e., improving / enhancing) an immune response in a subject comprising administering an adjuvant composition described herein. The term "enhance" or "enhancing," as used herein, means to increase or prolong the potency or duration of a desired effect.

[0198] In one embodiment, the immunogenic composition of the present disclosure provides increased antibody titers (i.e., amounts of antibodies) against one or more antigens when compared to the antibody titers of the immunogenic composition in the absence of the adjuvant composition.

[0199] In one embodiment, the immunogenic composition of the present disclosure exhibits increased CD4 expression against one or more antigens provided by the composition compared to the immunogenic composition in the absence of the adjuvant composition. + Provides a T cell response.

[0200] In one embodiment, a method is provided for enhancing an immune response in a host comprising administering to a subject an effective amount of an adjuvant composition described herein and one or more antigens, wherein the composition provides an increased amount of antibody titer (i.e., amount of antibody) against one or more antigens when compared to the antibody titer of the immunogenic composition in the absence of the adjuvant composition.

[0201] In one embodiment, there is provided a method of enhancing an immune response in a host comprising administering to a host an effective amount of an adjuvant composition described herein and one or more antigens, wherein the composition provides an enhanced antibody response to the one or more antigens when compared to the antibody response in the absence of the adjuvant composition described herein.

[0202] In one embodiment, a method for detecting CD4+ deficiency in a host comprising administering to the host an effective amount of an adjuvant composition described herein and one or more antigens, ... + Compared with T cell responses, the host's CD4 + Methods for enhancing T cell polyfunctionality are provided. In one embodiment, enhanced CD4 + T cell polyfunctionality is defined as the expression of T cells in which the host expresses at least three cytokines. + It refers to the ability to generate T cells. In one embodiment, the at least three cytokines are selected from INFγ+IL2+TNF+.

[0203] In one embodiment, a method for the treatment of CD4+ deficiency in a host is provided, comprising administering to the host an effective amount of an adjuvant composition described herein and one or more antigens. + A method for enhancing T cell polyfunctionality is provided, wherein CD4 + At least 40% of the T cells produce at least INFγ+IL2+TNF+ or INFγ+IL13+TNF+. + At least 50% of the T cells produce INFγ+IL2+TNF+. In some embodiments, CD4 + At least 50% of T cells produce INFγ+IL13+TNF+.

[0204] In one embodiment there is provided the use of an adjuvant composition described herein in the manufacture of a medicament for adjuvanting an immune response in a subject. In one embodiment, there is provided a combination of the adjuvant composition described herein with a vaccine formulation comprising an antigen. In one embodiment, a kit is provided that comprises (i) an adjuvant composition described herein, and an antigen.

[0205] The present disclosure also provides a delivery device pre-filled with the adjuvant composition described herein.A sterile container (e.g., a vial) containing the adjuvant composition described herein (e.g., containing a unit dose or a plurality of unit doses) is also provided.The present disclosure also provides a sealed container containing the adjuvant or immunogenic composition disclosed herein. EXAMPLES

[0206] The present invention will now be illustrated by the following non-limiting examples. Although specific embodiments of the present invention are described below, those skilled in the art will understand that various changes or modifications can be made. Reference to preparations carried out in a similar manner to other preparations, or in their general manner, can include variations in routine parameters such as minor changes in time, temperature, test conditions, and amounts of reagents.

[0207] Reference example: Synthesis of STING agonists The STING agonists used in the present invention can be prepared using the methods disclosed in WO 2017 / 175147 (International Patent Application No. PCT / IB2017 / 051945), which can be readily adapted using the knowledge of a trained organic chemist to prepare other compounds of the present invention. For example, (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide can be prepared according to Example 10 of WO 2017 / 175147. (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide can be prepared according to Example 13 of WO 2017 / 175147.

[0208] 3-(((E)-6-Carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate can be prepared according to Example 19 of WO 2017 / 175147. (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazole-5-carboxamide can be prepared according to Example 39 of WO 2017 / 175147. (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide can be prepared according to Example 43 of WO 2017 / 175147. (E)-4-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)butanoic acid can be prepared according to Example 52 of WO 2017 / 175147.

[0209] Example 1: Preformulation Study Exemplary immunogenic compositions of the present invention were tested to evaluate (i) the compatibility between the antigen and the STING agonist, and (ii) the adsorption of the antigen and the STING agonist to alum, respectively. The HSV2 gE-gl heterodimer tested here consisted of HSV2 gE (ectodomain) having the amino acid sequence shown in SEQ ID NO:5 associated in a non-covalent complex with HSV2 gl (ectodomain) having the amino acid sequence shown in SEQ ID NO:6. The alum used was aluminum hydroxide (Al(OH)3). The STING agonist used was 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate (hereinafter referred to as Compound 1):

[0210] [ka]

[0211] [Table 1]

[0212] The compatibility between the antigen and the STING agonist was evaluated by determining the amount of STING agonist recovered (using HPLC) in the supernatant after centrifugation of the formulations described in groups 2, 3 and 4 above on the one hand, and by determining the amount and profile of the antigen recovered (using HPLC) in the supernatant after centrifugation of the formulations described in groups 2, 3 and 4 above on the other hand. The adsorption of STING agonist and antigen on Al(OH)3 was similarly evaluated by determining the amount of STING agonist and the amount and profile of antigen recovered in the supernatant after centrifugation of the formulations described in groups 5, 6, 7 and 8 above, respectively. The concentration of HSV-2 gE-gI antigen was 20 μg / mL in each group. The exact methodology for preparing the STING agonist and Al(OH)3 formulations was as follows: a solution of Al(OH)3 in water for injection was prepared, the required amount of STING agonist was added, and the required amount of HSV-2 gE-gI was added to achieve 20 μg / mL in the final solution. The solution was then mixed for 30 minutes at room temperature. NaCl was added and the mixture was mixed for 5 minutes.

[0213] result Considering groups 2, 3 and 4 (STING agonist in aqueous formulation without added Al(OH)3), the same amount of STING agonist was found in the uncentrifuged samples and in the supernatant of the centrifuged samples, indicating that no aggregation or precipitation occurred in the presence of antigen. Similarly, the same profile of gE-gI was observed for the uncentrifuged samples and in the supernatant of the centrifuged samples by both SDS PAGE and SEC HPLC, indicating that no aggregation or precipitation occurred in the presence of STING agonist.

[0214] Considering groups 5, 6, 7 and 8 (STING agonist + Al(OH)3), after centrifugation of the samples, no STING agonist was found in the supernatant, indicating that the entire amount of STING agonist was adsorbed to Al(OH)3 in the presence of antigen. Similarly, for groups 5, 6 and 7, after centrifugation of the samples, no antigen was detected in the supernatant, indicating that the entire amount of antigen was adsorbed to Al(OH)3 in the presence of STING agonist. Only the group containing a relatively small amount of Al(OH)3 (group 8) showed reduced adsorption (30% of the antigen was adsorbed).

[0215] 1.2 Next, we determined the minimum amount of Al(OH)3 required to completely adsorb the STING agonist. Compound 1 was used. To test different ratios of STING agonist:Al(OH)3, the amount of STING agonist was fixed at 45 μg / mL and Al(OH)3 was added at 0–200 μg / mL (Al 3+ The adsorption of STING agonist was varied with respect to the amount of STING agonist adsorbed. The different formulations (as shown in Table 2) were mixed under stirring for 1 h. The samples were then centrifuged at 8000 rpm for 10 min. The supernatants of the centrifuged samples were then analyzed to determine the amount of unadsorbed STING agonist. Quantification was performed by fluorescence method. The results are shown in Figure 1.

[0216] [Table 2]

[0217] result It has been demonstrated that STING agonists are generally well adsorbed to Al(OH)3. This study indicates that the maximum ratio of STING agonist to aluminum hydroxide that allows optimal adsorption is approximately 1.5. Ratios between 0.45 and 1.5 allow for near-complete adsorption. This indicates that even with relatively high concentrations of STING agonist, very low doses of Al(OH)3 can be used (less than 500 μg typically found with alum-based vaccines).

[0218] Example 2: Immunogenicity assessment of a recombinant HSV-2 gE-gI antigen model adjuvanted with STING agonists and aluminum hydroxide The same recombinantly expressed HSV-2 gE-gI antigen was formulated with different doses of soluble forms of STING agonists alone or with Al(OH)3. Total HSV-2 gE and gI IgG titers, total HSV-1 gE-gI IgG titers, HSV-2 gE and gI CD4 + T cell responses, HSV-1 gE and gI CD4 + T cell responses, as well as antibody functionality, were examined and compared with the same HSV-2 gE-gI antigen adjuvanted with AS01 (a liposome-based adjuvant containing saponin and a TLR4 agonist; Adjuvant system AS01: helping to overcome the challenges of modern vaccines, Expert Rev Vaccines. 2017 Jan;16(1):55-63.).

[0219] Research design: In this study, nine groups of naive female CB6F1 mice (n=8 per group, except for n=4 in group 1 (control)), aged 6-8 weeks at the start of the study, were injected intramuscularly (IM) at 2-week intervals in the left gastrocnemius with 50 μL of unadjuvanted HSV-2 gE-gI protein, or different doses of STING agonist-adjuvanted gE-gI or STING agonist / Al(OH)3 formulations containing HSV-2 gE-gI, or AS01-adjuvanted HSV-2 gE-gI.

[0220] Serum samples were collected 14 days after one or two immunizations (Gp1-Gp9) to assess total anti-HSV-2 gE&gI antibody responses. Cross-reactive HSV-1 gE-gI-specific antibody responses and antibody function (competitive ELISA and mFcγRIV avidity / ADCC-like assays) were assessed only after two immunizations. Spleens were collected 14 days after two immunizations (Gp1-Gp9) to assess anti-HSV-2 gE&gI-specific CD4+ cells expressing IL-2, TNF-α, IFN-γ, IL-13 and / or IL-17 cytokines. + / CD8 + The frequency of T cells was evaluated. The study design and formulation are outlined in the table below. Compound 1 was used as a STING agonist.

[0221] [Table 3]

[0222] 2.1 Detection of total anti-HSV-2 gE and gI IgG antibodies by ELISA Quantification of total HSV-2 gE or gI specific IgG antibodies was performed using indirect ELISA. Recombinant HSV-2 gE (approximately 51 kDa) (BMP1291) or HSV-2 gI protein (approximately 46 kDa) (BMP1292) was used as coating antigen. These proteins were produced using the ExpiHEK293FTM expression system.

[0223] Polystyrene 96-well ELISA plates (Nunc F96 Maxisorp Cat. No. 439454) were coated with 100 μL / well of antigen diluted at 2 μg / mL (HSV-2 gE) and 1 μg / mL (HSV-2 gI) in carbonate / bicarbonate 50 mM pH 9.5 buffer and incubated overnight at 4° C. After incubation, the coating solution was removed and the plates were blocked with 200 μL / well of Difkomilk 10% (blocking buffer) (Becton Dickinson, USA) diluted in PBS for 1 h at 37° C. The blocking solution was removed and 3x (serum 14dPI & 14dPII) serum dilutions (in PBS + 0.1% Tween 20 + 1% BSA buffer) were added to the coated plates and incubated at 37° C. for 1 h. The plates were washed four times with PBS 0.1% Tween20 (washing buffer) and peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L) (Jackson, USA) was used as the secondary antibody. 100 microliters per well of secondary antibody diluted at 1:500 in PBS+0.1% Tween20+1% BSA buffer was added to each well and the plates were incubated for 45 minutes at 37°C. The plates were then washed four times with washing buffer, twice with deionized water and incubated for 10 minutes at RT with 100 μL / well of a solution of 75% single component TMB peroxidase ELISA substrate (Bio-Rad, USA) diluted in sodium citrate 0.1M pH 5.5 buffer. The enzymatic development was stopped with 100 μL per well of 0.4 N sulfuric acid (H2SO4) and the plates were read at an absorbance of 450 / 620 nm using a Versamax ELISA reader.

[0224] Optical density (OD) was obtained and analyzed using SoftMaxPro GxP v5.3 software. A standard curve was generated by applying a four-parameter logistic regression fit to the reference standard results (pools 1.1-1.20 of mice immunized with reference standard anti-HSV-2 gE = 14PIII - 5 μg HSV-2 gE / AS01 / dose; pools 2.1-2.10 of mice immunized with reference standard anti-HSV-2 gI = 14PII - 5 μg HSV-2 gI / AS01 / dose). Antibody titers in samples were calculated by interpolation of the standard curve. Antibody titers of samples were obtained by averaging values ​​from dilutions that fell within the 20-80% dynamic range of the standard curve. To allow for titer comparison, ELISA titers were normalized to the same starting dilution.

[0225] 2.2 Detection of total anti-HSV-1 gE-gI specific IgG antibodies by ELISA Total anti-HSV-1 gE-gI specific IgG antibodies were assessed using an indirect ELISA. Recombinant gE-gI heterodimer protein from HSV-1 (BMP1299) was used as the coating antigen. This protein was purified using ExpiCHO TM It was produced using an expression system.

[0226] Except that 100 μl / well of recombinant HSV-1 gE-gI heterodimer was used to coat polystyrene 96-well ELISA plates, the remainder of the procedure was the same as that described above for quantification of total HSV-2 gE or gI-specific IgG antibodies.

[0227] result All vaccine formulations were immunogenic, as antibody responses were significantly higher than those observed with antigen alone (see Figures 2, 3, and 4). A clear enhancement of antibody responses was observed between the first and second doses of vaccine. No clear dose effect was observed with soluble STINGa or STINGa / Al(OH)3. Furthermore, no significant differences were observed in HSV-2 gE and HSV-2 gI antibody titers between the STINGa / Al(OH)3 ratios tested.

[0228] Comparison between the two STINGa-based vaccines after two immunizations suggests that the STINGa / Al(OH)3 formulation induced similar or higher HSV-2 gE-specific IgG antibody titers than the soluble STINGa formulation (Figure 3). For HSV-2 gI responses, higher antibody titers were observed with the STINGa / Al(OH)3 formulation when compared to the soluble STINGa formulation, with fold increases ranging from 3.3 to 4 (Figure 2).

[0229] The STINGa / Al(OH)3 formulation tended to induce higher HSV-2 gE-specific IgG titers compared to AS01 (Fig. 3). With regard to HSV-2 gI-specific titers, the STINGa formulation induced a similar response to the AS01 formulation (Fig. 2).

[0230] Similar observations were made for anti-HSV-1 gE-gI cross-reactive IgG antibody responses across the dose range (comparison within the two STINGa formulations and with AS01) (see Figure 4).

[0231] 2.3 Anti-HSV-2 and HSV-1 gE and gI CD4 by Intracellular Cytokine Staining (ICS) + Assessment of T cell responses Vaccine-specific CD4 producing IL-2 and / or IFN-γ and / or TNF-α and / or IL-13 and / or IL-17 +The frequency of was assessed in splenocytes harvested 14 days after the second immunization following ex vivo stimulation with HSV-2 gE or gI peptide pools or HSV-1 gE or gI peptide pools.

[0232] Isolation of splenocytes : Spleens were collected from individual mice 14 days after the second immunization and placed in RPMI 1640 medium (=RPMI / additives) supplemented with RPMI supplements (glutamine, penicillin / streptomycin, sodium pyruvate, non-essential amino acids & 2-mercaptoethanol). A cell suspension was prepared from each spleen using a tissue grinder. The splenocyte suspension was passed through a filter (cell strainer 100 μm) followed by rinsing the filter with 35 mL of cold RPMI / additives. After centrifugation (335 g, 10 min at 4° C.), the cells were resuspended in 5 mL of cold RPMI / additives. The splenocyte suspension was passed again through a filter (cell strainer 100 μm) followed by a second washing step as described above and finally the cells were resuspended in 2 mL of RPMI / additives supplemented with 5% FCS. The cell suspension was then diluted 20-fold (10 μL) in PBS buffer (190 μL) for cell counting (using a MACSQuant analyzer). After counting, the cells were centrifuged (335 g, 10 min at RT) and diluted to 10 μL in RPMI / supplements supplemented with 5% FCS. 7 Resuspended at 100 cells / mL.

[0233] Cell preparation Fresh splenocytes were cultured at 10 6 Cells were seeded into round-bottom 96-well plates at 100 μL per well. The cells were then transferred to 100 μL of either: - A 15mer overlapping peptide pool covering the sequence of the gE protein from HSV-2 (1 μg / mL per peptide per well) - A 15mer overlapping peptide pool covering the sequence of the gI protein from HSV-2 (1 μg / mL per peptide per well) - A 15mer overlapping peptide pool covering the sequence of the gE protein from HSV-1 (1 μg / mL per peptide per well) - A 15mer overlapping peptide pool covering the sequence of the gI protein from HSV-1 (1 μg / mL per peptide per well) - 15mer overlapping peptide pools covering the sequence of the human β-actin protein (1 μg / mL per peptide per well) (irrelevant stimuli) - RPMI / supplemented medium (as a negative control for the assay) - PMA - ionomycin solution at working concentrations of 0.25 μg / mL and 2.5 μg / mL, respectively (as positive control for the assay) The cells were stimulated with anti-CD28 (clone 37.51) and anti-CD49d antibodies (clone 9C10 (MFR4.B)) at 1 μg / mL per well containing 100 μg / mL of 100% MFR4.

[0234] After 2 h of ex vivo stimulation, brefeldin (GolgiPlug, BD Bioscience) diluted 1 / 200 in RPMI / additives supplemented with 5% FCS and monensin (BD GolgiStop, BD Bioscience) diluted 1 / 300 in RPMI / additives supplemented with 5% FCS were added for an additional 4 h to inhibit cytokine secretion, and plates were then transferred to 4°C for overnight incubation.

[0235] Intracellular cytokine staining After overnight incubation at 4°C, cells were transferred to a V-bottom 96-well plate, centrifuged (189g, 5 min at 4°C) and washed with 250 μL cold PBS + 1% FCS (flow buffer). After a second centrifugation (189g, 5 min at 4°C), cells were resuspended in 50 μL flow buffer containing anti-CD16 / 32 antibody (clone 2.4G2) diluted 1 / 50 to block non-specific antibody binding (10 min at 4°C). Subsequently, mouse anti-CD4-A700 antibody (clone RM4-5, diluted 1 / 100) and Live / Dead antibody (Clone RM4-5, diluted 1 / 100) were added. TM50 μL of flow buffer containing a fixable near-IR dead cell dye (diluted 1 / 500) was added for 30 min in the dark at 4° C. After incubation, 100 μL of flow buffer was added to each well and the cells were subsequently centrifuged (189 g for 5 min at 4° C.). A second washing step was performed with 200 μL of flow buffer and after centrifugation the cells were fixed and permeabilized by adding 200 μL of Cytofix-Cytoperm solution for 20 min at 4° C. in the dark. After centrifugation of the plates (500g, 5 min at 4°C), the cells were washed with 200 μL Perm / Wash buffer, centrifuged (500g, 5 min at 4°C) and resuspended in 50 μL Perm / Wash buffer containing mouse anti-IL2-FITC (clone JES6-5H4, 1 / 400 dilution), anti-IFN-γ-APC (clone XMG1.2, 1 / 200 dilution) and anti-TNF-α-PE (clone MP6-XT22, 1 / 700 dilution) and anti-IL-13 PeCy7 (clone ebio13A, 1 / 50 dilution) and anti-IL-17 BV605 (clone TC11-18H10, 1 / 100 dilution) antibodies for 1 h at 4°C in the dark. After incubation, 100 μL of Perm / Wash buffer was added to each well, and the cells were then washed a final time with 200 μL of Perm / Wash buffer (centrifugation 500 g, 5 min at 4° C.) and resuspended in 220 μL PBS.

[0236] Cell acquisition and analysis Stained cells were acquired by flow cytometry and analyzed using FlowJo software. Live / Dead staining was used to identify viable cells, followed by lymphocyte isolation based on forward / side scatter (FSC / SSC) gating. Approximately 20,000 CD4 + Acquisition was performed for T cell events. IFN-γ + / - IL-2 + / - TNF-α + / - IL-13 + / - and IL-17 + / - The percentage of CD4 producing cells was +and calculated for the cell population. For each sample, the non-specific signal detected after medium stimulation was subtracted from the specific signal detected after peptide pool stimulation.

[0237] result CD4+ cells expressing at least one of the cytokines IL-2, TNF-α, IFN-γ, IL-13, or IL-17 observed after two injections of HSV-2 gE-gI adjuvanted with soluble STINGa, STINGa / Al(OH)3, and AS01 + The individual frequencies of T cells are shown in FIG.

[0238] No dose-dependence was observed for soluble STINGa, but an inverse dose range was observed with the STINGa / Al(OH)3 formulation, with higher CD4+ T cells at lower STINGa doses (0.74 μg). + T cell responses were observed. In addition, HSV-2 gE and gI CD4 + No significant differences in terms of specific T cells could be observed between the two Al(OH)3 ratios (Figures 5 and 6, respectively).

[0239] Compared to AS01, HSV-2 gE-gI adjuvanted with STINGa (0.74 μg) / Al(OH)3 (5.55 μg) also induced significantly higher HSV-2 gE- and gI-specific CD4 + T cell responses were observed (Figures 5 and 6, respectively). However, no differences were observed between the soluble STINGa preparations and AS01 for any of the antigens tested.

[0240] Finally, with respect to dose range, anti-HSV-1 gI and gE cross-reactive CD4 + Similar observations were made for T cell responses within the two STINGa formulations and compared with AS01 (Figures 7 and 8, respectively).

[0241] 2.4 CD4 + Assessment of T cell polyfunctional profiles Next, we investigated T cell polyfunctionality. CD4 T cells producing one or more cytokines + T cell potency was assessed and T cell profiles were compared between the different formulations. The 0.74μg STINGa dose elicited the highest levels of CD4 + This dose was used for comparison because it induced a T cell response. HSV-2 gE-specific CD4+ T cells expressing one, two, three, or four cytokines after in vitro stimulation were + The percentage of T cells was determined and is shown in Figure 9. The pie chart shows the percentage of total HSV-2 specific CD4 + Cells expressing a single marker among T cells and any combination of IFN-γ, IL-2, TNF-α, IL-13, and IL-17 markers positive CD4 + The mean percentage of T cells is represented.

[0242] result It was observed that the profiles obtained with AS01 and soluble STINGa preparations were similar, with a predominance of double-positive cells. In contrast, with STINGa / Al(OH)3, the proportion of cells expressing the three cytokines was relatively high, which was consistent with the increased CD4+ expression by STINGa / Al(OH)3 compared to soluble STINGa preparations and AS01. + Similar results were obtained for HSV-2 gI (Figure 10), suggesting T cell polyfunctionality.

[0243] Example 3: Evaluation of innate immune responses induced by a STINGa-adjuvanted vaccine To assess whether STINGa / Al(OH)3 formulations reduce possible systemic effects, the kinetics and strength of the systemic innate immune response induced by STINGa formulations (soluble and alum-formulated) were also evaluated. Recombinant gE protein of VZV (SEQ ID NO: 7) was used as the antigen. The data were compared to AS01 formulation. STINGa was compound 1.

[0244] research design Four groups of female 6-8 week old C57BL / 6JOlaHsd mice were injected intramuscularly (IM) into the left gastrocnemius muscle on day 0 with 50 μL / site containing 5 μg VZV gE alone (negative control group), 5 μg VZV gE formulated with either soluble STINGa, STINGa / Al(OH)3 or AS01 (positive control group). To evaluate the kinetics of the innate response induced by the different vaccine formulations, serum was obtained at different time points (3 h, 6 h, 24 h and 48 h) after immunization (n=5 mice / group / time point). For the AS01 group (Gp1), responses were evaluated only at 6 h, corresponding to the peak of the response. To define a baseline for each cytokine measured, 30 mice were bled prior to vaccination, resulting in 5 pools of 6 mice.

[0245] result Innate cytokine responses induced by the two STINGa formulations (soluble and alum-formulated) peaked at 6 hours for the majority of innate markers tested and returned to baseline within 24 or 48 hours (IL-6, IP-10, and IFN-γ in FIG. 11). For type 1 IFNs (IFN-a and IFN-b), the peak was at 3 hours (FIG. 12).

[0246] Adsorption of STINGa to Al(OH)3 reduced innate cytokine responses in serum compared to those observed with soluble STINGa preparations, an effect observed primarily with IFN-a and IFN-b (Figure 12) and IL-6 and IFN-γ (Figure 11). When compared to AS01 (6 h), both STINGa formulations induced: High levels of type 1 IFN (IFN-a and IFN-b) (STINGa specific) were not induced by AS01 (Figure 12). relatively low levels of IFN-γ and IL-6 (Figure 11), and · Similar or slightly higher levels of IP-10 (Figure 11).

[0247] Collectively, these results suggest that adsorption of STINGa to Al(OH)3 reduces STINGa-induced systemic cytokine responses. Both STINGa formulations induced relatively low levels of markers known to be associated with systemic reactogenicity when compared to AS01, suggesting an acceptable tolerogenicity profile for the two STINGa formulations.

[0248] Example 4: Determination of immunogenicity induced by STINGa-adjuvanted vaccines compared to alum alone and AS01 The same recombinantly expressed HSV-2 gE-gI antigen was used to evaluate adjuvant compositions including a STING agonist (using compound 1) adsorbed to Al(OH)3 compared to Al(OH)3 alone and AS01.

[0249] research design In this study, five groups of naive female CB6F1 mice (n=13 per group, except for group 1 (control), n=4), aged 6-8 weeks at the start of the study, were injected intramuscularly (IM) into the left gastrocnemius muscle at 2-week intervals with 50 μL of either unadjuvanted gE-gI protein, Al(OH)3-formulated gE-gI protein, different doses of STING agonist / Al(OH)3-adjuvanted gE-gI, or AS01-adjuvanted gE-gI.

[0250] Serum samples were collected 14 days after the second immunization to assess total anti-HSV-2 gE-gI antibody levels and function (competitive ELISA and mFcγRIII binding activity / ADCC-like assay). Spleens were collected 14 days after the second immunization to assess anti-HSV-2 gE&gI-specific CD4+ cells expressing IL-2, TNF-αIFN-γ, IL-13 and / or IL-17 cytokines. + / CD8 + The frequency of T cells was assessed. The study design and formulation are outlined in the table below. Compound 1 was used as a STING agonist.

[0251] [Table 4] Detection of HSV-2 gE or gI-specific IgG antibodies and HSV-1 gE-gI-specific IgG antibodies was performed as previously described.

[0252] result All adjuvanted vaccine formulations were immunogenic, as antibody responses were much higher than those observed with antigen alone (Figures 13 and 14). Superiority of the adjuvant formulations containing STING agonist and Al(OH)3 compared to Al(OH)3 alone was demonstrated in terms of HSV-2 gE or gI-specific IgG titers, Th1 profile, and T cell polyfunctionality with geometric mean ratios (GMR) within 3.4 and 4 (see Figures 13-17).

[0253] overall results Overall, the results showed good immunogenicity of recombinant HSV-2 gE-gI protein adjuvanted with different doses of STINGa (soluble or alum-formulated) after one and / or two injections, as well as an acceptable tolerogenicity profile.

[0254] Higher functional antibodies and higher CD4 than both AS01 and soluble STINGa preparations + The benefit of the STINGa / Al(OH)3 formulation in inducing T cell responses was demonstrated. Furthermore, the STINGa / Al(OH)3 formulation induced more polyfunctional CD4 + T cells were induced. Total IgG, functional antibody or CD4 + For T cells, no differences were observed between the two Al(OH)3 ratios tested.

[0255] These results demonstrate that the adjuvant composition of the invention has superior immunogenicity when combined with an antigen and exhibits certain advantages over both soluble STINGa formulations and alternative adjuvants such as AS01.

[0256] Embodiments of the present invention: The present invention relates to the following embodiments: 1. (i) A STING agonist of formula (I) or a pharma- ceutical acceptable salt thereof [ka] [In the formula, X is -halo(C1-C5)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl; R 1 and R 9 are independently H, halogen, hydroxyl, -OP(O)(OH), -OP(O)(R I 2) optionally substituted C1-C6 alkyl or optionally substituted C1-C6 alkyloxy; In this case, the optionally substituted group includes hydroxyl, -OP(O)(OH), -OP(O)(R I )2, C1-C4 alkoxyl, -N(R A )2, -CO2(R B ), optionally substituted phenyl, and optionally substituted 5- to 6-membered heterocycloalkyl, wherein the optionally substituted phenyl or the optionally substituted 5- to 6-membered heterocycloalkyl is selected from the group consisting of halogen, hydroxy, -OP(O)(OH), -OP(O)(R I )2, amino, (C1-C6 alkyl)amino-, (C1-C6 alkyl)(C1-C6 alkyl)amino-, halo(C1-C6 alkyl), hydroxy-(C1-C4 alkyl)-, -(C1-C4 alkyl)-OP(O)(OH)2, -(C1-C4 alkyl)-OP(O)(R I )2, halo(C1-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)-OP(O)(OH)2, -(C2-C4 alkoxy)-OP(O)(R I)2, -(C1-C6 alkyl)-NH2, -C1-C4 alkyl-(C1-C4 alkoxyl) and C1-C4 alkoxy-(C1-C4 alkoxy)-, wherein R A and R B are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 2 and R 7 are each independently hydrogen, -CON(R C )2, -COOH, or CO2(R D ) or R 2 and R 7 One of the -CON(R C )(R D ) and the other is H, -COOH, or CO2(R E ), where R C and R D are each independently selected from hydrogen, -C1-C4 alkyl, -CO(C1-C4 alkyl), -OCO(C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-C1-C4 alkoxyl, or -CO2(C1-C4 alkyl); R 3 and R 8 are each independently H, halo(C1-C6 alkyl), halo(C1-C6 alkoxy)-, hydroxy, -OP(O)(OH), -OP(O)(R I )2, -NR C R D , -COR C , -CO2R C , -N(R D )COR C , -N(R D )SO2R C , -N(R g )SO2(C1-C2 alkyl)-N(R h )(R f), -N(R g )CO(C1-C2 alkyl)-N(R h )(R f ) and; R e , R f , R g , and R h are each independently H or C1-C4 alkyl; R 4 , R 5 , R 11 and R 12 are each independently H or C1-C4 alkyl; R 6 and R 10 are each C1-C4 alkyl; and R I each occurrence is independently C1-C6 alkyloxy-; and (ii) Aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, or a combination thereof. 13. An adjuvant composition comprising:

[0257] 2. The adjuvant composition according to embodiment 1, wherein the STING agonist has the structure of formula (II) or a pharma- ceutically acceptable salt thereof: [ka] [where X, R 1 , R 5 , R 6 , R 9 , R 10 and R 11 is as defined in embodiment 1].

[0258] 3. R 1 and R 9are each independently H, halogen, an optionally substituted (C1-C6 alkyl), or an optionally substituted (C1-C6 alkyl)oxy-, and the C1-C6 alkyl of the optionally substituted (C1-C6 alkyl), optionally substituted (C1-C6 alkyl)oxy- is hydroxyl, -OP(O)(OH), -OP(O)(R I )2, -N(R e )(R f ), C1-C4 alkoxyl, phenyl, and optionally substituted 5- to 6-membered heterocycloalkyl containing at least one nitrogen or oxygen ring member; e is independently selected from H, (C1-C4 alkyl), -(C1-C4 alkyl)-NH2, or -(C1-C4 alkyl)-C1-C4 alkoxy, and each R f The adjuvant composition according to embodiment 1 or 2, wherein is independently H or (C1-C4 alkyl).

[0259] 4. R 1 and R 9 One of the groups is -OP(O)(OH)2, -OP(O)(R I )2, -OP(O)(OH)2 or -OP(O)(R I The adjuvant composition according to embodiment 1 or 2, wherein the C1-C6 alkyl or C1-C6 alkyloxy group is substituted with 2. 5. R 3 and / or R 9 The adjuvant composition according to any one of embodiments 1-4, wherein 6. R 4 and / or R 12 The adjuvant composition according to any one of embodiments 1-5, wherein 7. R 6 and / or R 10 The adjuvant composition according to any one of embodiments 1-6, wherein is ethyl.

[0260] 8. R 1 and R 9 At least one of the following groups: [ka] [wherein a is a number from 2 to 6];

[0261] [ka] [wherein b is a number from 1 to 6];

[0262] [ka] [wherein c is a number from 1 to 6];

[0263] [ka] [In the formula, d is a number from 1 to 6, and R J and R K is C1-C3 alkyl; or

[0264] [ka] [In the formula, e is a number from 1 to 6, and Q is O or N(R X ), wherein R X is a C1-C6 alkyl group. The adjuvant composition according to any one of embodiments 1 to 7, selected from:

[0265] 9. The STING agonist (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0266] 4-(((E)-6-Carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)butanoic acid;

[0267] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-7-(3-(dimethylamino)propoxy)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0268] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-(4-(2-hydroxyethyl)piperazin-1-yl)propoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0269] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0270] (E)-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamide)-7-(3-morpholinopropoxy)-1H-benzo[d]imidazole-5-carboxamide;

[0271] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0272] (E)-1-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy)-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazole-5-carboxamide;

[0273] 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate; or Pharmaceutically acceptable salts thereof The adjuvant composition according to any one of embodiments 1 to 8, selected from the group consisting of:

[0274] 10. The STING agonist has the following formula: [ka]

[0275] The adjuvant composition according to any one of embodiments 1 to 9, wherein the adjuvant composition is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate as represented by 11. The adjuvant composition according to any one of embodiments 1 to 10, comprising aluminum hydroxide. 12. The adjuvant composition according to embodiment 11, wherein said STING agonist is adsorbed onto aluminium hydroxide.

[0276] 13. The adjuvant composition according to embodiment 12, wherein the amount of the STING agonist adsorbed onto aluminum hydroxide is greater than 80%, 85%, 90%, or 95%. 14. The adjuvant composition according to any one of embodiments 11-13, comprising aluminum hydroxide in an amount of 50-500 μg per dose. 15. The adjuvant composition according to any one of embodiments 1-14, comprising an amount of STING agonist between 0.5 and 250 μg per dose.

[0277] 16. An adjuvant composition according to any one of embodiments 1-15 comprising an amount of STING agonist of 1-100 μg per dose and aluminum hydroxide in an amount of 50-400 μg per dose.

[0278] 17. The adjuvant composition according to embodiment 16 comprising an amount of STING agonist of 1 to 50 μg per dose and aluminum hydroxide in an amount of 100 to 400 μg per dose.

[0279] 18. The adjuvant composition according to embodiment 17 comprising an amount of STING agonist of 3.1 μg per dose and aluminum hydroxide in an amount of 375 μg per dose.

[0280] 19. The adjuvant composition according to embodiment 17 comprising an amount of STING agonist of 6.3 μg per dose and aluminum hydroxide in an amount of 375 μg per dose.

[0281] 20. The adjuvant composition according to embodiment 17 comprising an amount of STING agonist of 12.5 μg per dose and aluminum hydroxide in an amount of 375 μg per dose.

[0282] 21. The adjuvant composition according to embodiment 17 comprising an amount of STING agonist of 25 μg per dose and aluminum hydroxide in an amount of 375 μg per dose.

[0283] 22. The adjuvant composition according to embodiment 17 comprising an amount of STING agonist of 50 μg per dose and aluminum hydroxide in an amount of 375 μg per dose. 23. An adjuvant composition according to any one of embodiments 1-10, comprising aluminum phosphate. 24. The adjuvant composition according to embodiment 23, wherein the STING agonist is adsorbed onto aluminum phosphate.

[0284] 25. The adjuvant composition according to embodiment 24, wherein the amount of the STING agonist adsorbed onto aluminum phosphate is greater than 80%, 85%, 90%, or 95%. 26. An adjuvant composition according to any one of embodiments 1 to 10, comprising aluminum oxyhydroxide. 27. The adjuvant composition according to embodiment 26, wherein said STING agonist is adsorbed onto aluminum oxyhydroxide.

[0285] 28. The adjuvant composition according to embodiment 27, wherein the amount of the STING agonist adsorbed onto aluminum oxyhydroxide is greater than 80%, 85%, 90%, or 95%. 29. An adjuvant composition according to any one of embodiments 1 to 10, comprising aluminum hydroxyphosphate. 30. The adjuvant composition according to embodiment 29, wherein the STING agonist is adsorbed onto aluminum hydroxyphosphate.

[0286] 31. The adjuvant composition according to embodiment 30, wherein the amount of the STING agonist adsorbed onto aluminum hydroxyphosphate is greater than 80%, 85%, 90%, or 95%. 32. (i) an adjuvant composition according to any one of embodiments 1 to 31; and (ii) Antigen 20. An immunogenic composition comprising: 33. The immunogenic composition according to embodiment 32, wherein the antigen is derived from a cancer cell. 34. The immunogenic composition according to embodiment 32, wherein the antigen is derived from a human pathogen.

[0287] 35. The immunogenic composition according to embodiment 34, wherein the antigen is derived from a human pathogen selected from the group consisting of bacteria, viruses, fungi, parasitic microorganisms and multicellular parasites. 36. The immunogenic composition according to any one of embodiments 32 to 35, wherein the antigen is a polypeptide.

[0288] 37. The immunogenic composition according to embodiment 35, wherein said human pathogen is selected from coronavirus, herpes simplex virus (HSV), HIV, hepatitis B virus, hepatitis C virus, meningitis B, Haemophilus influenzae type B, pertussis, diphtheria, tetanus, influenza virus, RSV, HPV, measles, rubella virus, mumps virus, HCMV, VZV, dengue virus, poliovirus, Ebola virus and rotavirus, or any combination of two or more thereof.

[0289] 38. The immunogenic composition according to embodiment 37, wherein the antigen is a VZV antigen. 39. The immunogenic composition according to embodiment 37, wherein the antigen is an HSV antigen. 40. The immunogenic composition according to embodiment 39, wherein said antigen is an HSV-2 gE-gI antigen. 41. The immunogenic composition according to embodiment 37, wherein the antigen is derived from at least one coronavirus. 42. The immunogenic composition according to embodiment 41, wherein the SARS-CoV-2 antigen is the SARS-CoV-2 S protein. 43. The immunogenic composition according to embodiment 42, wherein the SARS-CoV-2 S protein is a prefusion stabilized S protein.

[0290] 44. The immunogenic composition according to embodiment 43, wherein the SARS-CoV-2 S protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1.

[0291] 45. The immunogenic composition according to any of embodiments 32-44, wherein the adjuvant composition comprises aluminum hydroxide and the antigen is adsorbed onto the aluminum hydroxide. 46. ​​The immunogenic composition according to embodiment 45, wherein the antigen and STING agonist are adsorbed onto the same aluminum hydroxide. 47. The immunogenic composition according to embodiment 45, wherein the antigen and STING agonist are adsorbed onto different aluminum hydroxides.

[0292] 48. The immunogenic composition according to any one of embodiments 45 to 47, wherein the amount of the antigen bound to the aluminum hydroxide is more than 80%, 85%, 90%, or 95%.

[0293] 49. The immunogenic composition according to any of embodiments 32-44, wherein the adjuvant composition comprises aluminum phosphate and the antigen is adsorbed onto the aluminum phosphate. 50. The immunogenic composition according to embodiment 49, wherein the antigen and STING agonist are adsorbed onto the same aluminum phosphate. 51. The immunogenic composition according to embodiment 49, wherein the antigen and STING agonist are adsorbed onto different aluminium phosphates.

[0294] 52. The immunogenic composition according to any one of embodiments 49 to 51, wherein the amount of the antigen bound to aluminum phosphate is more than 80%, 85%, 90%, or 95%.

[0295] 53. The immunogenic composition according to any one of embodiments 31-44, wherein the adjuvant composition comprises aluminum oxyhydroxide and the antigen is adsorbed onto the aluminum oxyhydroxide. 54. The immunogenic composition according to embodiment 53, wherein the antigen and STING agonist are adsorbed onto the same aluminum oxyhydroxide. 55. The immunogenic composition according to embodiment 53, wherein the antigen and STING agonist are adsorbed onto different aluminum oxyhydroxides.

[0296] 56. The immunogenic composition according to any one of embodiments 53 to 55, wherein the amount of the antigen bound to aluminum oxyhydroxide is more than 80%, 85%, 90%, or 95%.

[0297] 57. The immunogenic composition according to any of embodiments 32-44, wherein the adjuvant composition comprises aluminum hydroxyphosphate and the antigen is adsorbed onto the aluminum hydroxyphosphate. 58. The immunogenic composition according to embodiment 57, wherein the antigen and STING agonist are adsorbed onto the same aluminum hydroxyphosphate. 59. The immunogenic composition according to embodiment 57, wherein the antigen and STING agonist are adsorbed onto different aluminum hydroxyphosphates.

[0298] 60. The immunogenic composition according to any one of embodiments 59 to 59, wherein the amount of said antigen bound to aluminum hydroxyphosphate is more than 80%, 85%, 90%, or 95%. 61. An immunogenic composition according to any one of embodiments 32 to 59, for use in therapy.

[0299] 62. An adjuvant composition according to any one of embodiments 1 to 31 for use in a method of immunizing a host, comprising administering to the host the adjuvant composition of any one of embodiments 1 to 31 and an antigen. 63. A method of immunizing a host, comprising administering to the host an adjuvant composition according to any one of embodiments 1 to 31 and an antigen. 64. The method of embodiment 56, wherein the antigen is formulated in a composition separate from the adjuvant composition and administered separately. 65. A method of immunizing a host, comprising the step of administering to the host an immunogenic composition according to any one of embodiments 32 to 60.

[0300] 66. A combination of an adjuvant composition according to any one of embodiments 1 to 31 with a vaccine formulation comprising an antigen. 67. A method of supporting an immune response in a host, comprising administering an adjuvant composition according to any one of embodiments 1 to 31. 68. Use of an adjuvant composition according to any one of embodiments 1 to 31 in the manufacture of a medicament for supporting an immune response in a subject.

[0301] 69. A kit comprising (i) a first container comprising an adjuvant composition according to any one of embodiments 1 to 31, and (ii) a second container comprising an antigen as defined in embodiments 26 to 44. TIFF2024540888000031.tif250144TIFF2024540888000032.tif250144TIFF2024540888000033.tif250144 TIFF2024540888000034.tif250144TIFF2024540888000035.tif250144TIFF2024540888000036.tif251141 TIFF2024540888000037.tif251141TIFF2024540888000038.tif251141TIFF2024540888000039.tif251141 TIFF2024540888000040.tif251141TIFF2024540888000041.tif251141TIFF2024540888000042.tif251141 TIFF2024540888000043.tif251141TIFF2024540888000044.tif251141TIFF2024540888000045.tif251141 TIFF2024540888000046.tif252140TIFF2024540888000047.tif252140TIFF2024540888000048.tif252140 TIFF2024540888000049.tif252140TIFF2024540888000050.tif252140TIFF2024540888000051.tif243140 TIFF2024540888000052.tif251142TIFF2024540888000053.tif251142TIFF2024540888000054.tif243143

Claims

1. (i) STING agonists: - the following formula: 【Chemical 1】 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate, as represented by (E)-3-((5-carbamoyl-1-(4-(5-carbamoyl-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-7-methoxy-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-(1-ethyl-3-methyl-1H-pyrazole-5-carboxamido)-1H-benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate; and / or - 3-(((Z)-6-Carbamoyl-3-((E)-4-((Z)-5-carbamoyl-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-1H-benzo[d]imidazol-1-yl)but-2-en-1-yl)-2-((1-ethyl-3-methyl-1H-pyrazole-5-carbonyl)imino)-2,3-dihydro-1H-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate or a pharmaceutically acceptable salt thereof; and (ii) aluminum hydroxide, aluminum phosphate, aluminum oxyhydroxide, or aluminum hydroxyphosphate, or a combination thereof; 1. An adjuvant composition comprising:

2. 10. The adjuvant composition of claim 1, comprising aluminum hydroxide.

3. 3. The adjuvant composition of claim 2, wherein the STING agonist is adsorbed onto aluminum hydroxide.

4. containing aluminum hydroxide in an amount of 50-500 μg per dose; and / or comprising an amount of STING agonist of 0.5 to 250 μg per dose; 3. The adjuvant composition of claim 2.

5. (i) an adjuvant composition according to any one of claims 1 to 4; and (ii) Antigen An immunogenic composition comprising:

6. the antigen is derived from a cancer cell; or the antigen is derived from a human pathogen; or The antigen is derived from a human pathogen selected from the group consisting of bacteria, viruses, fungi, parasitic microorganisms and multicellular parasites. The immunogenic composition of claim 5.

7. 7. The immunogenic composition of claim 6, wherein the antigen is derived from a human pathogen, and the human pathogen is selected from coronavirus, herpes simplex virus (HSV), HIV, hepatitis B virus, hepatitis C virus, meningitis B, Haemophilus influenzae type B, pertussis, diphtheria, tetanus, influenza virus, RSV, HPV, measles, rubella virus, mumps virus, HCMV, VZV, dengue virus, poliovirus, Ebola virus, and rotavirus, or any combination of two or more thereof.

8. the antigen is a VZV antigen; or the antigen is an HSV antigen; or the antigen is an HSV-2 gE-gI antigen; The immunogenic composition of claim 7.

9. the adjuvant composition comprises aluminum hydroxide and the antigen is adsorbed onto the aluminum hydroxide; or the antigen and STING agonist are adsorbed onto the same aluminum hydroxide; or The antigen and the STING agonist are adsorbed onto different aluminum hydroxides. The immunogenic composition of claim 5.

10. A combination of an adjuvant composition described in any one of claims 1 to 4 and a composition comprising an antigen, used to immunize a host, wherein the adjuvant composition and the composition comprising the antigen are administered separately to the host.

11. The immunogenic composition of claim 5, which is administered to a host to immunize the host.

12. 1. A kit comprising: (i) a first container comprising an adjuvant composition as defined in any one of claims 1 to 4; and (ii) a second container comprising an antigen derived from a cancer cell or from a human pathogen selected from the group consisting of bacteria, viruses, fungi, parasitic microorganisms and multicellular parasites.

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