ADJUVANT COMPOSITION COMPRISING STING

FIELD OF THE INVENTION

The present application relates to the field of adjuvant compositions, immunogenic compositions including the adjuvant composition, their use in a method of immunising a subject, and to related aspects.

BACKGROUND TO THE INVENTION

Vaccine adjuvants are included in formulations to enhance humoral and cellular immune responses, particularly in the case of poorly immunogenic subunit vaccines or patients with challenging immune status (e.g., infants, elderly adults, and immune comprised patients). Similar to natural infections by pathogens, adjuvants rely on the activation of the innate immune system to promote long-lasting adaptive immunity. As simultaneous activation of multiple innate immune pathways is a feature of natural infections, adjuvants may combine multiple immunostimulants in order to promote adaptive immune responses to vaccination. The Adjuvant System 01 (AS01) is of particular interest due to its ability to promote antigen-specific CD4+ T cells, as well as antigen-specific antibodies. The AS01 adjuvant system relies on the synergistic activities of two immunoenhancers, 3-O-desacyl-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 dependent from a natural source. It is therefore desirable to develop adjuvant systems that induce similar or even better immune responses and/or show a similar or improved reactogenicity profile, but are easier to prepare.

Recently developed STING (STimulator of InterferoN Genes) agonists have been proposed as vaccine adjuvants. Specifically, WO 2017/175147 (PCT/IB2017/051945) discloses a series of dimeric amidobenzimidazole (diABZI) based compounds and their use as vaccine adjuvants. However, there remains a need to develop STING agonists formulations that are capable of providing adjuvant compositions having an improved immunogenicity and/or reduced reactogenicity.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides an adjuvant composition comprising:

(i) a STING agonist of Formula (I) or a pharmaceutically acceptable salt thereof wherein

X is - halo(Ci-Cs)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl;

R ¹ and R ⁹ are independently H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, optionally substituted Ci-Ce alkyl or optionally substituted Ci-Ce alkyloxy, wherein optionally substituted means substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, C1-C4 alkoxyl, -N(R ^A )2, -CO2(R ^B ), optionally substituted phenyl, and optionally substituted 5-6 membered heterocycloalkyl, wherein said optionally substituted phenyl, or optionally substituted 5-6 membered heterocycloalkyl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(R ^I )2, amino, (Ci-Ce alkyl)amino-, (Ci-Ce alkyl)(Ci-Ce alkyl)amino-, halo(Ci-Ce alkyl), hydroxy-(Ci-C ₄ alkyl)-, -(C1-C4 alkyl)-O-P(O)(OH) ₂ , -(Ci-C ₄ alkyl)-O-P(O)(R ^I )2, ha lo(Ci-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)- O-P(O)(OH) ₂ , -(C2-C4 alkoxy)-O-P(O)(R ^I ) ₂ , -(Ci-C ₆ alkyl)-NH ₂ , -C1-C4 alkyl-(Ci- C4 alkoxyl) and C1-C4 alkoxy-(Ci-C4 alkoxy)-, wherein R ^A and R ^B are each independently selected from hydrogen, -C1-C4 alkyl, -CC C1-C4 alkyl), - OCC C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CO2(Ci- C ₄ alkyl),

R ² and R ⁷ are each independently hydrogen, -CON(R ^C )2, -COOH, or CC>2(R ^D ), or one of R ² and R ⁷ is -CON(R ^C )(R ^D ) and the other is H, -COOH, or CO2(R ^E ), wherein R ^c and R ^D are each independently selected from hydrogen, -C1-C4 alkyl, -CC C1-C4 alkyl), - OCC C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CC>2(Ci-C4 alkyl); R ³ and R ⁷ are each independently H, halo(Ci-Cealkyl), halo(Ci-Cealkoxy)-, hydroxy, -O-P(O)(OH) ₂ , -O-P(O)(R ^I ) ₂ , -NR ^C R ^D , -COR ^C , -CO ₂ R ^C , -N(R ^D )COR ^C , -N(R ^D )SO 2R ^D , -N(R9)SO2(Ci-C ₂ alkyl)-N(R ^h )(R ^f ), -N(R9)CO(Ci-C ₂ alkyl)-N(R ^h )(R ^f );

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 each occurrence of R ¹ is independently Ci-Ce alkyloxy-; and

(ii) aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate, or combinations thereof.

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 the adjuvant composition of the invention for use in therapy.

In a further aspect, the present 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 present invention and an antigen.

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.

In a further aspect, the present invention provides a method of adjuvanting an immune response in a subject, said method comprising administering to the subject the adjuvant composition of the invention and an antigen.

In a further aspect, the present 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 the adjuvant composition of the invention, and ii) a second container comprising an antigen.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by reference to the accompanying drawings, which are nonlimiting.

Fig. 1 represents the adsorption of a STING agonist on AI(OH)3 (by reference to Al ³⁺ ) at different ratios of STING agonist to AI(OH)3 tested (as indicated).

Fig. 2 represents the HSV-2 gl specific IgG antibody response induced by different vaccine formulations including the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist and AI(OH)s at different ratios (as indicated) in mice. IgG titers were measured 2 weeks post-immunization I (Post I) and 2 weeks post-immunization II (Post II). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Confidence Intervals (Cis) are provided.

Fig. 3 represents the HSV-2 gE specific IgG antibody response induced by different vaccine formulations including the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated) in mice. IgG titers were measured 2 weeks post-immunization I (Post I) and 2 weeks post-immunization II (Post II). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV- 2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 4 represents the HSV-1 gE-gl specific IgG antibody response induced by different vaccine formulations including the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated) in mice. IgG titers were measured 2 weeks post-immunization II (Post II). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 5 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-2 gE peptide pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 6 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-2 gl peptides pool or with HSV-1 gE or gl pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 7 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-1 gl peptide pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 8 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-1 gE peptide pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 9 represents the HSV-2 gE specific CD4+ T cell polyfunctionality analysis after in vitro stimulation. Proportions of HSV-2 gE specific CD4+ T cells expressing 1, 2, 3 or 4 cytokines are represented. Pie charts represent the mean proportions of cells expressing single markers and any combination of INF-g, IL-2, TNF-a, IL-13 and IL-17 marker-positive CD4+ T cells out of the total HSV- 2 specific CD4+ T cells. The vaccines used in this analysis were as follows (as indicated): HSV-2 gE- gl antigen adjuvanted with either (i) AS01, (ii) 0.74 pg of soluble STING agonist, or (iii) 0.74 pg STING agonist and 5.55 pg AL(OH)s.

Fig. 10 represents the HSV-2 gl specific CD4+ T cell polyfunctionality analysis after in vitro stimulation. Proportions of HSV-2 gl specific CD4+ T cells expressing 1, 2, 3 or 4 cytokines are represented. Pie charts represent the mean proportions of cells expressing single markers and any combination of INF-g, IL-2, TNF-a, IL-13 and IL-17 marker-positive CD4+ T cells out of the total HSV- 2 specific CD4+ T cells. The vaccines used in this analysis were as follows (as indicated): HSV-2 gE- gl antigen adjuvanted with either (i) AS01, (ii) 0.74 pg of soluble STING agonist, or (iii) 0.74 pg STING agonist and 5.55 pg AL(OH)s.

Fig. 11 represents an analysis of cytokines (IL-6, IP10 and IFN-y, as indicated) measurement in the serum of mice, 3h, 6h, 24h and 48 h post-immunization I with either the VZV gE antigen alone, VZV gE adjuvanted with soluble STING agonist or adjuvanted with STING agonist and AI(OH)s. A vaccine formulation including VZV gE adjuvanted with AS01 was used as a comparator.

Fig. 12 represents an analysis of cytokines (IFN-a, IFN-b and IFN-y, as indicated) measurement in the serum of mice, 3h, 6h, 24h and 48 h post-immunization I with either the VZV gE antigen alone, VZV gE adjuvanted with soluble STING agonist or adjuvanted with STING agonist and AI(OH)s. A vaccine formulation including VZV gE adjuvanted with AS01 was used as a comparator.

Fig. 13 represents the HSV-2 gE specific IgG antibody response induced by different vaccine formulations including the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist and AI(OH)s at different ratios (as indicated), AS01 or alum in mice. IgG titers were measured 2 weeks post-immunization I (Post I) and 2 weeks postimmunization II (Post II). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Confidence Intervals (Cis) are provided.

Fig. 14 the HSV-2 gl specific IgG antibody response induced by different vaccine formulations including the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist and AI(OH)s at different ratios (as indicated) in mice. IgG titers were measured 2 weeks post-immunization I (Post I) and 2 weeks post-immunization II (Post II). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Confidence Intervals (Cis) are provided.

Fig. 15 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-1 gE peptide pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

Fig. 16 represents the frequencies 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 post-immunization II after ex vivo stimulation with HSV-1 gl peptide pools in mice. The vaccines administered included the antigen HSV-2 gE-gl adjuvanted either with different doses of a soluble STING agonist or different doses of STING agonist to AI(OH)s at different ratios (as indicated). The antigen HSV-2 gE-gl alone was used as a control. A vaccine formulation including HSV-2 gE-gl adjuvanted with AS01 was used as a comparator. Individual geometric means and 95% Cis are provided.

BRIEF DESCRIPTION OF THE 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: Pre-fusion stabilised 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 gl.

SEQ ID NO. 7: Polypeptide sequence for VZV gE. DETAILED DESCRIPTION OF THE EMBODIMENTS

As described above, in one aspect, there is provided an adjuvant composition comprising:

(i) a STING agonist of Formula (I) or a pharmaceutically acceptable salt thereof wherein

X is — halo(Ci-Cs)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl;

R ¹ and R ⁹ are independently H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, optionally substituted Ci-Ce alkyl or optionally substituted Ci-Ce alkyloxy, wherein optionally substituted means substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, C1-C4 alkoxyl, -N(R ^A )2, -CC>2(R ^B ), optionally substituted phenyl, and optionally substituted 5-6 membered heterocycloalkyl, wherein said optionally substituted phenyl, or optionally substituted 5-6 membered heterocycloalkyl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(R ^I )2, amino, (Ci-Ce alkyljamino-, (Ci-Ce alkyl)(Ci-Ce alkyl)amino-, halo(Ci-Ce alkyl), hydroxy-(Ci-C ₄ alkyl)-, -(C1-C4 alkyl)-O-P(O)(OH) ₂ , -(Ci-C ₄ alkyl)-O-P(O)(R ^I )2, ha lo(Ci-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)- O-P(O)(OH) ₂ , -(C2-C4 alkoxy)-O-P(O)(R ^I ) ₂ , -(Ci-C ₆ alkyl)-NH ₂ , -C1-C4 alkyl-(Ci- C4 alkoxyl) and C1-C4 alkoxy-(Ci-C4 alkoxy)-, wherein R ^A and R ^B are each independently selected from hydrogen, -C1-C4 alkyl, -CC C1-C4 alkyl), - OCC C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CO2(Ci- C ₄ alkyl); R ² and R ⁷ are each independently hydrogen, -CON(R ^C )2, -COOH, or CC>2(R ^D ), or one of R ² and R ⁷ is -CON(R ^C )(R ^D ) and the other is H, -COOH, or CO2(R ^E ), wherein R ^c and R ^D are each independently selected from hydrogen, -C1-C4 alkyl, -CO(Ci-C4 alkyl), - OCO(Ci-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CO2(Ci-C4 alkyl); R ³ and R ⁸ are each independently H, halo(Ci-Cealkyl), halo(Ci-Cealkoxy)-, hydroxy, -O-P(O)(OH) ₂ , -O-P(O)(R ^I ) ₂ , -NR ^C R ^D , -COR ^C , -CO ₂ R ^C , -N(R ^D )COR ^C , -N(R ^D )SO 2R ^C , -N(R9)SO2(Ci-C ₂ alkyl)-N(R ^h )(R ^f ), -N(R9)CO(Ci-C ₂ alkyl)-N(R ^h )(R ^f );

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 each occurrence of R ¹ is independently Ci-Ce alkyloxy-; and

(ii) aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate, or combinations thereof.

DEFINITIONS

As used herein, the term "STING agonist" (also referred to as "STINGa" herein) refers to a compound of Formula (I) that is capable of binding to and activating the STING receptor and STING signaling. For example, the STING agonist, upon contact with the STING receptor causes one or more of the following: (1) stimulates or activates the STING receptor, (2) upregulates IRF3 and NFkB signaling pathways and/or (3) induces 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, measurement of cell signaling, cell proliferation, immune cell activation markers, cytokine production. STING agonist activity can also be measured in vivo by various assays that measure surrogate end points such as, but not limited to the measurement of T-cell proliferation or innate immunity-related cytokine production, in particular type I interferon.

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

It will be appreciated by those skilled in the art that said compounds may exist in other tautomeric forms including zwitterionic forms, or isomeric forms. All tautomeric (including zwitterionic forms) and isomeric forms of Formula (I) and compounds described herein are intended to be encompassed within the scope of the present disclosure.

For example, it will be appreciated by those skilled 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).

The chemical names provided for the intermediate compounds and/or the compounds for use described herein may refer to any one of the tautomeric representations of such compounds (in some instances, such alternate names are provided with the experimental). It is to be understood that any reference to a named compound (an intermediate compound or a compound of the disclosure) or a structurally depicted compound (an intermediate compound or a compound of the disclosure) is intended to encompass all tautomeric forms including zwitterionic forms of such compounds and any mixture thereof.

As used herein, the term "alkyl" represents a saturated, straight or branched hydrocarbon group having the specified number of carbon atoms. The term "CiOalkyl" refers to a straight or branched alkyl moiety containing from 1 to 4 carbon atoms. Exemplary alkyls include, but are not limited to methyl, ethyl, /7-propyl, isopropyl, /7-butyl, isobutyl, s-butyl, f-butyl, pentyl and hexyl.

When a substituent term such as "alkyl" is used in combination with another substituent term, for example as in "hydroxy(Ci-C4alkyl)", the linking substituent term (e.g., alkyl) is intended to encompass a divalent moiety, wherein the point of attachment is through that linking substituent. Examples of "hydroxy(Ci-C4alkyl)" groups include, but are not limited to, hydroxymethyl, hydroxyethyl, and hydroxyisopropyl.

As used herein, the term "halo(alkyl)" represents a saturated, straight or branched hydrocarbon group having the specified number (n) of carbon atoms and one or more (up to 2n+l) halogen atoms. For example, the term "halo(Ci-Csalkyl)" represents a group having one or more halogen atoms, which may be the same or different, at one or more carbon atoms of an alkyl moiety containing from 1 to 5 carbon atoms. Examples of "halo(Ci-Csalkyl)" groups include, but are not limited to, -CF3 (trifluoromethyl), -CCI3 (trichloromethyl), 1,1-difluoroethyl, 2,2,2-trifluoroethyl, and hexafluoroisopropyl. "Alkenyl" refers to straight or branched hydrocarbon group having the specified number of carbon atoms and at least 1 and up to 3 carbon-carbon double bonds. Examples include ethenyl and propenyl.

"Alkoxy-" or "(alkyl)oxy-" refers to an "alkyl-oxy-" group, containing an alkyl moiety, having the specified number of carbon atoms, attached through an oxygen linking atom. For example, the term "Ci-C4alkoxy-" represents a saturated, straight or branched hydrocarbon moiety having at least 1 and up to 4 carbon atoms attached through an oxygen linking atom. Exemplary "Ci-Cqalkoxy-" or "(Ci-C4alkyl)oxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, s-butoxy, and t-butoxy. Exemplary "Ci-Cealkoxy-" or "Ci-Cealkyloxy-" groups include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, s-butoxy, and t-butoxy.

As used herein, the term "halo(alkoxy)-" represents a saturated, straight or branched hydrocarbon group having the specified number (n) of carbon atoms and one or more (up to 2n+l) halogen atoms, attached through an oxygen linking atom. For example, the term "halo(Ci-C4alkoxy)- " refers to a "haloalkyl-oxy-" group, containing a "halo(Ci-C4alkyl)" moiety attached through an oxygen linking atom. Exemplary "halo(Ci-C4alkoxy)-" groups include, but are not limited to, -OCHF2 (difluoromethoxy), -OCF3 (trifluoromethoxy), -OCH2CF3 (trifluoroethoxy), and -OCH(CF3)2 (hexafluoroisopropoxy).

A heterocyclic group or moiety is a cyclic group or moiety having, as ring members, atoms of at least two different elements, which cyclic group or moiety may be saturated, partially unsaturated (non-aromatic) or fully unsaturated (aromatic).

"Heterocycloalkyl" refers to a non-aromatic, monocyclic or bicyclic group containing 3-10 ring atoms and containing one or more (generally one or two) heteroatom ring members independently selected from oxygen, sulfur, and nitrogen. The point of attachment of a heterocycloalkyl group may be by any suitable carbon or nitrogen atom.

The term "5-6 membered heterocycloalkyl" represents a saturated, monocyclic group, containing 5 or 6 ring atoms, which includes one or two heteroatoms selected independently from oxygen, sulfur, and nitrogen. Illustrative examples of 5-6 membered heterocycloalkyl groups include, but are not limited to pyrrolidinyl, tetra hydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperidinyl, piperazinyl, morpholinyl, and thiomorpholinyl.

The terms "halogen" and "halo" refers to a halogen radical, for example, a fluoro, chloro, bromo, or iodo substituent.

"Oxo" represents a double-bonded oxygen moiety; for example, if attached directly to a carbon atom forms a carbonyl moiety (C = O).

"Hydroxy" or "hydroxyl" is intended to mean the radical -OH.

As used herein, the term "cyano" refers to a nitrile group, -C=N.

As used herein, the term "optionally substituted" indicates that a group (such as an alkyl, cycloalkyl, alkoxy, heterocycloalkyl, aryl, or heteroaryl group) or ring or moiety may be un substituted, or the group, ring or moiety may be substituted with one or more substituent(s) as defined in the substituent definitions (A, R ³ , etc,) provided herein. In the case where groups may be selected from a number of alternative groups, the selected groups may be the same or different.

The term "independently" means that where more than one substituent is selected from a number of possible substituents, those substituents may be the same or different.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term "vaccine" is optionally substitutable with the term "immunogenic composition".

As used herein, the term "reactogenicity" refers to a subset of adverse events that is associated with the inflammatory response to the vaccination. The adverse events can be divided into both local (e.g. pain, swelling, erythma and induration) and systemic e.g. fever, nausea/vomiting, diarrhoea, headaches, fatigue and myalgia). Improving vaccines by reducing their reactogenicity may improve ease of access of vaccines to specific populations, for example by reducing pain in adolescents and fever in infants. Accordingly, reduced reactogenicity may improve vaccine uptake leading to greater population coverage and therefore reducing morbidity/mortality. Moreover, an excessive inflammation may also possibly negatively affect the quality of the immune response induced by a vaccine or an immunogenic composition. It is therefore an object of the disclosure to reduce the reactogenicity of vaccines.

As used herein, the term about is used to indicate a variance of ±10%.

Identity or homology with respect to a sequence is defined herein as the percentage of amino acid residues in the 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.

Sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see Dayhoff eta/., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the shorter sequences in order to align the two sequences. STING agonist

In one embodiment, the STING agonist is of Formula (II) or a pharmaceutically acceptable salt thereof wherein X, R ¹ , R ⁵ , R ⁶ , R ⁹ , R ¹⁰ and R ¹¹ are as defined in relation to Formula (I).

In one embodiment, R ¹ and R ⁹ in Formula (I) or (II) are each independently H, halogen, optionally substituted (Ci-Cealkyl), or optionally substituted (Ci-Cealkyl)oxy-, and the Ci-Cealkyl of said optionally substituted (Ci-Cealkyl), optionally substituted (Ci-Cealkyl)oxy- is optionally substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, - O-P(O)(R ^I )2, -N(R ^e )(R ^f ), Ci-C4alkoxyl, phenyl, optionally substituted 5-6 membered heterocycloalkyl containing at least one nitrogen or oxygen as a member of the ring, each R ^e is independently selected from H, (Ci-C4alkyl), -(CiC4alkyl)-NH2, or -(CiC4alkyl) CiOalkoxy and each R ^f is independently H or (Ci-C ₄ alkyl).

In one embodiment, the compound of Formula (I) or (II) comprises at least one phosphate group. In one embodiment, one of R ¹ and R ⁹ in Formula (I) or (II) is -O-P(O)(OH)2, -O-P(O)(R ^I )2, Ci-C ₆ alkyl substituted with -0-

P(O)(OH)2 or -O-P(O)(R ^I )2, or a Ci-Ce alkyloxy group. In one embodiment, one of R ¹ and R ⁹ in Formula (I) or (II) is -O-P(O)(OH)2, -O-P(O)(R ^I )2, Ci-Ce alkyl substituted with -O-P(O)(OH)2 or - O-P(O)(R ^I )2. In one embodiment, one of -O-P(O)(OH)2, -O-P(O)(R ^I )2, Ci-Ce alkyl substituted with - O-P(O)(OH)2 or -O-P(O)(R ^I )2, and the other is H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, optionally substituted Ci-Ce alkyl or optionally substituted Ci-Ce alkyloxy, wherein optionally substituted means substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, C1-C4 alkoxyl, -N(R ^A )2, -CC>2(R ^B ). In one embodiment, one of R ¹ and R ⁹ in Formula (I) or (II) is -O-P(O)(OH)2, -O-P(O)(R ^T )2, Ci-Ce alkyl substituted with -0-P(0)(0H)2 or -0-P(0)(R ^I )2, and the other is H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, optionally substituted Ci-Ce alkyl or optionally substituted Ci-Ce alkyloxy, wherein optionally substituted means substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, -N(R ^A )2, -CC>2(R ^B ). In one embodiment, one of R ¹ and R ⁹ in Formula (I) or (II) is -O-P(O)(OH)2, -O-P(O)(R ^I )2, Ci-Ce alkyl substituted with - O-P(O)(OH)2 or -O-P(O)(R ^I )2, and the other is H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, Ci-C ₆ alkyl or Ci-Ce alkyloxy.

In one embodiment, R ³ in Formula (I) or (II) is H. In one embodiment, R ⁸ in Formula (I) or (II) is H. In one embodiment, R ³ and R ⁸ in Formula (I) or (II) are H.

In one embodiment, R ⁴ in Formula (I) or (II) is H. In one embodiment, R ¹² in Formula (I) or (II) is H. In one embodiment, R ⁴ and R ¹² in Formula (I) or (II) are H.

In one embodiment, R ⁶ in Formula (I) or (II) is ethyl. In one embodiment, R ¹⁰ in Formula (I) or (II) is ethyl. In one embodiment, R ⁶ and R ¹⁰ in Formula (I) or (II) are ethyl.

In one embodiment, at least one of R ¹ and R ⁹ in Formula (I) and (II) is selected from the following groups: ,

0 and N(R ^X ) wherein R ^x is a Ci-Ce alkyl. In one embodiment, the STING agonist is selected from the group consisting of (E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-carb oxamido)-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxam ido)-7-(3-hydroxypropoxy)-lH- benzo[d]imidazole-5-carboxamide;

(E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyr azole-5-carbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH -pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-lH-benzo[d]imidazole-5-carboxami de;

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

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

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-7-methoxy-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-7-(3-(dimethylamino)pro poxy)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide;

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

(E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyr azole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-e n-l-yl)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d] imidazole-5-carboxamide;

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-lH-benzo[d]imidazole-5-carboxamide;

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

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

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-7-(3- morpholinopropoxy)-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2 -(l-ethyl-3-methyl-lH-pyrazole-5- carboxamido)-7-methoxy-lH-benzo[d]imidazole-5-carboxamide; (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazo le-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but- 2-en-l-yl)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d] imidazole-5-carboxamide;

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

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

3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl -3-methyl-lH-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d] imidazol-4-yl)oxy)propyl dihydrogen phosphate; or a pharmaceutically acceptable salt thereof.

In one embodiment, the STING agonist is 4-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2- ((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro -lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-d ihydro-lH-benzo[d]imidazol-4- yl)oxy)butanoic acid as shown below or a pharmaceutically acceptable salt thereof: . This compound may also be labelled as (E)-4-((5- carbamoyl-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole- 5-carboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-lH- benzo[d]imidazol-7-yl)oxy)butanoic acid or 4-(((Z)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l- ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-

2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-di hydro-lH-benzo[d]imidazol-4- yl)oxy)butanoic acid depending on its isomeric/tautomeric form.

In one embodiment, the STING agonist is (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl- lH-pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo [d]imidazol-l-yl)but-2-en-l-yl)-7-(3- (dimethylamino)propoxy)-2-((l-ethyl-3-methyl-lH-pyrazole-5-c arbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazole-5-carboxamide as represented by the below structure: . The compound may be labelled as (E)-l-(4-(5- carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-7-m ethoxy-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-7-(3-(dimethylamino)propoxy)-2-(l-ethyl-3- methyl-lH-pyrazole-5-carboxamido)- lH-benzo[d]imidazole-5-carboxamide depending on its isomeric/tautomeric form.

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

In one embodiment, the STING agonist is (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl- lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazo l-l-yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy) -2,3-dihydro-lH-benzo[d]imidazole- 5-carboxamide (as shown below) or a pharmaceutically acceptable salt thereof: The compound may also be labelled as (£)-l-(4-(5-

Carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)- lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-7-(3-morp holinopropoxy)-lH- benzo[d]imidazole-5-carboxamide or (Z)-l-((E)-4-((Z)-5-carbamoyl-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-l -yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-7-(3-morpholinopropoxy) -2,3-dihydro-lH-benzo[d]imidazole- 5-carboxamide depending on its isomeric/tautomeric form.

In one embodiment, the STING agonist is (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-l -yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2, 3-dihydro-lH-benzo[d]imidazole-5- carboxamide (as shown by the structure below) or a pharmaceutically acceptable salt thereof: . This compound may also be labelled (E)-l-(4-

(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamid o)-lH-benzo[d]imidazol-l-yl)but-2-en- l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido)-7-(3-hy droxypropoxy)-lH-benzo[d]imidazole- 5-carboxamide or (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazo le-5-carbonyl)imino)-

2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l -ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro-lH-benzo[d] imidazole-5-carboxamide depending on its isomeric/tautomeric form.

In one embodiment, the STING agonist is (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro- lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-7-met hoxy-2,3-dihydro-lH- benzo[d]imidazole-5-carboxamide as represented by the below formula or a pharmaceutically This compound may also be labelled

(E)-l-(4-(5-Carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-7-(3-hydroxypropoxy)-lH- benzo[d]imidazol-l-yl) but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH-pyrazole-5-carboxamido )-7-methoxy- lH-benzo[d]imidazole-5-carboxamide or (Z)-l-((E)-4-((Z)-5-carbamoyl-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2,3-dihydro- lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-7-met hoxy-2,3-dihydro-lH- benzo[d]imidazole-5-carboxamide depending on the isomeric/tautomeric form. In one embodiment, the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-

((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-7-methox y-2,3-dihydro-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbony l)imino)-2,3-dihydro-lH- benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate as represented by the below formula or a pharmaceutically acceptable salt thereof: carbamoyl-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole- 5-carboxamido)-7-methoxy-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-lH- benzo[d]imidazol-7-yl)oxy)propyl dihydrogen phosphate or 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5- carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino) -7-methoxy-2,3-dihydro-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH -pyrazole-5-carbonyl)imino)-2,3- dihydro-lH-benzo[d]imidazol-4-yl)oxy)propyl dihydrogen phosphate depending on the isomeric/tautomeric form.

In one embodiment, the STING agonist of Formula (I) or (II) in free base or free acid form.

As used herein, the STING agonist 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, for example a pharmaceutically acceptable salt thereof) and any physical form thereof (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 mono-, di- and hemi- hydrates, and mixtures of various forms).

Accordingly, included for use herein are the compounds of Formula (I) or Formula (II), as defined herein, in any salt or non-salt form and any physical form thereof, and mixtures of various forms. While such are included for use within the present 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 varying levels of activity, different bioavailabilities and different handling properties for formulation purposes.

Since the STING agonist of Formula (I), or a pharmaceutically acceptable salt thereof, is intended for use in immunogenic compositions it will readily be understood that it is preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of a compound may be used for preparing the more pure form used in the immunogenic compositions.

The STING agonists for use herein may contain one or more asymmetric centers (also referred to as a chiral center), such as a chiral carbon, or a chiral -SO- moiety. Said STING agonists may contain one or more chiral centers may be present as racemic mixtures, diastereomeric mixtures, enantiomerically enriched mixtures, diastereomerically enriched mixtures, or as enantiomerically or diastereomerically pure individual stereoisomers.

The stereochemistry of the chiral center present in compounds used herein is generally represented in the compound names and/or in the chemical structures illustrated. Where the stereochemistry of a chiral center present in a compound of the present disclosure, or in any chemical structure illustrated herein, is not specified, the structure is intended to encompass any stereoisomer and all mixtures thereof. Accordingly, all STING agonists of Formula (I) or (II) and salts thereof, whether as individual isomers isolated such as to be substantially free of the other isomer (i.e. pure) or as mixtures (i.e. racemates and racemic mixtures) are encompassed for use herein. An individual isomer isolated such as to be substantially free of the other isomer (i.e. pure) may be isolated such that less than 10%, particularly less than about 1%, for example less than about 0.1% of the other isomer is present. Individual stereoisomers of the STING agonists may be resolved (or mixtures of stereoisomers may be enriched) using methods known to those skilled in the art. For example, such resolution may be carried out (1) by formation of diastereoisomeric salts, complexes or other derivatives; (2) by selective reaction with a stereoisomer-specific reagent, 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 with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired stereoisomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired form. Alternatively, specific stereoisomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to the other by asymmetric transformation.

The definition of the STING agonist used herein also includes various deuterated forms of the compounds. Each available hydrogen atom attached to a carbon atom may be independently replaced with a deuterium atom. A person of ordinary skill in the art will know how to synthesize deuterated forms of the compounds used herein. For example, a-deuterated a-amino acids are commercially available or may be prepared by conventional techniques (see for example: Elemes, Y. and Ragnarsson, U. J. Chem. Soc., Perkin Trans. 1, 1996, 6, 537-40). Employing such compounds may allow for the preparation of compounds in which the hydrogen atom at a chiral center is replaced with a deuterium atom. Other commercially available deuterated starting materials may be employed in the preparation of deuterated analogs of the compounds (see for example: methyl- ds-amine available from Aldrich Chemical Co., Milwaukee, WI), or they may be synthesized using conventional techniques employing deuterated reagents (e.g. by reduction using lithium aluminum deuteride or sodium borodeuteride or by metal-halogen exchange followed by quenching with D2O or methanol-ds).

In one embodiment, the STING agonist of Formula (I) or (II) is used in the form of a pharmaceutically acceptable salt.

Suitable pharmaceutically acceptable salts of the STING agonist of Formula (I) or (II) can include acid addition salts or base addition salts. For reviews of suitable pharmaceutically acceptable salts see Berge et ai., J. Pharm. Sci., 66: 1-19, (1977) and P. H. Stahl and C. G. Wermuth, Eds., Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich:Wiley- VCH/VHCA (2002).

Salts of the STING agonist of Formula (I) or (II) containing a basic amine or other basic functional group may 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 pharmaceutically acceptable salts so formed include acetate, adipate, ascorbate, aspartate, benzenesulfonate, benzoate, camphorate, camphor-sulfonate (camsylate), caprate (decanoate), caproate (hexanoate), caprylate (octanoate), carbonate, bicarbonate, cinnamate, citrate, cyclamate, dodecylsulfate (estolate), ethane-l,2-disulfonate (edisylate), ethanesulfonate (esylate), formate, fumarate (hemi-fumarate, etc.), galactarate (mucate), gentisate (2,5-dihydroxybenzoate), glucoheptonate (gluceptate), gluconate, glucuronate, glutamate, glutarate, glycerophosphorate, glycolate, hippurate, hydrobromide, hydrochloride (dihydrochloride, etc.), hydroiodide, isobutyrate, lactate, lactobionate, laurate, maleate, malate, malonate, mandelate, methanesulfonate (mesylate), naphthalene-1,5- disulfonate (napadisylate), naphtha lene-sulfonate (napsylate), nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, phosphate (diphosphate, etc.), proprionate, pyroglutamate, salicylate, sebacate, stearate, succinate, sulfate, tartrate, thiocyanate, -toluenesulfonate (tosylate), undecylenate, l-hydroxy-2-naphthoate, 2,2-dichloroacetate, 2-hydroxyethanesulfonate (isethionate), 2 -oxoglutarate, 4-acetamidobenzoate, and 4-aminosalicylate.

Salts of the disclosed compounds containing a carboxylic acid or other acidic functional group can be prepared by reacting with a suitable base. Such a pharmaceutically acceptable salt may be made with a base which affords a pharmaceutically acceptable cation, which includes alkali metal salts (especially sodium and potassium), alkaline earth metal salts (especially calcium and magnesium), aluminum salts and ammonium salts, as well as salts made from physiologically acceptable organic bases such as trimethylamine, triethylamine, morpholine, pyridine, piperidine, picoline, dicyclohexylamine, /V,/V-dibenzylethylenediamine, 2-hydroxyethylamine, bis- 2- hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine, dibenzylpiperidine, dehydroabietylamine, /V,/Wbisdehydroabietylamine, glucamine, /V-methylglucamine, collidine, choline, quinine, quinoline, and basic amino acids such as lysine and arginine.

The present disclosure includes within its scope all possible stoichiometric and non- stoichiometric forms of the salts (e.g., hydrobromide, dihydrobromide, fumarate, hemi-fumarate, etc.) of the STING agonist of Formula (I) or (II).

When a disclosed compound or its salt is named or depicted by structure, it is to be understood that the compound or salt, including solvates (particularly, hydrates) thereof, may exist in crystalline forms, non-crystalline forms or a mixture thereof. The compound or salt, or solvates (particularly, hydrates) thereof, may also exhibit polymorphism (i.e. the capacity to occur in different crystalline forms). These different crystalline forms are typically known as "polymorphs." It is to be understood that the present disclosure includes all polymorphs of any compound herein, e.g., all polymorphic forms of any compound named or depicted by structure herein, including any salts and/or solvates (particularly, hydrates) thereof.

Polymorphs have the same chemical composition but differ in packing, geometrical arrangement, and other descriptive properties of the crystalline solid state. Polymorphs, therefore, 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, which may be used for identification. It will be appreciated that different polymorphs may be produced, for example, by changing or adjusting the conditions used in crystallizing/recrystallizing the compound. Polymorphic forms may 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), thermogravi metric analysis (TGA) and solid state nuclear magnetic resonance (SSNMR).

The skilled artisan will appreciate that pharmaceutically acceptable solvates (particularly, hydrates) of the STING agonist, including pharmaceutically acceptable solvates of a pharmaceutically acceptable salt of the STING agonist, may be formed when solvent molecules are incorporated into the crystalline lattice during crystallization. Solvates may involve non-aqueous solvents such as ethanol, or they may involve water as the solvent that is incorporated into the crystalline lattice. Solvates wherein water is the solvent that is incorporated into the crystalline lattice are typically referred to as "hydrates."

The STING agonist defined herein includes within its scope all possible stoichiometric and non-stoichiometric salt and/or hydrate forms.

Salts and solvates (e.g. hydrates and hydrates of salts) of the STING agonist used in the present disclosure includes all which are suitable for use in medicine are those wherein the counterion or associated solvent is pharmaceutically acceptable. Salts having non-pharmaceutically acceptable counterions are within the scope, for example, for use as intermediates in the preparation of other compounds.

Typically, a pharmaceutically acceptable salt may be readily prepared by using a desired acid or base as appropriate. The resultant salt may crystallize or precipitate from solution, or form by trituration, and may be recovered by filtration, or by evaporation of the solvent.

The present disclosure includes all prodrugs of the STING agonists of Formula (I), which upon administration to the recipient are capable of providing (directly or indirectly) a compound of Formula (I), or an active metabolite or residue thereof. Such derivatives are recognisable to those skilled in the art, without undue experimentation. Nevertheless, reference is made to the teaching of Burger's Medicinal Chemistry and Drug Discovery, 5 ^th Edition, Vol 1: Principles and Practice, which is incorporated herein by reference to the extent of teaching such derivatives.

It is to be further understood that the present disclosure includes within its scope all tautomeric or isomer forms of any free base form of the compounds as well as all possible stoichiometric and non-stoichiometric salt forms.

ALUMINIUM COMPOUND

The adjuvant composition comprises aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate, or a combination thereof.

Suitable forms of aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate for adjuvant use are well known to the skilled person. In one embodiment, the adjuvant composition comprises aluminium phosphate, aluminium hydroxide or a combination thereof. Suitable forms of aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate include but are not limited to Rehydragel™ HS, Alhydrogel™ 85, Rehydragel™ PM, Rehydragel™ AB, Rehydragel™ HPA, Rehydragel™ LV, Alhydrogel™ or a combination thereof.

In one embodiment, the adjuvant composition comprises aluminium hydroxide. In one embodiment, the adjuvant composition comprises aluminium hydroxide and does not include (i.e. is substantially free from) aluminium oxyhydroxide, or aluminium hydroxyphosphate.

In one embodiment, the adjuvant composition comprises aluminium phosphate.

In one embodiment, the adjuvant composition comprises aluminium oxyhydroxide.

In one embodiment, the adjuvant composition comprises aluminium hydroxyphosphate.

In one embodiment, the adjuvant composition comprises aluminium hydroxide. In one embodiment, the adjuvant composition comprises aluminium hydroxide, and a STING agonist of Formula (I) selected from the group consisting of (E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-carb oxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-(3- hydroxypropoxy)-lH-benzo[d]imidazole-5-carboxamide;

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

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

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

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-7-methoxy-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-7-(3-(dimethylamino)pro poxy)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-lH-benzo[d]imidazole-5-carboxamide;

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

(E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyr azole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-e n-l-yl)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d] imidazole-5-carboxamide;

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-lH-benzo[d]imidazole-5-carboxamide;

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

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

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-7-(3- morpholinopropoxy)-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2 -(l-ethyl-3-methyl-lH-pyrazole-5- carboxamido)-7-methoxy-lH-benzo[d]imidazole-5-carboxamide;

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

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

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

3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl -3-methyl-lH-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d] imidazol-4-yl)oxy)propyl dihydrogen phosphate; or a pharmaceutically acceptable salt thereof.

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

In one embodiment, the aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate (e.g., the aluminium hydroxide) may have a protein adsorption capacity of between 2.5 and 3.5, between 2.6 and 3.4, between 2.7 and 3.3, or between 2.9 and 3.2, between 2.5 and 3.7, between 2.6 and 3.6, between 2.7 and 3.5, or between 2.8 and 3.4 protein (BSA)/mL Al ³⁺ . In one embodiment, the aluminium hydroxide has a protein adsorption capacity of between 2.9 and 3.2 mg BSA/mg Al ³⁺ . Protein adsorption capacity of the aluminium compound can be measured by any means known to the skilled person. The protein adsorption capacity of the aluminium compound may be measured using the method as described in Example 1 of WO 12/136823 (which utilises BSA) or variations thereof. Aluminium hydroxide described herein (i.e. having the protein adsorption capacity described herein) may have a crystal size of between 2.8 and 5.7nm as measured by X-ray diffraction, for example 2.9 to 5.6nm, 2.8 to 3.5nm, 2.9 to 3.4nm or 3.4 to 5.6nm or 3.3 and 5.7nm as measured by X-ray diffraction. X-ray diffraction is well known to the skilled person. In a particular embodiment the crystal size is measured using the method described in Example 1 of WO 12/136823 or variations thereof.

In embodiments wherein the adjuvant composition further comprises an antigen, the antigen may be adsorbed onto the aluminium compound. This can be done prior to mixing with the STING agonist or adjuvant composition.

In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to the same aluminium hydroxide. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to different aluminium hydroxide.

In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to the same aluminium phosphate. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to different aluminium phosphate.

In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to the same aluminium oxyhydroxide. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to different aluminium oxyhydroxide.

In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to the same aluminium hydroxyphosphate. In one embodiment, the antigen and STING agonist of the immunogenic composition are adsorbed on to different aluminium hydroxyphosphate.

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

The meaning of "adsorbed STING agonist" is for example taken to mean greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% adsorbed. The amount of the adsorbed STING agonist can be measured using HPLC (e.g., by centrifuging the composition and conducting HPLC on the supernatant). In one embodiment, the adjuvant composition comprises a ratio of STING agonist to aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate of 1:2.5-250. In one embodiment, the adjuvant composition contains the STING agonist defined herein and aluminium hydroxide in an amount of 1: 1-250. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:2.5-100.

In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1: 1-10. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:1. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:5.

In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:10-100. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:10. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:25. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:50. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:75. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:50-100. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:50-125. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:100. Alternatively, the ratio of STING agonist to aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium 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.

In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1: 100-200. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1: 150. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:175. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:200. In one embodiment, the ratio of STING agonist to aluminium hydroxide is 1:250. Alternatively the ratio of STING agonist to aluminium hydroxide 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.

In one embodiment, the adjuvant composition comprises aluminium hydroxide, and the ratio of antigen to aluminium hydroxide is 1:1 to 1:50. In one embodiment, the adjuvant composition comprises aluminium hydroxide, and the ratio of antigen to aluminium hydroxide is 1:1 to 1:25. In one embodiment, the adjuvant composition comprises aluminium hydroxide, and the ratio of antigen to aluminium hydroxide is 1:1 to 1: 10. In one embodiment, the adjuvant composition comprises aluminium hydroxide, and the ratio of antigen to aluminium hydroxide is 1:1 to 1:5. In one embodiment, the ratio of antigen to aluminium hydroxide is 1:3.125. FORMULATIONS

The adjuvant compositions or immunogenic compositions may be administered via various suitable routes, including parenteral, such as intramuscular or subcutaneous administration. In one embodiment, the adjuvant composition is adapted for intramuscular administration. In one embodiment, the adjuvant composition is adapted for subcutaneous administration. In one embodiment, the adjuvant composition is adapted for intranasal administration.

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

The adjuvant composition may be administered with an antigen. In embodiments where the adjuvant composition is administered in combination with an antigen, the antigen may be administered separately from the adjuvant composition or may be administered within the same composition as the adjuvant composition. The antigen may also be administered via a different route to the adjuvant composition, when co-administered separately.

In one embodiment, the adjuvant composition is in aqueous form.

In one embodiment, the adjuvant composition is in non-aqueous form.

The pH of the adjuvant composition can be adjusted in view of the components and necessary suitability for administration to the 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 may be less than 9, less than 8, less than 7.5 or less than 7.

In one embodiment, the pH of the adjuvant composition is between 4 and 9, between 5 and 8, such as between 5.5 and 8. In a further embodiment, a buffer is added to the formulation. It is well known that for parenteral administration solutions should have a pharmaceutically acceptable osmolality to avoid cell distortion or lysis. A pharmaceutically acceptable osmolality will generally mean that solutions will have an osmolality which is approximately isotonic or mildly hypertonic. Suitably the adjuvant compositions will have an osmolality in the range of 250 to 750 mOsm/kg, for example, the osmolality may be in the range of 250 to 550 mOsm/kg, such as in the range of 280 to 500 mOsm/kg.

Osmolality may be measured according to techniques known in the art, such as by the use of a commercially available osmometer, for example the Advanced Model 2020 available from Advanced Instruments Inc. (USA). A desired osmolality may be achieved by the inclusion of salts or through the use of non-ionic isotonicity agents. In one embodiment, suitable non-ionic isotonicity agents are polyols, sugars (in particular sucrose, fructose, dextrose or glucose) or amino acids such as glycine. In one embodiment the polyol is a sugar alcohol especially a C3-6 sugar alcohol. Exemplary sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, sorbitol, mannitol, dulcitol and iditol. In a specific example of this embodiment, a suitable non-ionic isotonicity agent is sorbitol. In a

T1 particular embodiment the non-ionic isotonicity agent in the formulations is sucrose and/or sorbitol. Parenteral compositions are suitably sterile.

In one embodiment, the adjuvant composition is in the form of a parenteral dosage forms suitable to be administered in the form of sterile or sterilizable injectable solutions, suspensions, dry and/or lyophilized products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection (reconstitutable powders) and emulsions. Vehicles used in such dosage forms include, but are not limited to, Water for Injection USP; aqueous vehicles such as, but 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 not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

ANTIGEN

The adjuvant composition may be administered in combination with an antigen. The antigen may be formulated separately or in the same formulation (i.e., the antigen may be administered as part of the same formulation or a separate formulation).

The term "antigen" refers to any molecule capable of raising an immune response in a human or animal. The immune response is a protective immune response, e.g. reducing partially or completely the severity of one or more symptoms and/or time over which one or more symptoms are experienced by a subject, reducing the likelihood of developing an established infection after challenge and/or slowing progression of an associated illness (e.g. extending survival).

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

The immune response may be raised against a pathogen, such as for example, viruses, bacteria, parasites or fungus. That is, 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 bacteria, virus, fungi, parasitic microorganisms and multicellular parasites. In one embodiment, the antigen is derived from a combination of two or more bacteria, virus, fungi, parasitic microorganisms and multicellular parasites.

Alternatively, the antigen may be derived from a tumor cell (i.e., the antigen may be a tumor- associated antigen), and the adjuvant composition may be useful for the immunotherapeutic treatment of cancers.

In the sense, "an antigen derived from an organism" encompasses, in particular, the organism as a whole (whole-organisms, such as for example a whole-virus or a whole-bacterium), or one or more molecules only from the organism. The antigen may be the naturally occurring whole-organism, and the one or more molecules for instance one or more polypeptides, from the organism may be isolated and purified from such naturally occurring whole-organism.

Alternatively, the antigen may be artificially produced, for example, using recombinant technology or using chemical synthesis. Such recombinant antigens may be in a wild-type form, i.e. their nucleotide sequence, or amino acid sequence, is identical to the sequence of the corresponding antigens derived from the naturally occurring whole-organism. Alternatively, said recombinant antigens may advantageously comprise one or more mutations, i.e. their nucleotide sequence, or amino acid sequence, comprises one or more mutations, as compared with the sequence of the corresponding wild type antigens. Whole-organisms may be live attenuated or killed/inactivated. Inactivation processes using physical and/or chemical means are known to the skilled person. Such recombinant/modified/designed antigens are considered to be within the definition of "derived from" an organism within the context of the present disclosure.

In some embodiments, the antigen comprises at least one B or T cell epitope, an antigen comprises B and T cell epitopes. The elicited immune response may be an antigen specific B cell response which produces neutralizing antibodies. The elicited immune response may be an antigen specific T cell response, which may be a systemic and/or a local response. The antigen specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing a plurality of cytokines, e.g. IFN gamma, TNF alpha and/or IL2. Alternatively, or additionally, the antigen specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing a plurality of cytokines, e.g., IFN gamma, TNF alpha and/or IL2.

Suitably the encoded antigen contains 3000 residues or fewer, especially 2000 residues or fewer, in particular 1500 residues or fewer. The encoded antigen may contain 1000 residues or fewer, 800 residues or fewer, 600 residues or fewer, 400 residues or fewer or 200 residues or fewer.

Suitably the antigen contains 50 residues or more, especially 100 residues or more, in particular 150 residues or more.

Suitably the antigen contains 50 to 3000 residues, especially 100 to 1500 residues, in particular 200 to 1000 residues.

VIRAL ANTIGENS

The antigen used either in or with the adjuvant composition may derive from a virus. Accordingly, in particular embodiments, the antigen derives from a virus. In particular, the antigen may be a whole-virus. The whole virus may be live attenuated or killed/inactivated. Alternatively, the antigen may be a polypeptide derived from a virus.

Suitable viruses are from the families Orthomyxoviridae, such as for instance influenza viruses, Paramyxoviridae, such as for instance respiratory syncytial viruses (RSV), mumps virus or measles, Togaviridae, such as for instance rubella virus, Papovaviridae, such as for instance human papillomaviruses (HPV), Herpesviridae, such as for instance herpes simplex viruses (HSV), human cytomegaloviruses (HCMV), Epstein-Barr viruses (EBV), or varicella-zoster viruses (VZV), Picornaviridae, such as for instance enteroviruses, rhinoviruses, polioviruses, Fiaviviridae, such as for instance Dengue viruses or hepatitis C virus (HCV), Hepadnaviridae, such as for instance hepatitis B virus (HBV), Retroviridae, such as for instance human immunodeficiency viruses (HIV), Reoviridae, such as for example rotaviruses, Rhabdoviridae, such as for instance rabies viruses, or Flloviridae, such as for example Ebola virus. In one embodiment, the antigen used either in or with the adjuvant composition derives from a virus selected from the group consisting of influenza virus, RSV, HPV, measles, rubella virus, mumps virus, HCMV, VZV, Dengue virus, poliovirus, HIV, HBV, Ebola virus and rotavirus, or any combination of two or more thereof.

In a particular embodiment, the antigen derives from HCMV. Suitably, the HCMV antigen is the glycoprotein gB, which may lack the transmembrane domain (as disclosed in EP0802979), optionally in combination with one or more of the HCMV proteins pp65, IE1, pUL131, gL, gH, pUL128, and pUL130. Suitably, 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.

In one embodiment, the antigen is a varicella-zoster viruses (VZV) antigen. In one embodiment, the VZV antigen is a gE antigen. Suitably, the VZV antigen is the glycoprotein gE, which may be deleted from its transmembrane domain, as disclosed in EP0405867 Bl. In one embodiment, the VZV antigen is a protein derived Varicella Zoster Virus which is one of gpl, gpll or g pill as defined on pages 5-7 of EP 0 405 867 Bl and is missing from 4-20 percent of the total amino acid residues of the full length glycoprotein at the carboxy terminal end.

In a further embodiment, the antigen derives from RSV. Suitably, the RSV antigen is a polypeptide selected from the group consisting of the fusion protein (F), the attachment protein (G), the matrix protein (M2) and the nucleoprotein (N). Particularly suitable as an RSV polypeptide antigen to be included in or administered with the adjuvant composition are conformationally constrained F polypeptides. Conformationally constrained F polypeptides have previously been described in both the prefusion (PreF) and postfusion (PostF) conformations. Exemplary F protein antigens conformationally constrained in the prefusion conformation have been described in the art and are disclosed in detail in e.g. WO 09/079796, WO 10/149745, WO 11/008974 and WO 12/158613. Likewise, F protein antigens conformationally constrained in the postfusion conformation are also well known in the art and can be used in or administered with the adjuvant composition. Examples of postfusion conformationally constrained F protein polypeptides are disclosed in details in e.g. WO 11/008974, and Swanson et al. (PNAS, 2011, Vol. 108: 9619-9624). In particular embodiments, the adjuvant composition comprises an antigen polypeptide derived from RSV selected from the group consisting of: F protein, preF protein, N protein and M2 protein.

In a further embodiment, the antigen derives from HBV. Suitably, the antigen is the Hepatitis B surface antigen (HBS) In one embodiment, the antigen is derived from an RNA virus. In one embodiment, the antigen is derived from a coronavirus, particularly from SARS-CoV-2.

A plurality of antigens maybe encoded. Consequently, in some embodiments the antigen is derived from at least one coronavirus, for example from SARS-CoV-2. In some embodiments the antigen is derived from more than one coronavirus (such as 2, 3, 4 or 5), for example from SARS- CoV-2 (such as a plurality of SARS-CoV-2 variant antigens).

SARS-CoV-2 makes use of a densely glycosylated spike (S) protein to gain entry into host cells. In coronaviruses, the S protein is a trimeric class I fusion protein which exists in a metastable prefusion conformation that undergoes a substantial structural rearrangement 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 P The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol. 2003 Aug;77(16):8801-ll.).

A coronavirus protein of use is a fragment or variant of a native coronavirus protein which is capable of eliciting neutralising antibodies and/or a T cell response (such as a CD4 or CD8 T cell response) to a coronavirus, suitably a protective immune response.

A SARS-CoV-2 S protein of use comprises, such as consists of, a fragment or variant of a native SARS-CoV-2 S protein which is capable of eliciting neutralising antibodies and/or a T cell response (such as a CD4 or CD8 T cell response) to SARS-CoV-2, suitably a protective immune response.

The encoded SARS-CoV-2 S protein may comprise, such as consist of, a full length S protein (such as SEQ ID NO:1). Alternatively, the encoded SARS-CoV-2 S protein may comprise, such as consist of, an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:1. The encoded SARS-CoV-2 S protein may comprise, such as consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 1, especially at least 98% identity to the amino acid sequence set forth in SEQ ID NO:1, in particular at least 99% identity to the amino acid sequence set forth in SEQ ID NO:1, such as 100% identity to the amino acid sequence set forth in SEQ ID NO:1.

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

The encoded SARS-CoV-2 S protein may comprise, such as 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 may comprise, such as consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 2, especially at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 2, in particular at least 99% identity to the amino acid sequence set forth in SEQ ID NO:2, such as 100% identity to the amino acid sequence set forth in SEQ ID NO:2.

The encoded SARS-CoV-2 S protein may comprise, such as 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 may comprise, such as consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO: 3, especially at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 3, in particular at least 99% identity to the amino acid sequence set forth in SEQ ID NO:3, such as 100% identity to the amino acid sequence set forth in SEQ ID NO:3.

Suitably the encoded SARS-CoV-2 S protein is pre-fusion stabilised to facilitate appropriate presentation to the immune system. For example, Wrapp and colleagues (Wrapp et al., Science 367, 1260-1263 (2020)) produced a recombinant prefusion S ectodomain using a stabilization strategy that proved effective for other beta coronavirus S proteins (Pallesen eta/., Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7348-E7357; Kirchdoerfer, R.N. et al. Sci Rep 8, 15701 (2018)). To this end, starting with the SARS-CoV-2 polynucleotide sequence (GenBank accession number MN908947.3), a gene encoding residues 1 to 1208 of SARS-CoV-2 S protein (UniProt accession number P0DTC2 version 1 dated 22 April 2020) with proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site (residues 682-685) a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an 8XHisTag was synthesized and cloned into the mammalian expression vector paH.

Residues 1 to 1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987, a "GSAS" substitution at the furin cleavage site are provided in SEQ ID NO:4, which is an example of a pre-fusion stabilized ectodomain of SARS-CoV-2 S protein.

The encoded SARS-CoV-2 S protein may comprise, such as 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 may comprise, such as consist of, an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:4, especially at least 98% identity to the amino acid sequence set forth in SEQ ID NO:4, in particular at least 99% identity to the amino acid sequence set forth in SEQ ID NO:4, such as 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 stabilised protein.

In one embodiment, the SARS-CoV-2 S protein is the stabilized recombinant prefusion S ectodomain disclosed by Wrapp et al., Science 367, 1260-1263 (2020).

The SARS-CoV-2 S protein (such as a pre-fusion stabilized SARS-CoV-2 S protein) may desirably be in the form of a trimer and consequently may comprise a trimerization motif, such as a T4 fibritin trimerization motif, more suitably a C-terminal T4 fibritin trimerization motif. Alternative trimerization motifs include, for example, a domain derived from collagen called 'Trimer-Tag' such as disclosed in Liu eta/., 2017, or a molecular clamp, such as that disclosed in W02018/176103.

An encoded SARS-CoV-2 S protein is desirably 1800 residues or fewer in length, especially 1500 residues or fewer, in particular 1400 residues or fewer, such as 1300 residues or fewer.

An encoded SARS-CoV-2 S protein is desirably 150 residues or more in length, especially 200 residues or more, in particular 400 residues or more, such as 600 residues or more.

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 a hemagglutinin or a neuraminidase.

In one embodiment the antigen is an Epstein-Barr virus (EBV) antigen.

In one embodiment, the antigen is an Herpes simplex virus (HSV) antigen, such as a gE and/or a gl antigen. Suitable antigens are disclosed in WO 2021/013798. In one embodiment, the antigen comprises i) a HSV2 gE antigen or HSV1 gE antigen, for example as defined in Figure 1 of WO 2021/013798, and/or ii) an HSV2 gl or HSV2 gl antigen, for example as defined in Figure 2 of WO 2021/013798. In a particular embodiment, the antigen comprises a HSV2 gE and gl heterodimer or an immunogenic fragment thereof.

BACTERIAL ANTIGEN

The antigen used either in or with the adjuvant composition may derive from a bacterium. Accordingly, in particular embodiments, the antigen derives from a bacterium. In one embodiment, the antigen is from a bacterium selected from the group consisting of: B. pertussis, S. Pneumoniae, and N. meningitidis, or any combination of two or more thereof.

The antigen may be a whole-bacterium and may be killed/inactivated or live attenuated. Particular whole-bacterium antigens for use in the present compositions are Bordetella pertussis. In one embodiment, the B. pertussis antigen is the 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). Pw antigen may be inactivated by several known methods, including mercury-free methods. Such methods may include heat, formaldehyde, glutaraldehyde, acetone-I, or acetone-IO inactivation (see for example Gupta et al. , 1987, j. Biol. Stand. 15:87; Gupta et al. , 1986, Vaccine, 4: 185). Methods of preparing inactivated Pw antigen suitable for use in the formulations are disclosed in WO 93/24148. In one embodiment of a Pw antigen-comprising adjuvant composition of the disclosure, the Pw component of the formulation elicits reduced reactogenicity. Reactogenicity of Pw vaccines is primarily caused by lipo-oligosaccharide ('LOS'), which is the endotoxin from the bacterial outer membrane. The lipid A part of the LOS is mainly responsible for the reactogenicity. In order to produce a less reactogenic Pw antigen-containing vaccine (relative to 'traditional' Pw vaccines such as produced by the above-discussed inactivation procedures), the endotoxin can be genetically or chemically detoxified and/or extracted from the outer membrane. In one embodiment, the B. pertussis antigen used in or with the adjuvant composition comprises a "low reactogenicity" Pw antigen in which the LOS has been genetically or chemically detoxified and/or extracted. For example, the Pw antigen may 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).

In alternative embodiments, 'low reactogenicity' is achieved by deriving the Pw antigen from a B. pertussis strain genetically engineered to produce a less toxic LOS. WO 06/065139 discloses genetic 3-O-deacylation and detoxification of B. pertussis LOS, resulting in strains comprising at least partially 3-O-deacylated LOS. The B. pertussis antigen used in or with the adjuvant composition may therefore be a Pw antigen derived from a strain of B. pertussis which has been engineered to express a lipid A-modifying enzyme, such as a de-O-acylase. In particular, such a strain may express the 3-O- deacylase PagL as described in WO 06/065139, as well as 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 engineering, lacks the ability to modify its lipid A phosphate groups with glucosamine, has a lipid A diglucosamine backbone substituted at the C-3' position with C10-OH or C12-OH and/or express molecular LOS species that 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).

Further particular bacterial antigens for use in or with the adjuvant composition may derive from Streptococcus pneumoniae. At least one streptococcal protein and/or at least one streptococcal capsular saccharide, optionally conjugated to a carrier protein, can be suitably included as antigens in the adjuvant composition or can be administered with such formulation. Suitable protein and saccharide antigens derived from Streptococcus pneumoniae are described in WO 14/060385. In some embodiments, the at least one Streptococcus pneumoniae protein is selected from the group consisting of Poly Histidine Triad family (PhtX), Choline Binding Protein Family (CbpX), CbpX truncates, LytX (autolytic enzyme) family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins, PcpA (pneumococcal choline binding protein A), PspA (Pneumococcal Surface Protein A), PsaA (pneumococcal surface adhesion protein A, Spl28 (Streptococcus pneumoniae 128), Spl01(Streptococcus pneumoniae 101), Spl30 (Streptococcus pneumoniae 130), SP125 (Streptococcus pneumoniae 125) and SP133 (Streptococcus pneumoniae 133).

In one embodiments, the adjuvant composition comprises or is administered with 1 or more (e.g. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23) Streptococcus pneumoniae capsular saccharide, optionally conjugated to a carrier protein. In particular embodiments, the 1 or more Streptococcus pneumoniae capsular saccharide, optionally conjugated to a carrier protein, included in or administered with the adjuvant composition comprises saccharides derived from serotypes 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 and 33F.

The term "saccharide" may indicate a polysaccharide or oligosaccharide and includes both. Polysaccharides are isolated from bacteria and may be sized to some degree by known methods (see, for example, EP0497524 and EP0497525) and optionally by microfluidisation. Polysaccharides can be sized in order to reduce viscosity in polysaccharide samples and/or to improve filterability for conjugated products. The terms "conjugate" relate to a capsular saccharide covalently bonded to a carrier protein. The carrier protein may be any peptide or protein. Suitable carrier proteins are described in WO 14/060385. The carrier protein may be tetanus toxoid (TT), tetanus toxoid fragment C, non-toxic mutants of tetanus toxin, diphtheria toxoid (DT), CRM197, other non-toxic mutants of diphtheria toxin, such as CRM 176, CRM228, CRM 45; CRM 9, CRM 45, CRM 102, CRM 103 and CRM 107 (where CRM stands for Cross Reacting Material), pneumococcal pneumolysin, OMPC (outer membrane protein C), heat shock proteins, pertussis proteins, cytokines, lymphokines, growth factors or hormones, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen derived antigens, such as N19 protein, pneumococcal surface protein PspA, iron uptake proteins, toxin A or B of C. difficile, H. influenzae Protein, pneumococcal PhtA (poly histidine triad protein A), pneumococcal PhtD (poly histidine triad protein D, pneumococcal PhtB (poly histidine triad protein B), or PhtE (poly histidine triad protein E). In one embodiment the at least one Streptococcus pneumoniae capsular saccharide conjugate is conjugated to a carrier protein selected from the group consisting of tetanus toxoid (TT), fragment C of TT, diphtheria toxoid, CRM 197 (cross reacting material 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. Further particular bacterial antigens for use in the present disclosure are derived from Neisseria meningitidis. In some embodiments, the antigen of the is a N. meningitidis 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. Indeed, these saccharides may suitably be conjugated to any of the carrier protein described above in relation to streptococcal saccharides. In some embodiments, the antigen is a N. meningitidis serogroup A capsular saccharide (MenA), N. meningitidis serogroup C capsular saccharide (MenC), N. meningitidis serogroup Y capsular saccharide (MenY), and N. meningitidis serogroup W-135 capsular saccharide (MenW), optionally conjugated to the carrier protein CRM197 or the carrier protein TT.

Further particular bacterial antigens derived from Neisseria meningitidis for use herein are derived from N. meningitidis serogroup B ("MenB"). Suitable antigens for eliciting anti-MenB responses include polypeptides, lipo-oligosaccharide and/or membrane vesicles. The adjuvant composition may include or be administered with one or more serogroup B meningococcal polypeptide antigen(s). In some embodiments, the antigen is a N. 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'), or any combination of two or more thereof, optionally in combination with a N. meningitidis serogroup B-derived OMV. These antigens will usefully be present as purified polypeptides, e.g. recombinant polypeptides. Suitable forms of these antigens are disclosed in WO 04/032958. The five antigens may be present in the formulation as five separate proteins, or suitably at least two of the antigens are expressed as a single polypeptide chain (a 'hybrid' protein) e.g. such that the five antigens form fewer than five polypeptides, as described in WO 04/032958. In some embodiments, the adjuvant composition comprises or is administered with at least NadA protein, NHBA protein, fHBP protein, GNA1030 protein and GNA2091 protein. In particular embodiments, the adjuvant composition comprises or is administered with comprise 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 an N. meningitidis serogroup B-derived OMV, as described below.

Further particular bacterial antigens are outer membrane vesicles (OMV). These include any proteo-liposomic vesicle obtained by disruption of or blebbling from an outer membrane to form vesicles therefrom that include protein components of the outer membrane. Gram-negative bacteria, such as Neisseria secrete OMV during active growth. The primary immunogenic components of the OMV are the outer membrane proteins (OMPs) and the membrane-bound lipo-polysaccharides (LPS). OMVs may be prepared from any Gram-negative bacterium, 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 poorly immunogenic. Accordingly, in some embodiments, the adjuvant composition comprises or is administered with an OMV derived from a N. meningitidis serogroup B strain, optionally in combination with any of the above-described serogroup B meningococcal polypeptide antigens. OMVs are prepared artificially from bacteria, and may be prepared using detergent treatment (e.g. with deoxycholate), or by non-detergent means, as described in WO 12/020326, for example.

PARASITE ANTIGEN

The antigen used in or administered with the adjuvant composition may derive from a parasite. In some embodiments, the antigen may derive from parasites causing Malaria. Accordingly, in some embodiments, the antigen in or administered with the adjuvant composition is derived from parasites that cause 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 via four amino acids of the preS2 portion of Hepatitis B surface antigen to the surface (S) antigen of Hepatitis B virus. TUMOR- ASSOCIATED ANTIGENS

The antigen in the immunogenic composition or administered with the adjuvant composition may be a tumor-associated antigen. Suitably, the antigen may be a tumor rejection antigen, such as those for prostate, breast, colorectal, lung, pancreatic, renal or melanoma cancers. Exemplary, nonlimiting, antigens include MAGE 1, 3 and MAGE 4 or other MAGE antigens, such as disclosed in WO 99/40188.

ADDITIONAL ANTIGENS

The present compositions may involve a plurality of antigenic components, for example with the objective to elicit a broad immune response e.g. to a pathogen or to elicit responses to multiple pathogens. Consequently, more than one antigen may be present. Polysaccharides such as polysaccharide conjugates, may also be present.

DOSAGES

The therapeutically effective amount will vary depending on, among others, the disease indicated, 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, the daily dosage of a STING agonist of Formula (I), satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5mg/kg per body weight.

The amount of conjugate antigen in each immunogenic composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. The content of each protein antigen will typically be in the range 1-200 pg, suitably 1-100 pg, suitably 5-50 pg. The content of each saccharide antigen will typically be in the range 0.1-50 pg, suitably 0 1-10 pg, suitably 1-5 pg.

A dose which is in a volume suitable for human use is generally between 0.25 and 1.5 ml, although, for administration to the skin a lower volume of between 0.05 ml and 0.2 ml may be used. In one embodiment, a human dose is 0 5 ml. In a further embodiment, a human dose is higher than 0.5 ml, for example 0.6, 0.7, 0.8, 0.9 or 1 ml. In a further embodiment, a human dose is between 1 ml and 1.5 ml In another embodiment, in particular when the immunogenic composition is for the paediatric population, a human dose may be less than 0.5 ml such as between 0.25 and 0.5 ml.

In certain embodiments, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of between 0.5 to 250 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of between 1.0 to 100 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 1.0 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 10.0 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 25.0 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 50.0 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 75.0 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist of Formula (I) in an amount of 100.0 pg per dose.

In certain embodiments, the adjuvant composition comprises aluminium hydroxide in an amount of between 50 to 500 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of between 100 to 400 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 50 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 75 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 100 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 125 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 150 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 175 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 200 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 225 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 250 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 275 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 300 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 325 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 350 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 375 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 400 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 425 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 450 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 475 pg per dose. In some embodiment, the adjuvant composition comprises aluminium hydroxide in an amount of 500 pg per dose.

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-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)- 7-methoxy-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl )-2-((l-ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-4-yl)oxy)pro pyl dihydrogen phosphate, Compound 1) in an amount of between 0.5 to 250 pg per dose and aluminium hydroxide in an amount of 50 to 500 pg per dose. In some embodiment, the adjuvant composition comprises a STING agonist Compound 1 in an amount of between 1.0 to 100 pg per dose and aluminium hydroxide in an amount of 50 to 500 pg per dose (e.g. 100-400 pg per dose). In one embodiment, the adjuvant composition comprises a STING agonist Compound 1 in an amount of between 1.0 pg per dose and aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition includes a STING agonist Compound 1 in an amount of between 25 pg per dose and aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises a STING agonist Compound 1 in an amount of between 50 pg per dose and aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises a STING agonist Compound 1 in an amount of between 75 pg per dose and aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises a STING agonist Compound 1 in an amount of between 100 pg per dose and aluminium hydroxide in an amount of 375 pg per dose.

In one embodiment, the adjuvant composition comprises an amount of the STING agonist (e.g., Compound 1) in an amount of between 1 and 50 pg per dose, and comprises aluminium hydroxide in an amount of 100 to 400 pg per dose.

In one embodiment, the adjuvant composition comprises an amount of the STING agonist of 3.1 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises an amount of the STING agonist of 6.3 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises an amount of the STING agonist of 12.5 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises an amount of the STING agonist of 25 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose. In one embodiment, the adjuvant composition comprises an amount of the STING agonist of 50 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose.

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

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

In one embodiment, the immunogenic composition comprises an amount of the STING agonist of 6.3 pg per dose, aluminium hydroxide in an amount of 375 pg per dose, and 80 pg per dose of HSV2 gE-gl heterodimer of a HSV2 gE having the amino acid sequence shown in SEQ ID NO: 5 (ectodomain) associated in a non-covalent complex with the HSV2 gl having the amino acid sequence shown in SEQ ID NO: 6 (ectodomain). In one embodiment, the immunogenic composition comprises an amount of the STING agonist of 12.5 pg per dose, aluminium hydroxide in an amount of 375 pg per dose, and 80 pg per dose of HSV2 gE-gl heterodimer of a HSV2 gE having the amino acid sequence shown in SEQ ID NO: 5 (ectodomain) associated in a non-covalent complex with the HSV2 gl having the amino acid sequence shown in SEQ ID NO: 6 (ectodomain).

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

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

METHODS OF USE

The adjuvant composition described herein may 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 a vaccine composition.

The adjuvant composition described herein may be used in conjunction with a vaccine to improve the immunogenicity of the vaccine, or may be used as a vaccine when including an antigen in the adjuvant composition.

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

In one embodiment, the immunogenic composition of the present disclosure provides an increased antibody titer (i.e., amount of antibody) to one or more antigens when compared to the antibody titer of an immunogenic composition in the absence of the adjuvant composition.

In one embodiment, the immunogenic composition of the present disclosure provides an increased CD4+ T cell response to one or more antigens provided by the composition compared to an immunogenic composition in the absence of the adjuvant composition.

In one embodiment, there is provided a method of enhancing an immune response in a host comprising administering to the 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) to the one or more antigens when compared to the antibody titer of an immunogenic composition in the absence of the adjuvant composition. In one embodiment, there is provided a method of enhancing an immune response in a host comprising administering to the 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 an antibody response in the absence of the the adjuvant composition described herein.

In one embodiment, there is provided a method of enhancing the CD4+ T cell polyfunctionality in a host comprising administering to the host an effective amount of an adjuvant composition described herein and one or more antigens in comparison to the CD4+ T cell response provided by a composition in the absence of the adjuvant composition . In one embodiment, the enhanced CD4+ T cell polyfunctionality refers to the capacity for the host to produce CD4+ T cells expressing at least three cytokines. In one embodiment, the at least three cytokines are selected from INFy+ IL2+ TNF+.

In one embodiment, there is a method of enhancing CD4+ T cell polyfunctionality in a host comprising comprising administering to the host an effective amount of an adjuvant composition described herein and one or more antigens, wherein at least 40% of CD4+ T cells produce at least INFy+ IL2+ TNF+ or INFy+ IL13+ TNF+. In some embodiments, at least 50% of CD4+ T cells produce INFy+ IL2+ TNF+. In some embodiments, at least 50% of CD4+ T cells produce INFy+ IL13+ TNF+.

In one embodiment, provided is 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, provides is a combination of the adjuvant composition of described herein, and a vaccine formulation comprising an antigen.

In one embodiment, provided is a kit comprising i) the adjuvant composition as described herein, and an antigen.

The present disclosure also provides a delivery device pre-filled with the adjuvant composition described herein. Also provided is a sterile container (e.g., a vial) containing the adjuvant composition described herein (e.g., containing a unit dose or a multiple of unit doses). The present disclosure also provides a hermetically sealed container containing an adjuvant or immunogenic composition disclosed herein.

EXAMPLES

The invention will now be illustrated by way of the following non-limiting examples. While particular embodiments of the invention are described below a skilled artisan will appreciate that various changes and modifications can be made. References to preparations carried out in a similar manner to, or by the general method of, other preparations, may encompass variations in routine parameters such as time, temperature, work-up conditions, and minor changes in reagent amounts, etc. Reference Example: Synthesis of the STING aqonist

The STING agonists used in the present invention can be prepared using the methods disclosed in WO 2017/175147 (International patent application number PCT/IB2017/051945), which can be readily adapted to prepare other compounds of the invention by drawing on the knowledge of a skilled organic chemist. For example, (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-l -yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-7-(3-hydroxypropoxy)-2, 3-dihydro-lH-benzo[d]imidazole-5- carboxamide can be prepared according to Example 10 of WO'147. (E)-l-((E)-4-((E)-5-carbamoyl-2- ((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro -lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-7-(3- morpholinopropoxy)-2,3-dihydro-lH- benzo[d]imidazole-5-carboxamide can be prepared according to Example 13 of WO'147. 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)- 7-methoxy-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl )-2-((l-ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-4-yl)oxy)pro pyl dihydrogen phosphate can be prepared according to Example 19 of WO'147. (E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-methoxy-lH-benzo[d]imidazol-l-yl)b ut-2-en-l-yl)-7-(3- (dimethylamino)propoxy)-2-(l-ethyl-3-methyl-lH-pyrazole-5-ca rboxamido)-lH-benzo[d]imidazole-5- carboxamide can be prepared according to Example 39 of WO'147. (E)-l-((E)-4-((E)-5-carbamoyl-2- ((l-ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-7-(3-(4-(2- hydroxyethyl)piperazin-l-yl)propoxy)- 2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-et hyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d]imidazole-5 -carboxamide can be prepared according to Example 43 of WO'147. (E)-4-((5-carbamoyl-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH - pyrazole-5-carboxamido)-lH-benzo[d]imidazol-l-yl)but-2-en-l- yl)-2-(l-ethyl-3-methyl-lH-pyrazole- 5-carboxamido)-lH-benzo[d]imidazol-7-yl)oxy)butanoic acid can be prepared according to Example 52 of WO'147.

Example 1: Pre-formulation studies

An exemplified immunogenic composition of the invention has been tested to assess (i) the compatibility between the antigen and the STING agonist and (ii) the adsorption of the antigen and the STING agonist on alum, respectively. The HSV2 gE-gl heterodimer tested herein consisted of the HSV2 gE having the amino acid sequence shown in SEQ ID NO: 5 (ectodomain) associated in a non- covalent complex with the HSV2 gl having the amino acid sequence shown in SEQ ID NO: 6 (ectodomain). The alum used was aluminium hydroxide (AI(OH)s). The STING agonist used was 3- (((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-me thyl-lH-pyrazole-5-carbonyl)imino)-7- methoxy-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)- 2-((l-ethyl-3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-4-yl)oxy)pro pyl dihydrogen phosphate (referred to as Compound 1 hereafter):

Table 1

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

Results

Considering groups 2, 3 and 4 (STING agonist in aqueous formulation, with no added AI(OH)s), the same amount of STING agonist was found in the non-centrifuged samples and in the supernatant of centrifuges samples indicating that no aggregation or precipitation occurs in the presence of the antigen. Likewise, the same profile of gE-gl was observed by both SDS PAGE and SEC HPLC on noncentrifuged samples and in the supernatant of centrifuges samples, indicating that no aggregation or precipitation occurs in the presence of the STING agonist.

Considering the groups 5, 6, 7 and 8 (STING agonist + AI(OH)s), after centrifugation of the samples, no STING agonist was found in the supernatant, indicating that the entire amount of the STING agonist was adsorbed on AI(OH)s in the presence of the antigen. Likewise, for the groups 5, 6 and 7, after centrifugation of the samples, no antigen was detected in the supernatant, indicating that the entire amount of the antigen was adsorbed on AI(OH)s in the presence of the STING agonist. Only the group with a lower amount of AI(OH)s (group 8) showed a reduced adsorption (with 30% of the antigen being adsorbed).

1.2 Next, the minimal amount of AI(OH)s needed to fully adsorb the STING agonist was determined. Compound 1 was used. The amount of the STING agonist was fixed at 45 pg/ml and AL(OH)s varied from 0 to 200 pg/ml (by reference to Al ³⁺ ), so that different ratios STING agonist:AI(OH)3 were tested. The different formulations (as indicated in Table 2) were mixed under agitation for lh. Samples were then centrifuged for 10 min at 8000 rpm. The supernatant of the centrifuged samples was then analysed to characterize the amount of STING agonist which was not adsorbed. The quantification was performed by fluorometry. Results are presented in Fig. 1.

Table 2

Results

It was demonstrated that the STING agonist is generally well adsorbed on AI(OH)s. This study shows that the maximum ratio STING agonist to aluminium hydroxide allowing for an optimal adsorption is about 1.5. Ratios between 0.45 and 1.5 allow a quasi-complete adsorption. This indicates that, even at a higher concentration of STING agonist, a very low dose of AI(OH)s may be used (lower than the 500 pg that is typically found in alum-based vaccines).

Example 2: Evaluation of the immunogenicity of a recombinant HSV-2 qE-ql antigen model adiuvanted with STING agonist and aluminium hydroxide

The same HSV-2 gE-gl antigen recombinantly expressed was formulated with different doses of either a soluble form of STING agonist alone or formulated with AI(OH)3. Total HSV-2 gE and gl IgG titers, Total HSV-1 gE-gl IgG titers, HSV-2 gE and gl CD4+ T cell responses, HSV-1 gE and gl CD4+ T cell responses, and antibody functionality were looked at, and compared with the same HSV- 2 gE-gl antigen adjuvanted with AS01 (a liposome-based adjuvant comprising a saponin and a TLR4 agonist; Adjuvant system AS01: helping to overcome the challenges of modern vaccines, Expert Rev Vaccines. 2017 Jan;16(l):55-63.). Study design:

In this study, 9 groups of naive female CB6F1 mice (n=8 per group, except group 1 (control) n=4), 6 to 8 weeks old at study start were injected intra-muscularly (IM) at 2 weeks interval in left gastrocnemius muscle with 50pL of either non-adjuvanted HSV-2 gE-gl protein, or different doses of STING agonist-adjuvanted gE-gl or different doses of STING agonist/AI(OH)3 formulations with HSV- 2 gE-gl or ASOl-adjuvanted HSV-2 gE-gl.

Serum samples were collected 14 days (Gpl-Gp9) post one and two immunizations to evaluate the total anti- HSV-2 gE & gl antibody response. Cross-reactive HSV-1 gE-gl specific antibody responses and the antibody function (Competitive ELISA and mFcyRIV binding activity/ADCC like assay) were only assessed after two immunizations. Spleen were collected 14 days (Gpl-Gp9) after two immunizations to assess the frequencies of anti-HSV-2 gE & gl-specific CD4+/CD8+ T cells expressing IL-2, TNF-a IFN-y , IL-13 and/or IL-17 cytokines. A summary of the study design and formulation is shown in the below table. Compound 1 was used as the STING agonist.

Table 3: Study design

* "PI" = Post-first immunization / "14dPI" means 14 days post-first immunization

** "PH" = Post-second immunization /"14dpII" means 14 days post-second immunization

*** 5pg AS01 refers to a liposomal formulation containing 5pg MPL and 5pg QS__21

2.1 Detection of total anti-HSV-2 gE and al laG antibodies bv ELISA

Quantification of the total HSV-2 gE or gl-specific IgG antibodies was performed using indirect ELISA. Recombinant HSV-2 gE (~51kDa) (BMP1291) or HSV-2 gl proteins (~46kDa) (BMP1292) were used as coating antigens. These proteins were produced using the ExpiHEK293FTM expression system.

Polystyrene 96-well ELISA plate (Nunc F96 Maxisorp cat 439454) were coated with lOOpL/well of antigen diluted at a concentration of 2 pg/mL (HSV-2 gE) and 1 pg/mL (HSV-2 gl) in carbonate/bicarbonate 50mM pH 9.5 buffer and incubated overnight at 4°C. After incubation, the coating solution was removed and the plates were blocked with 200pL/well of Difkomilk 10% diluted in PBS (blocking buffer) (Becton Dickinson, USA) for 1 h at 37°C. The blocking solution was removed and a three-fold (sera 14dPI & 14dPII) sera dilutions (in PBS + 0.1% Tween20 + 1% BSA buffer) were added to the coated plates and incubated for lh at 37°C. The plates were washed four times with PBS 0.1% Tween20 (washing buffer) and Peroxydase conjugated AffiniPure Goat anti-mouse IgG (H+L) (Jackson, USA) was used as a secondary antibody. One hundred microliters per well of the secondary antibody diluted at a concentration of 1:500 in PBS + 0.1% Tween20 + 1% BSA buffer was added to each well and the plates were incubated for 45min at 37°C.The plates were then washed four times with washing buffer and 2 times with deionised water and incubated for lOmin at RT (room temperature) with 100 pL/well of a solution of 75% single-component TMB Peroxidase ELISA Substrate (Bio-Rad, USA) diluted in sodium Citrate 0.1M pH5.5 buffer. Enzymatic color development was stopped with lOOpL of 0,4N Sulfuric Acid (H2SO4) per well and the plates were read at an absorbance of 450/620nm using the Versamax ELISA reader.

Optical densities (OD) were captured and analysed using the SoftMaxPro GxP v5.3 software. A standard curve was generated by applying a 4-parameter logistic regression fit to the reference standard results (reference standard anti-HSV-2 gE = 14PIII - Pool of mice 1.1 to 1.20 immunized with 5pg of HSV-2 gE/ASOl /dose; reference standard anti-HSV-2 gl = 14PII - Pool of mice 2.1 to 2.10. immunized with 5pg of HSV-2 gl/ASOl/dose). Antibody titer in the samples was calculated by interpolation of the standard curve. The antibody titer of the samples was obtained by averaging the values from dilutions that fell within the 20-80% dynamic range of the standard curve. ELISA titers were normalized at the same starting dilution to allow titers comparisons.

2.2 Detection of the total anti-HSV-1 gE-gl-specific IgG antibodies bv ELISA

The total anti-HSV-1 gE-gl-specific IgG antibodies was assessed using indirect ELISA. Recombinant gE-gl heterodimer protein (BMP1299) from HSV-1 were used as coating antigen. This protein was produced using the ExpiCHO™ expression system.

The rest of procedure was the same as for the quantification of total HSV-2 gE or gl-specific IgG antibodies previously described, except that the polystyrene 96-well ELISA plate were coated with lOOpL/well of recombinant HSV-1 gE-gl heterodimer. Results

All vaccine formulations were immunogenic as antibody responses were significantly higher than the one observed with the antigen alone (see Fig. 2, Fig. 3 and Fig. 4). A clear boost in the antibody response was observed between the first and second dose of the vaccine. No clear dose effect was observed with soluble STINGa or STINGa/AI(OH)3. Moreover, no significant difference in terms of HSV-2 gE and HSV-2 gl antibody titers was observed between the STINGa/AI(OH)3 ratios tested.

The comparison between the two STINGa-based vaccines after 2 immunizations suggests that STINGa/AI(OH)3 formulation induced similar or higher HSV-2 gE specific IgG antibody titers than the soluble STINGa formulation (Fig. 3). For HSV-2 gl response, higher antibody titers were observed with STINGa/AI(OH)3 formulations, as compared with the soluble STINGa formulations, with a fold-increase ranging from 3.3 to 4 (Fig. 2).

STINGa/AI(OH)3 formulations tended to induce higher HSV-2 gE specific IgG titers in comparison to AS01 (Fig. 3). In terms of HSV-2 gl specific titers, STINGa formulations induced similar responses as the AS01 formulation (Fig. 2).

Similar observations were made for the anti-HSV-1 gE-gl cross-reactive IgG antibody response in terms of dose range (comparison within the two STINGa formulations and comparison with AS01 (see Fig. 4).

2.3 Evaluation of anti-HSV-2 and HSV-1 gE and gl CD4+ T cell responses bv Intracellular cytokine staining (ICS)

The frequencies of vaccine-specific CD4+ producing IL-2 and/or IFN-y and/or TNF-a and/or IL-13 and/or IL-17 were evaluated in splenocytes collected 14 days after second immunization after ex-vivo stimulation with HSV-2 gE or gl peptides pools or with HSV-1 gE or gl peptides pools.

Isolation of splenocytes: Spleens were collected from individual mouse 14 days after second immunization and placed in RPMI 1640 medium supplemented with RPMI additives (Glutamine, Penicillin/streptomycin, Sodium Pyruvate, non-essential amino-acids & 2- mercaptoethanol) (= RPMI/additives). Cell suspensions were prepared from each spleen using a tissue grinder. The splenic cell suspensions were filtered (cell Stainer 100pm) and then the filter was rinsed with 35mL of cold RPMI/additives. After centrifugation (335g, lOmin at 4°C), cells were resuspended in 5mL of cold RPMI/additives. Splenic cell suspensions were again filtered (cell Stainer 100pm) before a second washing step was performed, as previously described, and the cells were finally resuspended in 2mL of RPMI/additives supplemented with 5% FCS. Cell suspensions were then diluted 20x (lOpL) in PBS buffer (190pL) for cell counting (using MACSQuant Analyzer). After counting, cells were centrifuged (335 g, 10 min at RT) and resuspended at 10 ⁷ cells/mL in RPMI/additives supplemented with 5% FCS. Cell preparation: Fresh splenocytes were seeded in round bottom 96-well plates at 10 ⁶ cells/well (lOOpL). The cells were then stimulated for 6 hours (37°C, 5% CO2) with anti-CD28 (clone 37.51) and anti-CD49d antibodies (clone 9C10 (MFR4.B) at lpg/mL per well, containing lOOpL of either:

- 15 mers overlapping peptides pool covering the sequences of gE protein from HSV-2 (lpg/mL per peptide per well).

- 15 mers overlapping peptides pool covering the sequences of gl protein from HSV-2 (lpg/mL per peptide per well).

- 15 mers overlapping peptides pool covering the sequences of gE protein from HSV-1 (lpg/mL per peptide per well).

- 15 mers overlapping peptides pool covering the sequences of gl protein from HSV-1 (lpg/mL per peptide per well).

- 15 mers overlapping peptides pool covering the sequences of Human 0-actin protein (lpg/mL per peptide per well) (irrelevant stimulation).

- RPMI/additives medium (as negative control of the assay).

- PMA - ionomycin solution at working concentrations of 0.25 pg/mL and 2.5 pg/mL respectively (as positive control of the assay).

After 2 hours of ex vivo stimulation, Brefeldin A (Golgi plug,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 was added for 4 additional hours to inhibit cytokine secretion. Plates were then transferred at 4°C for overnight incubation.

Intracellular Cytokine Staining: After overnight incubation at 4°C, cells were transferred to V-bottom 96-well plates, centrifuged (189g, 5min at 4°C) and washed with 250pL of cold PBS +1% FCS (Flow buffer). After a second centrifugation (189g, 5min at 4°C), cells were resuspended to block unspecific antibody binding (10 min at 4°C) in 50pL of Flow buffer containing anti-CD16/32 antibodies (clone 2.4G2) diluted 1/50. Then, 50 pL Flow Buffer containing mouse anti-CD4-A700 antibodies (clone RM4-5, diluted at 1/100), and Live/Dead™ Fixable near-IR dead cell stain (diluted at 1/500) was added for 30min in obscurity at 4°C. After incubation, lOOpL of Flow buffer was added into each well and cells were then centrifuged (189g for 5 min at 4°C). A second washing step was performed with 200pL of Flow buffer and after centrifugation, cells were fixed and permeabilized by adding 200pL of Cytofix- Cytoperm solution for 20min at 4°C in the obscurity. After plates centrifugation (500g for 5 min at 4°C), cells were washed with 200pL of Perm/Wash buffer, centrifuged (500g for 5 min 4°C) and resuspended in 50pL of Perm/Wash buffer containing mouse anti-IL2-FITC (clone JES6-5H4, diluted 1/400), anti- IFN-y-APC (clone XMG1.2, diluted 1/200) and anti-TNF-a-PE (clone MP6-XT22, diluted 1/700) and anti-IL-13 PeCy7 (clone ebiol3A , diluted 1/50) and anti-IL-17 BV605 (clone TC11-18H10 , diluted 1/100) antibodies, for 1 hour at 4°C in the obscurity. After incubation, lOOpL of Perm/wash buffer was added into each well and cells were then finally washed with 200pL of Perm/Wash buffer (centrifugation 500g for 5 min a 4°C) and resuspended in 220pL PBS.

Cell acquisition and analysis Stained cells were acquired by flow cytometry and analyzed using the FlowJo software. Live cells were identified with the Live/Dead staining and then lymphocytes were isolated based on Forward/Side Scatter lights (FSC/SSC) gating. The acquisition was performed on ~ 20.000 CD4+T-cell events. The percentages of IFN-y ^+/_ IL-2 ^+/_ TNF-a ^+/_ IL-13 ^+/_ and IL-17 ^+/_ producing cells were calculated on CD4+ and cell populations. For each sample, unspecific signal detected after medium stimulation was removed from the specific signal detected after peptide pool stimulation.

Results

Individual frequencies of CD4+ T cell expressing at least one cytokine among IL-2, TFN-a, IFN-y, IL-13 or IL-17 observed after two injections of HSV-2 gE-gl adjuvanted with soluble STINGa, STINGa/AI(OH)3, and AS01 are presented in Fig. 5.

While for soluble STINGa there is no dose dependency, an inverse dose range was observed with the STINGa/AI(OH)3 formulations, with the higher CD4+ T cell responses observed with the lower STINGa dose (0.74 pg). In addition, no significant difference in terms of HSV-2 gE and gl CD4+ specific T cells can be observed between the two AI(OH)s ratios (Fig. 5 and Fig. 6, respectively).

When compared to AS01, HSV-2 gE-gl adjuvanted with STINGa (0.74 pg)/AI(OH)s (5.55pg) also showed significantly higher HSV-2 gE and gl specific CD4+ T cell responses (Fig. 5 and Fig. 6, respectively). However, no difference was observed between the soluble STINGa formulation and AS01 for any of the tested antigens.

Finally, similar observations were made for the anti-HSV-1 gl and gE cross-reactive CD4+ T cell responses in terms of dose range (comparison within the two STINGa formulations and comparison with AS01) (Fig. 7 and Fig. 8, respectively).

2.4 Evaluation of CD4+ T cell polvfunctionalitv profile

T-cell polyfunctionality was next looked at. The capacity of CD4+ T cells to produce one or multiple cytokines was assessed, and the T cell profile was compared between the different formulations. As the 0.74 pg STINGa dose induced the highest level of CD4+ T cells response, this dose was used for the comparison. Proportions of HSV-2 gE -specific CD4+ T cells expressing 1, 2, 3, or 4 cytokines after in vitro stimulation were determined and represented in Fig. 9. Pie charts represent the mean proportions of cells expressing single markers and any combination of IFN-y, IL-2, TNF-a, IL-13 and IL-17 marker- positive CD4+ T cells out of the total HSV-2 specific CD4+ T cells.

Results

It was observed that the profile obtained with AS01 and the soluble STINGa formulation were similar with a dominance of double positive cells. In contrast, with STINGa/AI(OH)3, the proportion of cells expressing 3 cytokines was higher suggesting an increased CD4+ T cell polyfunctionality with STINGa/AI(OH)3 as compared with soluble STINGa formulations and AS01. Similar results were obtained for HSV-2 gl (Fig. 10).

Example 3: Evaluation of the innate immune response induced bv a STINGa adiuvanted vaccine

The kinetic and the intensity of the systemic innate immune response induced by the STINGa formulations (soluble and alum-formulated) were also assessed to evaluate if STINGa/AI(OH)3 formulations reduce the potential systemic effect. The recombinant gE protein of VZV (SEQ ID No. 7) was used as the antigen. Those data were compared with an AS01 formulation. The STINGa was Compound 1.

Study design

4 groups of Female 6-8 week old C57BL/6JOIaHsd Mice were injected intra-muscularly (IM) in left gastrocnemius on days 0 with 50 pl/site containing 5 pg VZV gE only (negative control group), 5 pg VZV gE formulated either with soluble STINGa, with STINGa/AI(OH)3 or with AS01 (positive control group). To assess the kinetic of the innate response induced by the different vaccine formulations, sera were taken at different time points (3h, 6h, 24h and 48h) post-immunization (n= 5 mice/group/time point). For the AS01 group (Gp 1), the responses were evaluated at 6h only, which corresponds to the peak of the response. To define a baseline for each cytokine measured, a bleeding was performed on 30 mice prior to vaccination and 5 pools of 6 mice were realized.

Results

Innate cytokine responses induced by the two STINGa formulations (soluble and alum- formulated) peak at 6h for the majority of innate markers tested and returns to baseline within 24h or 48h (IL-6, IP-10 and IFN-y in Fig. 11). For Type 1 IFNs (IFN-a and IFN-b), the peak was at 3h (Fig. 12).

The adsorption of STINGa on AI(OH)s decreases the innate cytokine response in serum with respect to the ones observed with the soluble STINGa formulation. This effect was mainly observed for IFN-a and IFN-b (Fig. 12) and IL-6 and IFN-y (Fig. 11).

As compared with AS01 (6h), both STINGa formulations induced the following: ■ High level of Type 1 IFNs (IFN-a and IFN-b) which were not induced by AS01 (STINGa specificity) (Fig. 12).

■ Lower level of IFN-y and IL-6 and (Fig. 11).

■ Similar or slightly higher level of IP-10 (Fig. 11).

Altogether, these results suggest that the adsorption of STINGa on AI(OH)s decreases the systemic cytokine response induced by the STINGa. Both STINGa formulations, as compared with AS01, induce lower levels of markers known to be associated with systemic reactogenicity suggesting an acceptable tolerogenic profile for the two STINGa formulations.

Example 4: Determination of the immunogenicity induced bv a STINGa adiuvanted vaccine in comparison with alum alone and AS01

The same HSV-2 gE-gl antigen recombinantly expressed was used to evaluate an adjuvant composition comprising a STING agonist (Compound 1 was used) adsorbed on AI(OH)s) in comparison to AI(OH)s alone and AS01.

Study design

In this study, 5 groups of naive female CB6F1 mice (n=13 per group, except group 1 (control) n=4), 6 to 8 week-old at study start were injected intra-muscularly (IM) at 2 weeks interval in the left gastrocnemius muscle with 50pL of either non-adjuvanted gE-gl protein, AI(OH)3-formulated gE-gl protein, different doses of STING agonist/AI(OH)3 adjuvanted gE-gl or ASOl-adjuvanted gE-gl.

Serum samples were collected 14 days post two immunizations to evaluate the total anti- HSV- 2 gE-gl antibody levels and functions (Competitive ELISA and mFcyRIII binding activity/ADCC like assay). Spleen were collected 14 days after two immunizations to assess the frequencies of anti-HSV- 2 gE & gl-specific CD4+/CD8+ T cells expressing IL-2, TNF-a IFN-y, IL-13 and/or IL-17 cytokines.

A summary of the study design and formulation is shown in the below table. Compound 1 was used as the STING agonist.

* "PI" = Post-first immunization / "14dPI" means 14 days post-first immunization

** "PH" = Post-second immunization / "14dpII" means 14 days post-second immunization

*** 5pg AS01 refers to a liposomal formulation containing 5pg MPL and 5pg QS__21

Detection of HSV-2 gE or gl-specific IgG antibodies and HSV-1 gE-gl-specific IgG antibodies was performed as previously described.

Results

All adjuvanted vaccine formulations were immunogenic as antibody responses were much higher than the one observed with the antigen alone (Figures 13 and 14). Superiority of an adjuvant formulation comprising a STING agonist and AI(OH)s over AI(OH)s alone was demonstrated in terms of HSV-2 gE or gl specific IgG titers with a geometric mean ratios (GMR) within 3.4 and 4, Thl profile and T cell polyfunctionality (see Figures 13-17).

Overall Results

Altogether, the results showed a good immunogenicity of the recombinant HSV-2 gE-gl protein adjuvanted with different doses of STINGa (soluble or alum-formulated) after one and/or two injections, and an acceptable tolerogenic profile. A benefit of the STINGa/AI(OH)3 formulation was demonstrated, inducing higher functional antibodies and a higher CD4+ T cell response than both AS01 and soluble STINGa formulations. Moreover, STINGa/AI(OH)3 formulations induced CD4+ T cells with a higher polyfunctionality, as compared with AS01 and soluble STINGa formulations. No difference was observed between the two AI(OH)s ratios tested in terms of total IgG, functional antibodies or CD4+ T cells.

These results demonstrate that the adjuvant composition of the present invention has excellent immunogenicity when combined with an antigen, and presents some superiority in comparison to both soluble STINGa formulations and alternative adjuvants, such as AS01. Embodiments of the invention:

The present invention relates to the following embodiments:

1. An adjuvant composition comprising:

(i) a STING agonist of Formula (I) or a pharmaceutically acceptable salt thereof wherein

X is — halo(Ci-Cs)alkyl, unsubstituted -C1-C5 alkyl, or unsubstituted -C2-C5 alkenyl;

R ¹ and R ⁹ are independently H, halogen, hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, optionally substituted Ci-Ce alkyl or optionally substituted Ci-Ce alkyloxy, wherein optionally substituted means substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-P(O)(R ^I )2, C1-C4 alkoxyl, -N(R ^A )2, -CC>2(R ^B ), optionally substituted phenyl, and optionally substituted 5-6 membered heterocycloalkyl, wherein said optionally substituted phenyl, or optionally substituted 5-6 membered heterocycloalkyl is optionally substituted by 1-4 substituents each independently selected from halogen, hydroxy, -O-P(O)(OH)2, -O-P(O)(R ^I )2, amino, (Ci-Ce alkyl)amino-, (Ci-Ce alkyl)(Ci-Ce alkyl)amino-, halo(Ci-Ce alkyl), hydroxy-(Ci-C ₄ alkyl)-, -(C1-C4 alkyl)-O-P(O)(OH) ₂ , -(Ci-C ₄ alkyl)-O-P(O)(R ^I )2, ha lo(Ci-C4 alkoxy)-, C1-C4 alkoxy-, hydroxy-(C2-C4 alkoxy)-, -(C2-C4 alkoxy)- O-P(O)(OH) ₂ , -(C2-C4 alkoxy)-O-P(O)(R ^I ) ₂ , -(Ci-C ₆ alkyl)-NH ₂ , -C1-C4 alkyl-(Ci- C4 alkoxyl) and C1-C4 alkoxy-(Ci-C4 alkoxy)-, wherein R ^A and R ^B are each independently selected from hydrogen, -C1-C4 alkyl, -CC C1-C4 alkyl), - OCC C1-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CO2(Ci- C ₄ alkyl),

R ² and R ⁷ are each independently hydrogen, -CON(R ^C )2, -COOH, or CC>2(R ^D ), or one of R ² and R ⁷ is -CON(R ^C )(R ^D ) and the other is H, -COOH, or CO2(R ^E ), wherein R ^c and R ^D are each independently selected from hydrogen, -C1-C4 alkyl, -CO(Ci-C4 alkyl), - OCO(Ci-C4 alkyl), -(C1-C4 alkyl)-NH2, -(C1-C4 alkyl)-Ci-C4 alkoxyl, or -CO2(Ci-C4 alkyl); R ³ and R ⁸ are each independently H, halo(Ci-Cealkyl), halo(Ci-Cealkoxy)-, hydroxy, -O-P(O)(OH) ₂ , -O-P(O)(R ^I ) ₂ , -NR ^C R ^D , -COR ^C , -CO ₂ R ^C , -N(R ^D )COR ^C , -N(R ^D )SO 2R ^C , -N(R9)SO2(Ci-C ₂ alkyl)-N(R ^h )(R ^f ), -N(R ^g )CO(Ci-C ₂ alkyl)-N(R ^h )(R ^f );

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 each occurrence of R ¹ is independently Ci-Ce alkyloxy-; and

(ii) aluminium hydroxide, aluminium phosphate, aluminium oxyhydroxide, or aluminium hydroxyphosphate, or a combination thereof.

2. The adjuvant composition according to embodiment 1, wherein the STING agonist has the structure of Formula (II) or a pharmaceutically acceptable salt thereof

wherein X, R ¹ , R ⁵ , R ⁶ , R ⁹ , R ¹⁰ and R ¹¹ are as defined in embodiment 1. The adjuvant composition according to embodiment 1 or 2, wherein R ¹ and R ⁹ are each independently H, halogen, optionally substituted (Ci-Cealkyl), or optionally substituted (Ci- Cealkyljoxy-, and the Ci-Cealkyl of said optionally substituted (Ci-Cealkyl), optionally substituted (Ci-Cealkyl)oxy- is optionally substituted with 1-4 substituents each independently selected from the group consisting of hydroxyl, -O-P(O)(OH)2, -O-PCOXR ¹ ^, -N(R ^e )(R ^f ), Ci- C4alkoxyl, phenyl, optionally substituted 5-6 membered heterocycloalkyl containing at least one nitrogen or oxygen as a member of the ring, each R ^e is independently selected from H, (CiOalkyl), -(Ci-C4alkyl)-NH2, or -(Ci-C4alkyl) Ci-C4alkoxy and each R ^f is independently H or (Ci-C ₄ alkyl). The adjuvant composition according to embodiment 1 or 2, wherein one of R ¹ and R ⁹ is -0- P(O)(OH)2, -O-P(O)(R ^I )2, CI-C ₆ alkyl substituted with -O-P(O)(OH)2 or -O-P(O)(R ^I )2, or a Ci- Ce alkyloxy group. The adjuvant composition according to any one of embodiments 1 to 4, wherein R ³ and/or R ⁹ is H. The adjuvant composition according to any one of embodiments 1 to 5, wherein R ⁴ and/or R ¹² is H. 7. The adjuvant composition according to any one of embodiments 1 to 6, wherein R ⁶ and/or R ¹⁰ is ethyl.

8. The adjuvant composition according to any one of embodiments 1 to 7, wherein at least one of R ¹ and R ⁹ is selected from the following groups: ,

0 or N(R ^X ) wherein R ^x is a Ci-Ce alkyl group.

9. The adjuvant composition according to any one of embodiments 1 to 8 wherein the STING agonist is selected from the group consisting of:

(E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyr azole-5-carbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-((l-ethyl-3-methyl-lH -pyrazole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-lH-benzo[d]imidazole-5-carboxami de;

4-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl -3-methyl-lH-pyrazole-5- carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-e n-l-yl)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d]imidazol-4 -yl)oxy)butanoic acid; (E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyrazo le-5-carbonyl)imino)-7- methoxy-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-en-l-yl)- 7-(3-(dimethylamino)propoxy)-2-((l- ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazole-5-carboxamide;

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

(E)-l-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3-methyl-lH-pyr azole-5-carbonyl)imino)-7-(3- hydroxypropoxy)-2,3-dihydro-lH-benzo[d]imidazol-l-yl)but-2-e n-l-yl)-2-((l-ethyl-3-methyl-lH- pyrazole-5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d] imidazole-5-carboxamide;

(E)-l-(4-(5-carbamoyl-2-(l-ethyl-3-methyl-lH-pyrazole-5-c arboxamido)-lH- benzo[d]imidazol-l-yl)but-2-en-l-yl)-2-(l-ethyl-3-methyl-lH- pyrazole-5-carboxamido)-7-(3- morpholinopropoxy)-lH-benzo[d]imidazole-5-carboxamide;

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

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

3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl -3-methyl-lH-pyrazole-5- carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d]imidazol-l- yl)but-2-en-l-yl)-2-((l-ethyl-3- methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH-benzo[d] imidazol-4-yl)oxy)propyl dihydrogen phosphate; or a pharmaceutically acceptable salt thereof.

10. The adjuvant composition according to any one of embodiments 1 to 9, wherein the STING agonist is 3-(((E)-6-carbamoyl-3-((E)-4-((E)-5-carbamoyl-2-((l-ethyl-3- methyl-lH-pyrazole- 5-carbonyl)imino)-7-methoxy-2,3-dihydro-lH-benzo[d]imidazol- l-yl)but-2-en-l-yl)-2-((l- ethyl-3-methyl-lH-pyrazole-5-carbonyl)imino)-2,3-dihydro-lH- benzo[d]imidazol-4- yl)oxy)propyl dihydrogen phosphate as represented by the below formula:

11. The adjuvant composition according to any one of embodiments 1 to 10, wherein the adjuvant composition comprises aluminium hydroxide.

12. The adjuvant composition according to embodiment 11, wherein the STING agonist is adsorbed on the aluminium hydroxide.

13. The adjuvant composition according to embodiment 12, wherein an amount of the STING agonist adsorbed on the aluminium hydroxide is greater than 80%, 85%, 90%, or 95%.

14. The adjuvant composition according to any one of embodiments 11 to 13, wherein the adjuvant composition comprises aluminium hydroxide in an amount of 50 to 500 pg per dose.

15. The adjuvant composition according to any one of embodiments 1 to 14, wherein the adjuvant composition comprises an amount of the STING agonist between 0.5 to 250 pg per dose.

16. The adjuvant composition according to any of embodiments 1 to 15 wherein the adjuvant composition comprises an amount of the STING agonist between 1 and 100 pg per dose, and comprises aluminium hydroxide in an amount of 50 to 400 pg per dose.

17. The adjuvant composition according to embodiment 16 wherein the adjuvant composition comprises an amount of the STING agonist between 1 and 50 pg per dose, and comprises aluminium hydroxide in an amount of 100 to 400 pg per dose.

18. The adjuvant composition according to embodiment 17 wherein the adjuvant composition comprises an amount of the STING agonist of 3.1 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose. 19. The adjuvant composition according to embodiment 17 wherein the adjuvant composition comprises an amount of the STING agonist of 6.3 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose.

20. The adjuvant composition according to embodiment 17 wherein the adjuvant composition comprises an amount of the STING agonist of 12.5 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose.

21. The adjuvant composition according to embodiment 17 wherein the adjuvant composition comprises an amount of the STING agonist of 25 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose.

22. The adjuvant composition according to embodiment 17 wherein the adjuvant composition comprises an amount of the STING agonist of 50 pg per dose, and comprises aluminium hydroxide in an amount of 375 pg per dose.

23. The adjuvant composition according to any one of embodiments 1 to 10, wherein the adjuvant composition comprises aluminium phosphate.

24. The adjuvant composition according to embodiment 23, wherein the STING agonist is adsorbed on the aluminium phosphate.

25. The adjuvant composition according to embodiment 24, wherein an amount of the STING agonist adsorbed on the aluminium phosphate is greater than 80%, 85%, 90%, or 95%.

26. The adjuvant composition according to any one of embodiments 1 to 10, wherein the adjuvant composition comprises aluminium oxyhydroxide.

27. The adjuvant composition according to embodiment 26, wherein the STING agonist is adsorbed on the aluminium oxyhydroxide.

28. The adjuvant composition according to embodiment 27, wherein an amount of the STING agonist adsorbed on the aluminium oxyhydroxide is greater than 80%, 85%, 90%, or 95%.

29. The adjuvant composition according to any one of embodiments 1 to 10, wherein the adjuvant composition comprises aluminium hydroxyphosphate. 30. The adjuvant composition according to embodiment 29, wherein the STING agonist is adsorbed on the aluminium hydroxyphosphate.

31. The adjuvant composition according to embodiment 30, wherein an amount of the STING agonist adsorbed on the aluminium hydroxyphosphate is greater than 80%, 85%, 90%, or 95%.

32. An immunogenic composition comprising:

(i) the adjuvant composition according to any one of embodiments 1 to 31; and

(ii) an antigen.

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.

35. The immunogenic composition according to embodiment 34, wherein the antigen is derived from a human pathogen selected from the group consisting of bacteria, virus, 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.

37. The immunogenic composition according to embodiment 35, wherein the human pathogen is selected from coronavirus, Herpes simplex virus (HSV), HIV, hepatitis B virus, hepatitis C virus, meningitis B, Haemophilus influenza 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.

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 the antigen is an HSV- 2 gE-gl 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 a SARS-CoV-2 S protein.

43. The immunogenic composition according to embodiment 42, wherein the SARS-CoV-2 S protein is a pre-fusion stabilised S protein.

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.

45. The immunogenic composition according to any of embodiments 32 to 44, wherein the adjuvant composition comprises aluminium hydroxide and the antigen is adsorbed on the aluminium hydroxide.

46. The immunogenic composition according to embodiment 45, wherein the antigen and STING agonist are adsorbed on to the same aluminium hydroxide.

47. The immunogenic composition according to embodiment 45, wherein the antigen and STING agonist are adsorbed on to different aluminium hydroxide.

48. The immunogenic composition according to any one of embodiments 45 to 47, wherein an amount of the antigen bound to the aluminium hydroxide is greater than 80%, 85%, 90%, or 95%.

49. The immunogenic composition according to any of embodiments 32 to 44, wherein the adjuvant composition comprises aluminium phosphate and the antigen is adsorbed on the aluminium phosphate.

50. The immunogenic composition according to embodiment 49, wherein the antigen and STING agonist are adsorbed on to the same aluminium phosphate. 51. The immunogenic composition according to embodiment 49, wherein the antigen and STING agonist are adsorbed on to different aluminium phosphate.

52. The immunogenic composition according to any one of embodiments 49 to 51, wherein an amount of the antigen bound to the aluminium phosphate is greater than 80%, 85%, 90%, or 95%.

53. The immunogenic composition according to any of embodiments 31 to 44, wherein the adjuvant composition comprises aluminium oxyhydroxide and the antigen is adsorbed on the aluminium oxyhydroxide.

54. The immunogenic composition according to embodiment 53, wherein the antigen and STING agonist are adsorbed on to the same aluminium oxyhydroxide.

55. The immunogenic composition according to embodiment 53, wherein the antigen and STING agonist are adsorbed on to different aluminium oxyhydroxide.

56. The immunogenic composition according to any one of embodiments 53 to 55, wherein an amount of the antigen bound to the aluminium oxyhydroxide is greater than 80%, 85%, 90%, or 95%.

57. The immunogenic composition according to any of embodiments 32 to 44, wherein the adjuvant composition comprises aluminium hydroxyphosphate and the antigen is adsorbed on the aluminium hydroxyphosphate.

58. The immunogenic composition according to embodiment 57, wherein the antigen and STING agonist are adsorbed on to the same aluminium hydroxyphosphate.

59. The immunogenic composition according to embodiment 57, wherein the antigen and STING agonist are adsorbed on to different aluminium hydroxyphosphate.

60. The immunogenic composition according to any one of embodiment 59 to 59, wherein an amount of the antigen bound to the aluminium hydroxyphosphate is greater than 80%, 85%, 90%, or 95%.

61. The immunogenic composition according to any one of embodiments 32 to 59 for use in therapy. 62. The 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 the 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 separate composition to the adjuvant composition, and is administered separately.

65. A method of immunizing a host comprising administering to the host the immunogenic composition according to any one of embodiments 32 to 60.

66. A combination of the adjuvant composition according to any one of embodiments 1 to 31, and a vaccine formulation comprising an antigen.

67. A method of adjuvanting an immune response in host, said method comprising administering the adjuvant composition according to any one of embodiments 1 to 31.

68. Use of the adjuvant composition according to any one of embodiments 1 to 31 in the manufacture of a medicament for adjuvanting an immune response in a subject.

69. A kit comprising i) a first container comprising the 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.

SEQUENCE LISTINGS

< 110> GlaxoSmithKline Biologicals SA

< 120> Adjuvant composition

< 130> 70067EP01P

<210> 1

<211> 1273

<212> PRT

<213> SARS-CoV-2 S protein

<400> 1

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu

35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Ala He His Vai Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn He lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie

115 120 125

Lys Vai Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Vai Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr

145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Vai Phe Lys Asn He Asp Gly Tyr Phe Lys He Tyr Ser Lys His Thr

195 200 205

Pro lie Asn Leu Vai Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Vai Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala

275 280 285

Vai Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai

305 310 315 320

Gin Pro Thr Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

340 345 350

Trp Asn Arg Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe 385 390 395 400

Vai He Arg Gly Asp Glu Vai Arg Gin He Ala Pro Gly Gin Thr Gly

405 410 415

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

485 490 495

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gin Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai

565 570 575

Arg Asp Pro Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe

580 585 590

Gly Gly Vai Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai

595 600 605

Ala Vai Leu Tyr Gin Asp Vai Asn Cys Thr Glu Vai Pro Vai Ala lie

610 615 620 His Ala Asp Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser

625 630 635 640

Asn Vai Phe Gin Thr Arg Ala Gly Cys Leu He Gly Ala Glu His Vai

645 650 655

Asn Asn Ser Tyr Glu Cys Asp He Pro lie Gly Ala Gly lie Cys Ala

660 665 670

Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Vai Ala

675 680 685

Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Vai Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie

705 710 715 720

Ser Vai Thr Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai

725 730 735

Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr

755 760 765

Gly lie Ala Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin

770 775 780

Vai Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe lie Glu Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly

820 825 830

Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp

835 840 845

Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Vai Leu Pro Pro Leu 850 855 860

Leu Thr Asp Glu Met He Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr He Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie

885 890 895

Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr

900 905 910

Gin Asn Vai Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn

915 920 925

Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gin Asp Vai Vai Asn Gin Asn Ala Gin Ala Leu Asn

945 950 955 960

Thr Leu Vai Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Vai

965 970 975

Leu Asn Asp lie Leu Ser Arg Leu Asp Lys Vai Glu Ala Glu Vai Gin

980 985 990 lie Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai

995 1000 1005

Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys

1025 1030 1035

Arg Vai Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gin Ser Ala Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai

1055 1060 1065

Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His

1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin

1100 1105 1110

He He Thr Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai

1115 1120 1125

Vai lie Gly lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn

1160 1165 1170

Ala Ser Vai Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu

1175 1180 1185

Vai Ala Lys Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gin Tyr lie Lys Trp Pro Trp Tyr lie Trp Leu

1205 1210 1215

Gly Phe lie Ala Gly Leu lie Ala lie Vai Met Vai Thr lie Met

1220 1225 1230

Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys

1235 1240 1245

Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro

1250 1255 1260

Vai Leu Lys Gly Vai Lys Leu His Tyr Thr

1265 1270

<210> 2

<211> 1208 <212> PRT

<213> SARS-CoV-2 S protein ectodomain

<400> 2

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu

35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Ala He His Vai Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser Thr Glu

85 90 95

Lys Ser Asn He lie Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie

115 120 125

Lys Vai Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Vai Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Vai Phe Lys Asn lie Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr

195 200 205 Pro He Asn Leu Vai Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Vai Asp Leu Pro He Gly lie Asn lie Thr Arg Phe Gin Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala

275 280 285

Vai Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai

305 310 315 320

Gin Pro Thr Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys

325 330 335

Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

340 345 350

Trp Asn Arg Lys Arg lie Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe

385 390 395 400

Vai lie Arg Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly

405 410 415

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn 435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp He Ser Thr Glu He Tyr Gin Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

485 490 495

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560

Pro Phe Gin Gin Phe Gly Arg Asp lie Ala Asp Thr Thr Asp Ala Vai

565 570 575

Arg Asp Pro Gin Thr Leu Glu lie Leu Asp lie Thr Pro Cys Ser Phe

580 585 590

Gly Gly Vai Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai

595 600 605

Ala Vai Leu Tyr Gin Asp Vai Asn Cys Thr Glu Vai Pro Vai Ala lie

610 615 620

His Ala Asp Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser

625 630 635 640

Asn Vai Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Vai

645 650 655

Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala

660 665 670 Ser Tyr Gin Thr Gin Thr Asn Ser Pro Arg Arg Ala Arg Ser Vai Ala

675 680 685

Ser Gin Ser He He Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Vai Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie

705 710 715 720

Ser Vai Thr Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai

725 730 735

Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr

755 760 765

Gly lie Ala Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin

770 775 780

Vai Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe

785 790 795 800

Asn Phe Ser Gin lie Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe lie Glu Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly

820 825 830

Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp

835 840 845

Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Vai Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie

885 890 895

Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr 900 905 910

Gin Asn Vai Leu Tyr Glu Asn Gin Lys Leu He Ala Asn Gin Phe Asn

915 920 925

Ser Ala He Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gin Asp Vai Vai Asn Gin Asn Ala Gin Ala Leu Asn

945 950 955 960

Thr Leu Vai Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Vai

965 970 975

Leu Asn Asp lie Leu Ser Arg Leu Asp Lys Vai Glu Ala Glu Vai Gin

980 985 990 lie Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai

995 1000 1005

Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn

1010 1015 1020

Leu Ala Ala Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys

1025 1030 1035

Arg Vai Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gin Ser Ala Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai

1055 1060 1065

Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala lie Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin

1100 1105 1110 lie lie Thr Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai

1115 1120 1125 Vai He Gly He Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn

1160 1165 1170

Ala Ser Vai Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu

1175 1180 1185

Vai Ala Lys Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gin

1205

<210> 3

<211> 192

<212> PRT

<213> SARS-CoV-2 S protein receptor binding domain

<400> 3

Pro Asn lie Thr Asn Leu Cys Pro Phe Gly Glu Vai Phe Asn Ala Thr

1 5 10 15

Arg Phe Ala Ser Vai Tyr Ala Trp Asn Arg Lys Arg lie Ser Asn Cys

20 25 30

Vai Ala Asp Tyr Ser Vai Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe

35 40 45

Lys Cys Tyr Gly Vai Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr

50 55 60

Asn Vai Tyr Ala Asp Ser Phe Vai lie Arg Gly Asp Glu Vai Arg Gin

65 70 75 80 lie Ala Pro Gly Gin Thr Gly Lys lie Ala Asp Tyr Asn Tyr Lys Leu

85 90 95 Pro Asp Asp Phe Thr Gly Cys Vai He Ala Trp Asn Ser Asn Asn Leu

100 105 110

Asp Ser Lys Vai Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg

115 120 125

Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp He Ser Thr Glu lie Tyr

130 135 140

Gin Ala Gly Ser Thr Pro Cys Asn Gly Vai Glu Gly Phe Asn Cys Tyr

145 150 155 160

Phe Pro Leu Gin Ser Tyr Gly Phe Gin Pro Thr Asn Gly Vai Gly Tyr

165 170 175

Gin Pro Tyr Arg Vai Vai Vai Leu Ser Phe Glu Leu Leu His Ala Pro

180 185 190

<210> 4

<211> 1208

<212> PRT

<213> Pre-fusion stabilised SARS-CoV-2 S protein ectodomain

<400> 4

Met Phe Vai Phe Leu Vai Leu Leu Pro Leu Vai Ser Ser Gin Cys Vai

1 5 10 15

Asn Leu Thr Thr Arg Thr Gin Leu Pro Pro Ala Tyr Thr Asn Ser Phe

20 25 30

Thr Arg Gly Vai Tyr Tyr Pro Asp Lys Vai Phe Arg Ser Ser Vai Leu

35 40 45

His Ser Thr Gin Asp Leu Phe Leu Pro Phe Phe Ser Asn Vai Thr Trp

50 55 60

Phe His Ala lie His Vai Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp

65 70 75 80

Asn Pro Vai Leu Pro Phe Asn Asp Gly Vai Tyr Phe Ala Ser Thr Glu

85 90 95 Lys Ser Asn He He Arg Gly Trp lie Phe Gly Thr Thr Leu Asp Ser

100 105 110

Lys Thr Gin Ser Leu Leu lie Vai Asn Asn Ala Thr Asn Vai Vai lie

115 120 125

Lys Vai Cys Glu Phe Gin Phe Cys Asn Asp Pro Phe Leu Gly Vai Tyr

130 135 140

Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Vai Tyr

145 150 155 160

Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Vai Ser Gin Pro Phe Leu

165 170 175

Met Asp Leu Glu Gly Lys Gin Gly Asn Phe Lys Asn Leu Arg Glu Phe

180 185 190

Vai Phe Lys Asn lie Asp Gly Tyr Phe Lys lie Tyr Ser Lys His Thr

195 200 205

Pro lie Asn Leu Vai Arg Asp Leu Pro Gin Gly Phe Ser Ala Leu Glu

210 215 220

Pro Leu Vai Asp Leu Pro lie Gly lie Asn lie Thr Arg Phe Gin Thr

225 230 235 240

Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser

245 250 255

Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Vai Gly Tyr Leu Gin Pro

260 265 270

Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr lie Thr Asp Ala

275 280 285

Vai Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys

290 295 300

Ser Phe Thr Vai Glu Lys Gly lie Tyr Gin Thr Ser Asn Phe Arg Vai

305 310 315 320

Gin Pro Thr Glu Ser lie Vai Arg Phe Pro Asn lie Thr Asn Leu Cys 325 330 335

Pro Phe Gly Glu Vai Phe Asn Ala Thr Arg Phe Ala Ser Vai Tyr Ala

340 345 350

Trp Asn Arg Lys Arg He Ser Asn Cys Vai Ala Asp Tyr Ser Vai Leu

355 360 365

Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Vai Ser Pro

370 375 380

Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Vai Tyr Ala Asp Ser Phe

385 390 395 400

Vai He Arg Gly Asp Glu Vai Arg Gin lie Ala Pro Gly Gin Thr Gly

405 410 415

Lys lie Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys

420 425 430

Vai lie Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Vai Gly Gly Asn

435 440 445

Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe

450 455 460

Glu Arg Asp lie Ser Thr Glu lie Tyr Gin Ala Gly Ser Thr Pro Cys

465 470 475 480

Asn Gly Vai Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gin Ser Tyr Gly

485 490 495

Phe Gin Pro Thr Asn Gly Vai Gly Tyr Gin Pro Tyr Arg Vai Vai Vai

500 505 510

Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Vai Cys Gly Pro Lys

515 520 525

Lys Ser Thr Asn Leu Vai Lys Asn Lys Cys Vai Asn Phe Asn Phe Asn

530 535 540

Gly Leu Thr Gly Thr Gly Vai Leu Thr Glu Ser Asn Lys Lys Phe Leu

545 550 555 560 Pro Phe Gin Gin Phe Gly Arg Asp He Ala Asp Thr Thr Asp Ala Vai

565 570 575

Arg Asp Pro Gin Thr Leu Glu He Leu Asp lie Thr Pro Cys Ser Phe

580 585 590

Gly Gly Vai Ser Vai lie Thr Pro Gly Thr Asn Thr Ser Asn Gin Vai

595 600 605

Ala Vai Leu Tyr Gin Asp Vai Asn Cys Thr Glu Vai Pro Vai Ala lie

610 615 620

His Ala Asp Gin Leu Thr Pro Thr Trp Arg Vai Tyr Ser Thr Gly Ser

625 630 635 640

Asn Vai Phe Gin Thr Arg Ala Gly Cys Leu lie Gly Ala Glu His Vai

645 650 655

Asn Asn Ser Tyr Glu Cys Asp lie Pro lie Gly Ala Gly lie Cys Ala

660 665 670

Ser Tyr Gin Thr Gin Thr Asn Ser Pro Gly Ser Ala Ser Ser Vai Ala

675 680 685

Ser Gin Ser lie lie Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser

690 695 700

Vai Ala Tyr Ser Asn Asn Ser lie Ala lie Pro Thr Asn Phe Thr lie

705 710 715 720

Ser Vai Thr Thr Glu lie Leu Pro Vai Ser Met Thr Lys Thr Ser Vai

725 730 735

Asp Cys Thr Met Tyr lie Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu

740 745 750

Leu Leu Gin Tyr Gly Ser Phe Cys Thr Gin Leu Asn Arg Ala Leu Thr

755 760 765

Gly lie Ala Vai Glu Gin Asp Lys Asn Thr Gin Glu Vai Phe Ala Gin

770 775 780

Vai Lys Gin lie Tyr Lys Thr Pro Pro lie Lys Asp Phe Gly Gly Phe 785 790 795 800

Asn Phe Ser Gin He Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser

805 810 815

Phe He Glu Asp Leu Leu Phe Asn Lys Vai Thr Leu Ala Asp Ala Gly

820 825 830

Phe lie Lys Gin Tyr Gly Asp Cys Leu Gly Asp lie Ala Ala Arg Asp

835 840 845

Leu lie Cys Ala Gin Lys Phe Asn Gly Leu Thr Vai Leu Pro Pro Leu

850 855 860

Leu Thr Asp Glu Met lie Ala Gin Tyr Thr Ser Ala Leu Leu Ala Gly

865 870 875 880

Thr lie Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gin lie

885 890 895

Pro Phe Ala Met Gin Met Ala Tyr Arg Phe Asn Gly lie Gly Vai Thr

900 905 910

Gin Asn Vai Leu Tyr Glu Asn Gin Lys Leu lie Ala Asn Gin Phe Asn

915 920 925

Ser Ala lie Gly Lys lie Gin Asp Ser Leu Ser Ser Thr Ala Ser Ala

930 935 940

Leu Gly Lys Leu Gin Asp Vai Vai Asn Gin Asn Ala Gin Ala Leu Asn

945 950 955 960

Thr Leu Vai Lys Gin Leu Ser Ser Asn Phe Gly Ala lie Ser Ser Vai

965 970 975

Leu Asn Asp lie Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Vai Gin

980 985 990 lie Asp Arg Leu lie Thr Gly Arg Leu Gin Ser Leu Gin Thr Tyr Vai

995 1000 1005

Thr Gin Gin Leu lie Arg Ala Ala Glu lie Arg Ala Ser Ala Asn

1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Vai Leu Gly Gin Ser Lys

1025 1030 1035

Arg Vai Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro

1040 1045 1050

Gin Ser Ala Pro His Gly Vai Vai Phe Leu His Vai Thr Tyr Vai

1055 1060 1065

Pro Ala Gin Glu Lys Asn Phe Thr Thr Ala Pro Ala He Cys His

1070 1075 1080

Asp Gly Lys Ala His Phe Pro Arg Glu Gly Vai Phe Vai Ser Asn

1085 1090 1095

Gly Thr His Trp Phe Vai Thr Gin Arg Asn Phe Tyr Glu Pro Gin

1100 1105 1110

He lie Thr Thr Asp Asn Thr Phe Vai Ser Gly Asn Cys Asp Vai

1115 1120 1125

Vai lie Gly lie Vai Asn Asn Thr Vai Tyr Asp Pro Leu Gin Pro

1130 1135 1140

Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn

1145 1150 1155

His Thr Ser Pro Asp Vai Asp Leu Gly Asp lie Ser Gly lie Asn

1160 1165 1170

Ala Ser Vai Vai Asn lie Gin Lys Glu lie Asp Arg Leu Asn Glu

1175 1180 1185

Vai Ala Lys Asn Leu Asn Glu Ser Leu lie Asp Leu Gin Glu Leu

1190 1195 1200

Gly Lys Tyr Glu Gin

1205

<210> 5

<211> 419 <212> PRT

<213> Artificial Sequence

<220>

<223> Polypeptide sequence of HSV-2 gE P317R

<400> 5

Met Ala Arg Gly Ala Gly Leu Vai Phe Phe Vai Gly Vai Trp Vai Vai

1 5 10 15

Ser Cys Leu Ala Ala Ala Pro Arg Thr Ser Trp Lys Arg Vai Thr Ser

20 25 30

Gly Glu Asp Vai Vai Leu Leu Pro Ala Pro Ala Gly Pro Glu Glu Arg

35 40 45

Thr Arg Ala His Lys Leu Leu Trp Ala Ala Glu Pro Leu Asp Ala Cys

50 55 60

Gly Pro Leu Arg Pro Ser Trp Vai Ala Leu Trp Pro Pro Arg Arg Vai

65 70 75 80

Leu Glu Thr Vai Vai Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu

85 90 95

Ala He Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr

100 105 110

Ser Glu Leu Ala Trp Arg Asp Arg Vai Ala Vai Vai Asn Glu Ser Leu

115 120 125

Vai He Tyr Gly Ala Leu Glu Thr Asp Ser Gly Leu Tyr Thr Leu Ser

130 135 140

Vai Vai Gly Leu Ser Asp Glu Ala Arg Gin Vai Ala Ser Vai Vai Leu

145 150 155 160

Vai Vai Glu Pro Ala Pro Vai Pro Thr Pro Thr Pro Asp Asp Tyr Asp

165 170 175

Glu Glu Asp Asp Ala Gly Vai Ser Glu Arg Thr Pro Vai Ser Vai Pro

180 185 190 Pro Pro Thr Pro Pro Arg Arg Pro Pro Vai Ala Pro Pro Thr His Pro

195 200 205

Arg Vai He Pro Glu Vai Ser His Vai Arg Gly Vai Thr Vai His Met

210 215 220

Glu Thr Pro Glu Ala He Leu Phe Ala Pro Gly Glu Thr Phe Gly Thr

225 230 235 240

Asn Vai Ser lie His Ala lie Ala His Asp Asp Gly Pro Tyr Ala Met

245 250 255

Asp Vai Vai Trp Met Arg Phe Asp Vai Pro Ser Ser Cys Ala Glu Met

260 265 270

Arg lie Tyr Glu Ala Cys Leu Tyr His Pro Gin Leu Pro Glu Cys Leu

275 280 285

Ser Pro Ala Asp Ala Pro Cys Ala Vai Ser Ser Trp Ala Tyr Arg Leu

290 295 300

Ala Vai Arg Ser Tyr Ala Gly Cys Ser Arg Thr Thr Arg Pro Pro Arg

305 310 315 320

Cys Phe Ala Glu Ala Arg Met Glu Pro Vai Pro Gly Leu Ala Trp Leu

325 330 335

Ala Ser Thr Vai Asn Leu Glu Phe Gin His Ala Ser Pro Gin His Ala

340 345 350

Gly Leu Tyr Leu Cys Vai Vai Tyr Vai Asp Asp His lie His Ala Trp

355 360 365

Gly His Met Thr lie Ser Thr Ala Ala Gin Tyr Arg Asn Ala Vai Vai

370 375 380

Glu Gin His Leu Pro Gin Arg Gin Pro Glu Pro Vai Glu Pro Thr Arg

385 390 395 400

Pro His Vai Arg Ala Pro Pro Pro Ala Pro Ser Ala Arg Gly Pro Leu

405 410 415

Arg Leu Gly <210> 6

<211> 256

<212> PRT

<213> Artificial Sequence

<220>

<223> Polypeptide sequence of HSV-2 gl

<400> 6

Met Pro Gly Arg Ser Leu Gin Gly Leu Ala He Leu Gly Leu Trp Vai

1 5 10 15

Cys Ala Thr Gly Leu Vai Vai Arg Gly Pro Thr Vai Ser Leu Vai Ser

20 25 30

Asp Ser Leu Vai Asp Ala Gly Ala Vai Gly Pro Gin Gly Phe Vai Glu

35 40 45

Glu Asp Leu Arg Vai Phe Gly Glu Leu His Phe Vai Gly Ala Gin Vai

50 55 60

Pro His Thr Asn Tyr Tyr Asp Gly He lie Glu Leu Phe His Tyr Pro

65 70 75 80

Leu Gly Asn His Cys Pro Arg Vai Vai His Vai Vai Thr Leu Thr Ala

85 90 95

Cys Pro Arg Arg Pro Ala Vai Ala Phe Thr Leu Cys Arg Ser Thr His

100 105 110

His Ala His Ser Pro Ala Tyr Pro Thr Leu Glu Leu Gly Leu Ala Arg

115 120 125

Gin Pro Leu Leu Arg Vai Arg Thr Ala Thr Arg Asp Tyr Ala Gly Leu

130 135 140

Tyr Vai Leu Arg Vai Trp Vai Gly Ser Ala Thr Asn Ala Ser Leu Phe

145 150 155 160

Vai Leu Gly Vai Ala Leu Ser Ala Asn Gly Thr Phe Vai Tyr Asn Gly 165 170 175

Ser Asp Tyr Gly Ser Cys Asp Pro Ala Gin Leu Pro Phe Ser Ala Pro

180 185 190

Arg Leu Gly Pro Ser Ser Vai Tyr Thr Pro Gly Ala Ser Arg Pro Thr

195 200 205

Pro Pro Arg Thr Thr Thr Ser Pro Ser Ser Pro Arg Asp Pro Thr Pro

210 215 220

Ala Pro Gly Asp Thr Gly Thr Pro Ala Pro Ala Ser Gly Glu Arg Ala

225 230 235 240

Pro Pro Asn Ser Thr Arg Ser Ala Ser Glu Ser Arg His Arg Leu Thr

245 250 255

<210> 7

<211> 546

<212> PRT

<213 > Polypeptide sequence for VZV gE

<400> 7

Met Gly Thr Vai Asn Lys Pro Vai Vai Gly Vai Leu Met Gly Phe Gly

1 5 10 15

He He Thr Gly Thr Leu Arg lie Thr Asn Pro Vai Arg Ala Ser Vai

20 25 30

Leu Arg Tyr Asp Asp Phe His lie Asp Glu Asp Lys Leu Asp Thr Asn

35 40 45

Ser Vai Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp

50 55 60

Vai Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro

65 70 75 80

Tyr lie Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His

85 90 95

Glu His His Gly Vai Tyr Asn Gin Gly Arg Gly lie Asp Ser Gly Glu 100 105 110

Arg Leu Met Gin Pro Thr Gin Met Ser Ala Gin Glu Asp Leu Gly Asp

115 120 125

Asp Thr Gly He His Vai He Pro Thr Leu Asn Gly Asp Asp Arg His

130 135 140

Lys lie Vai Asn Vai Asp Gin Arg Gin Tyr Gly Asp Vai Phe Lys Gly

145 150 155 160

Asp Leu Asn Pro Lys Pro Gin Gly Gin Arg Leu lie Glu Vai Ser Vai

165 170 175

Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro lie Gin Arg lie Tyr

180 185 190

Gly Vai Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys

195 200 205

Thr Gly Asp Ala Ala Pro Ala lie Gin His lie Cys Leu Lys His Thr

210 215 220

Thr Cys Phe Gin Asp Vai Vai Vai Asp Vai Asp Cys Ala Glu Asn Thr

225 230 235 240

Lys Glu Asp Gin Leu Ala Glu lie Ser Tyr Arg Phe Gin Gly Lys Lys

245 250 255

Glu Ala Asp Gin Pro Trp lie Vai Vai Asn Thr Ser Thr Leu Phe Asp

260 265 270

Glu Leu Glu Leu Asp Pro Pro Glu lie Glu Pro Gly Vai Leu Lys Vai

275 280 285

Leu Arg Thr Glu Lys Gin Tyr Leu Gly Vai Tyr lie Trp Asn Met Arg

290 295 300

Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Vai Thr Trp Lys

305 310 315 320

Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Vai Thr Pro Gin Pro

325 330 335 Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Vai Phe Ser

340 345 350

Vai Gly Asp Thr Phe Ser Leu Ala Met His Leu Gin Tyr Lys He His

355 360 365

Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Vai Pro He Asp

370 375 380

Pro Thr Cys Gin Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro

385 390 395 400

Asn Ala Pro Gin Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr

405 410 415

Ser Pro His Leu Ala Gin Arg Vai Ala Ser Thr Vai Tyr Gin Asn Cys

420 425 430

Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly lie Ser His Met

435 440 445

Glu Pro Ser Phe Gly Leu lie Leu His Asp Gly Gly Thr Thr Leu Lys

450 455 460

Phe Vai Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Vai Phe Vai Vai

465 470 475 480

Tyr Phe Asn Gly His Vai Glu Ala Vai Ala Tyr Thr Vai Vai Ser Thr

485 490 495

Vai Asp His Phe Vai Asn Ala lie Glu Glu Arg Gly Phe Pro Pro Thr

500 505 510

Ala Gly Gin Pro Pro Ala Thr Thr Lys Pro Lys Glu lie Thr Pro Vai

515 520 525

Asn Pro Gly Thr Ser Pro Leu lie Arg Tyr Ala Ala Trp Thr Gly Gly

530 535 540

Leu Ala

545