The invention relates to glyco conjugates and vaccine compositions and the use of

glycoconjugates in the prevention and treatment of HIV infection and AIDS.

HIV-1 is thought to have infected up to 60 million people since its discovery over 20 years ago. Of those infected, more than 20 million have died, with the vast majority of individuals affected being from developing countries. An effective vaccine is, therefore, paramount to combat the epidemic. The HIV-1 envelope spike, critical for viral infectivity, consists of a compact, unstable trimer of the glycoproteins gpl20 and gp41 and is a key target for design of an antibody-based HIV-1 vaccine. The envelope spike undergoes rapid evolution in each individual patient, resulting in enormous sequence heterogeneity among individual isolates of HIV-1. Moreover, neutralization-sensitive epitopes on gpl20 and gp41 are either difficult to access or shielded from recognition by the immune system by an extensive display of host- derived N-glycans. Nevertheless, a small group of rare, broadly neutralizing antibodies (bl2, 2G12, 2F5, 4E10, Z13) against gpl20 and gp41 have been previously isolated from HIV-1- infected patients that provide protection against viral challenge in animal models as well as the more recent discovery of new highly potent human antibodies (PG9, PG16). Structural analyses have revealed how they broadly neutralize HIV-1 and the mechanism by which the virus normally evades detection by the immune system. Identification of antigens that could generate similar types of broadly neutralizing antibodies is, therefore, an important step in the development of an HIV-1 vaccine. From this small group, broadly neutralizing antibody 2G12 is uniquely capable of recognizing sugars on the immunologically "silent"

carbohydrate face of gpl20 (part of HIV's glycan shield) and escaping immune tolerance. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine.

Crystallisation studies have shown that the Dl arm of the (MangGlcNAc ₂ ) oligomannose (Fig. 1) is an epitope of the 2G12 antibody. However, glycans containing the natural sugar D-mannose have previously been thought to be poor candidates for immunogens. One major limitation to the recognition and, hence, immunogenicity of carbohydrate structures is their exhibition of microheterogeneity. A single protein may display many variations of carbohydrate structure (glyco forms) that can result in a polyclonal and reduced antigenic response. Carbohydrate-protein interactions tend also to be weaker than protein-protein interactions (μΜ rather that nM) further reducing affinities of antibodies to glycans. Large glycan structures can also shield underlying peptide epitopes, thereby further reducing the effectiveness of the immunogenic response. Furthermore, viruses typically rely on host glycosylation machinery; their glycosylation patterns are, therefore, inevitably similar to that of the host. As a result, host immune mechanisms such as 2G12 should normally recognize such sugars as self and display tolerance, thus not eliciting antibodies to host derived sugars such as D-mannose. The present inventors, however, have surprisingly found that a series of modified mannose- containing glycoconjugates are capable of eliciting an immune response to the HIV-1 surface glycoprotein gpl20. Thus, the present invention provides a glycoconjugate for use in treating or preventing an HIV infection or AIDS, wherein the glycoconjugate comprises an oligosaccharide group which is linked to a carrier protein, wherein the oligosaccharide group is of formula (I):

(I)

wherein:

- M3 is a modified mannose unit;

each M2 is the same or different and represents a mannose unit or a modified mannose

unit;

M l represents a mannose unit or a modified mannose unit, and the oligosaccharide moiety is linked to the carrier protein via Ml ;

p is 0, or 1;

q is 0 or 1 ;

SI is a saccharide unit or a modified saccharide unit;

r, s and t are zero or an integer from 1 to 5, wherein at least two of r, s and t are zero; wherein the or each modified mannose unit or modified saccharide unit is a mannose unit or saccharide unit having one or more modifications, the or each modification being independently selected from modifications A and B, wherein:

modification A is replacement of a hydrogen atom of the mannose unit or saccharide unit with a CI -3 alkyl group optionally substituted with a hydro xyl group; and

modification B is replacement of a hydroxyl group of the mannose unit or saccharide unit with a hydrogen atom.

The present invention also provides an oligosaccharide of formula (lb)

(lb)

wherein:

- Ml represents a mannose unit or a modified mannose unit, which modified

mannose unit is as defined above;

Each M2 and SI is as defined above;

- p and q are as defined above;

- r, and s and t are as defined above;

- M3 represents a modified mannose unit having (a) modification A at one or more of the CI to C4 and C6 positions, the modified C6 position having (R) configuration, and/or (b) modification B at one or more of the C2 to C5 positions; wherein modifications A and B are as defined above.

In one aspect, the present invention provides a vaccine composition comprising

glycoconjugate of the invention.

In another aspect, the present invention also provides use of a glycoconjugate or

oligosaccharide of the invention in the manufacture of a medicament or vaccine

treatment or prevention of an HIV infection or AIDS. The present invention further provides a method for treating or preventing, or for ameliorating or reducing the incidence of, an HIV infection or AIDS in a subject, which method comprises administering to the subject an effective amount of a glycoconjugate of the invention.

Brief Description of the Figures

Figure 1 depicts the structure of oligomannose (Man ₉ GlcNAc ₂ ) found on the "silent face" of gpl20. Dl, D2 and D3 arms are highlighted.

Figures 2a, 2b and 2c show the results of the gpl20/serum binding assay for rabbit serums 290, 304 and 379, respectively, reported in Example 16. Figures 3, 4 and 5 show the results of the conjugate/serum assay reported in Example 17.

Figure 6 shows the results of the CFG glycan array assay reported in Example 18.

Figures 7 to 9 show the results of the neutralisation assay reported in Example 19.

Detailed Description of the Invention

In the present invention, the glycoconjugate comprises a mono- or oligosaccharide linked to a carrier protein. The mono- or oligosaccharide may be linked covalently to the carrier protein via a linker moiety.

As used herein, the term oligosaccharide refers to a chain of linked monosaccharide units, typically a small number of monosaccharide units, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 units, for example 2, 3, 4, 9 or 11 units linked together to form a chain. The monosaccharide units are typically linked via glycosidic bonds. Examples of monosaccharides include ribulose, xylulose, ribose, arabinose, xylose, lyxose, deoxyribose, psicose, fructose, sorbose, tagatose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, fucose, fuculose, rhamnose, neuramininc acid and derivatives thereof including glucosamine, N-acetylglucosamine and N-acetylneuramininc acid (sialic acic). Monosaccharides having an anomeric (hemiacetal) carbon atom are typically given an a- or β- prefix, depending on the orientation of the hydroxyl group at the anomeric carbon atom. Monosaccharides are typically given a D- or L- prefix, depending on the stereochemistry at the C5 position.

The saccharides disclosed herein may exist as isomers or mixtures of isomers, for example α/β anomers, L/D stereoisomers and ring/straight-chain isomers. The present invention relates to all such isomers and mixtures of isomers of the saccharides disclosed herein, except where specified.

As used herein, a glycosidic bond is a bond from the anomeric carbon of a monosaccharide unit (the glycosyl donor) to a glycosyl acceptor. The glycosyl acceptor may be any molecule which contains a nucleophilic atom capable of forming a bond to the glycosyl donor. In the case of a glycosidic bond which links the monosaccharide unit to a second monosaccharide unit, the glycosyl acceptor is typically an oxygen atom of a hydroxyl group of the second monosaccharide unit. Further examples of glycosyl acceptors include any molecule which contains a nucleophilic oxygen atom, nitrogen atom, sulfur atom or carbon atom. Glycosidic bonds can be described using an arrow drawn from the glycosyl donor to the glycosyl acceptor. For example, Glu-al→2-Man indicates a glucose unit which is linked by a glycosidic bond from its anomeric (CI) carbon, in a orientation, to the oxygen atom bound to the C2 carbon of a mannose unit.

As used herein, the term CI -3 alkyl means an alkyl radical having one two or three carbon atoms. A CI -3 alkyl group may be a methyl, ethyl, n-propyl or isopropyl group. A CI -3 alkyl group is unsubstituted, except where specified.

As used herein, a CI to C6 alkylene group is a linear or branched, divalent, saturated, aliphatic hydrocarbon radical containing 1 to 6 carbon atoms. Examples include methylene, ethylene and propylene

As used herein, a C2 to C6 alkenylene group is a linear or branched, divalent, aliphatic, hydrocarbon radical containing 1 to 6 carbon atoms and one or more carbon-carbon double bond. Examples include ethenylene and propenylene. As used herein, a C2 to C6 alkynylene group is a linear or branched, divalent, aliphatic, hydrocarbon radical containing 1 to 6 carbon atoms and one or more carbon-carbon triple bond. Examples include ethynylene and propynylene. A said alkylene, alkenylene or alkynylene group may be unsubstituted or substituted by one or more groups selected from halogen atoms and hydroyl groups.

As used herein, a halogen atom is a fluorine, chlorine, bromine or iodine atom. Oligosaccharides

In one embodiment of the present invention, each mannose unit is an a-D-mannose unit, and each modified mannose unit is an α-D-mannose unit having one or more modifications as defined in claim 1. Where the oligosaccharide contains other monosaccharides or modified monosaccharides, these are typically also a-D-orientated. However, in another embodiment of the invention, the oligosaccharide contains some β-glycosidic bonds. Typically, in this embodiment, 25% or fewer of the glycosidic bonds in the oligosaccharide are β-glycosidic bonds, for example 20% or fewer, 15% or fewer, 10% or fewer or 5% or fewer Typically, each bond from one Ml , M2, M3 or S 1 unit to another Ml , M2, M3 or S 1 unit is a glycosidic bond. Preferably:

The glycosidic bond from M3 to M2 is an a-l→2 glycosidic bond (i.e. M3-a-l→2-M2);

Each glycosidic bond from one M2 to another M2 is an a-l→2 glycosidic bond

. M2-a-l→2-M2);

- the glycosidic bond from M2 to Ml is an a-l→3 glycosidic bond (i.e.

M2-a-l→3-Ml);

each glycosidic bond from one SI to another SI is an a-l→2 or a-l→3 glycosidic bond (i.e. Sl-a-l→2-Sl or Sl-a-l→3-Sl); and/or

each glycosidic bond from SI to Ml or M2 is an a-l→6 glycosidic bond (i.e. Sl-a-l→6-Ml or Sl-a-l→6-M2). Typically, in the glycoconjugate of the invention, the or each modified mannose unit has one, two or three modifications, preferably one or two modifications, more preferably one modification. The or each modification A in a modified mannose unit present in the glycoconjugate of the invention can be at the CI, C2, C3, C4, C5 or C6 position. For the avoidance of doubt, the or each modification A involves replacement of a hydrogen atom which is boded directly to the carbon backbone of the mannose unit. Thus, for example, the or each modification A does not involve replacement of a hydrogen atom which is part of a hydroxyl group of the mannose unit.

Typically the or each modification A in a modified mannose unit present in the

glycoconjugate of the invention is at the C3, C5 or C6 position, and more typically at the C5 position. Preferably, when a mannose unit is modified with modification A, the hydrogen atom of the mannose unit is replaced with a methyl, ethyl, hydro xymethyl or hydro xyethyl group, more preferably a methyl or hydroxymethyl group, and most preferably a methyl group.

The or each modification B in a modified mannose unit present in the glycoconjugate of the invention can be at the C2, C3, C4, C5 or C6 position. Preferably, the or each modification is at the C3, C5 or C6 position of the modified mannose unit. Typically, when a mannose unit is modified with modification B, at least the C6 position of the modified mannose unit is modified with modification B. In one embodiment, only the C6 position of the modified mannose unit is modified with modification B.

Typically, in the glycoconjugate of the invention, the sum of p and q is 1 or 2. Preferably, the sum of p and q is 2.

Typicaly, Ml represents a mannose unit (i.e. an unmodified mannose unit).

Typically, at least one M2 represents a mannose unit (i.e. an unmodified mannose unit). Preferably, all M2 units present in the glycoconjugate or oligosaccharide of the invention represent a mannose unit. Preferably, M3 represents a modified mannose unit having (a) modification A at one or more of the C3, C5 and C6 positions, and/or (b) modification B at one or more of the C3, C5 and C6 positions; preferably modification B is at the C6 position. Preferably, M3 contains a single modification.

When one or more SI groups are present, r and s are typically zero. When r, s or t is 3 or more, (Sl) _r , (Sl) _s , or (Sl) _t may represent a linear or branched oligosaccharide group.

Typically, r, s and t are from 0 to 5, preferably 0, 3 or 5. When one or more SI groups are present, for the avoidance of doubt, (Sl) _r , (Sl) _s , and (Sl) _t , when present, represent a single linear or branched moiety attached to a Ml or M2 moiety. Thus, for example, when r is greater than 1, (Sl) _r represents a single oligosaccharide group having r saccharide units, and does not represent a plurality of individual monosaccharide moieties each bonded directly to an M2 moiety. When more than one SI group is present, each SI group may be the same or different.

Typically, each SI is independently a modified or unmodified mannose, unmodified galactose, unmodified fucose, sialic acid or N-acylglucosamine unit. Prefereably each SI is independently a modified or unmodified mannose unit. When SI represents a modified saccharide unit, it is modified with one or more modification A and/or modification B, as defined above. Particularly preferred modifications at SI are the same as those for the modified mannose units described above. Preferably, each SI is independently a mannose unit or a modified mannose unit. Most preferably each SI is a mannose unit (i.e. an unmodified mannose unit). Preferably, when one or more SI units are present, (Sl) _t -Ml represents group (1) or (2):

(1) : Man-a-(l→2)-Man-a-(l→3)- _|

Man-a-( 1→2)-Man-a-( 1→6)-Man-a-( 1→6)-M 1

(2) : Man-a-( 1→2)-Man-a-( 1→6)-Man-a-( 1→6)-M 1

Most preferably, when one or more SI units are present, (Sl) _t -Ml represents group (1).

In one embodiment, when M3 represents a modified mannose unit having modification A at the C6 position, the modified C6 position has (R) configuration. In an alternative embodiment, when M3 represents a modified mannose unit having modification A at the C6 position, the modified C6 position has (S) configuration.

Typically, in said alternative embodiment:

(a) the carrier protein is BSA or CRM;

(b) Ml and/or one or more M2 is a modified mannose unit;

(c) one of r, s and t is an integer from 1 to 5; and/or

(d) M3 is modified at the CI, C2, C3, C4 or C5 position.

In one embodiment, the oligosaccharide is of formula (la):

(la) wherein R _{l s} R ₂ , R ₃ and R4 are independently a hydrogen atom or a CI -3 alkyl group optionally substituted with a hydroxyl group, and X is a hydrogen atom or a hydroxyl group, provided that X is a hydrogen atom when Ri, R ₂ , R ₃ and R4 all represent a hydrogen atom.

Typically, R _{l s} R ₂ , R ₃ and R4 each independently represent a hydrogen atom, a methyl group, an ethyl group, a hydroxymethyl group or a hydroxyethyl group; preferably a hydrogen atom, a methyl group or a hydroxymethyl group; and most preferably a hydrogen atom or a methyl group. Typically, R _{l s} R ₂ , R ₃ and R ₄ all represent a hydrogen atom, or only one of Ri, R ₂ , R ₃ and R ₄ is other than a hydrogen atom. Preferably, when one of Ri, R ₂ , R ₃ and R ₄ is other than a hydrogen atom, X is a hydroxyl group.

In one embodiment, when R ₃ is other than a hydrogen atom, R ₄ is a hydrogen atom.

Preferably, R ₄ is a hydrogen atom.

Particularly preferred oligosaccharides for use in the treatment or prevention of an HIV infection or AIDS include:

D-Rham-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1→3)Man;

[C6-(S)-Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1→3)Man;

[C3 -Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1→3)Man;

[C5 -Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1→3)Man; and

[C6-(R)-Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1→3)Man.

Particularly preferred among the glycoconjugates of the invention inlcude those containing, as the oligosaccharide:

D-Rham-a-( 1→2)-Man-a-( 1→2)-Man-a~( 1→3)Man;

[C3 -Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1 - →3)Man;

[C5 -Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a-( 1 -→3)Man; and

[C6-(R)-Methyl] -Man-a-( 1→2)-Man-a-( 1→2)-Man-a -(l→3)Man.

Linker moieties

The linker moiety can be any moiety capable of forming one or more covalent bonds to the oligosaccharide, and one or more covalent bonds to the carrier protein. For the avoidance of doubt, the linker moiety is a non- saccharide moiety. The use of such linker moieties in glycocojugates is well known in the art. Thus, the linker moieties which can be used are not particularly limited and one of skill in the art will be capable of selecting an appropriate linker.

Factors relevant to selecting an appropriate linker include its length and chemical stability/reactivity. The linker should not be so long that the oligosaccharide is too far away from the carrier protein for the glyco conjugate to be internalised by a B-cell. Typically, the linker can easily be bonded to the oligosaccharide and the carrier protein in high yield, and is typically stable in an aqueous environment. The linker moiety will typically be attached to the oligosaccharide moiety via the anomeric carbon atom of the Ml unit, and will typically be attached to the carrier protein via the side chain of an amino acid residue in said carrier protein, e.g. via an amine group.

Without prejudice to the generality of the linkers suitable for use in the glycoconjugates of the present invention, the linker moiety may be a moiety of Formula (II):

-L-Y-L ^* -

(II)

wherein:

L and L' are independently a single bond, or a group selected from CI to C6 alkylene, C2 to C6 alkenylene and C2 to C6 alkynylene; which group may be substituted or unsubstituted by one or more (e.g. 1, 2 or 3) substituents selected from halogen atoms and hydroxyl groups; and

- Y is a single bond or a group selected from -NR-, -C(O)-, -C(0)-0- -O-C(O)-,

-0-C(0)-0- -C(0)-NR- -NR-C(O)-, -NR-C(0)-NR'-, -C-(S)-, -C(S)-0- - O-C(S)-, -C(S)-NR- -NR-C(S)-, -NR-C(S)-NR'- and -C(NR)-NR'-; wherein

R and R' are independently hydrogen or a CI to C3 alkyl group;

provided that at least one of L, L' and Y does not represent a single bond.

Typically, each L and L' are independently a single bond or a CI to C5 alkylene group.

Typically, L and L' taken together contain up to 8 carbon atoms, for example up to 6 carbon atoms. For the avoidance of doubt, the left hand side of L is attached to Ml and the right hand side of L' is attached to the carrier protein.

Typically, Y is a group -NR-C(S)-NR- -C(NR)-NR'- or -C(0)-NR-. Preferably, Y is - NH-C(S)-NH- -C(NH)-NH- or -C(0)-NH-. For the avoidance of doubt, the left hand side of the Y groups depicted above is attached to L and the right hand side of the Y groups depicted above is attached to L'.

Particularly preferred linker moieties are

Carrier proteins

The use of carrier proteins in conjugate vaccines is well known in the art, and it will be evident to a skilled person that the carrier protein which can be used in the glycoconjugate of the present invention is not particularly limited. Thus, the carrier protein used in the glycoconjugate of the present invention may be any protein capable of eliciting T-cell help in the generation of 2G 12 antibodies.

The carrier protein of the invention comprises or consists of a sequence which is recognized by T-cells as a hapten when presented on surface of a B-cell as a peptide:MHC complex. T- cell recognition of that peptide epitope activates the B-cell and leads to the development of an immune response against HIV-1 (i.e. the generation of antibodies which bind the gpl20 epitope).

Suitable carrier proteins include LTB, KLH, tetanus toxin, QP, BSA, and CRM. Typically, the carrier protein in the glycoconjugate of the invention is QP, BSA, or CRM. Preferably, the carrier protein is CRM.

In the glycoconjugates of the invention, the carrier protein may carry more than one oligosaccharide of formula (I). Typically, the carrier protein carries from 1 to 30

oligosaccharides of formula (I), more typically from 1 to 20 and most typically from 1 to 15. However, the number of oligosaccharides presented on the surface of the carrier protein is not thought to affect the strength of the oligosaccharide-2G12 interactions.

Processes Glycoconjugates of formula (la) can be prepared by following the general process set out in Scheme (2):

Scheme (2) wherein, in formulae (2), (3) and (4), LG represents a leaving group and Ri', R ₂ ', R ₃ ' and R4' are independently a hydrogen atom or a CI -3 alkyl group optionally substituted with a group OPg, and X' is a hydrogen atom or a group OPg, provided that X' is a hydrogen atom when Ri', R ₂ ', R ₃ ' and R4' all represent a hydrogen atom, and Pg represents a hydroxyl protecting group; and in formula (5), Ri, R ₂ , R ₃ , R4 and X are as defined above for formula (la)

The skilled person will be familiar with leaving groups and protecting groups which could be used as LG and Pg, respectively. Examples of suitable leaving groups include thioester- forming groups such as -SPh and -SEt. Examples of suitable protecting groups include ester forming acyl groups such as -Ac, ether-forming alkyl or aralykyl groups such as -Bn, and divalent acetal or ketal- forming protecting groups such as >CH-Ph, which can be used to protecting two hydroxyl groups simultaneously. Step (i) involves activation of the LG group of glycosyl donor (2) and coupling with glycosyl acceptor (3) to form the tetrasaccharide-containing compound of formula (4). The skilled person will be able to select an appropriate leaving group as LG and a will be familiar with appropriate activating agents for any given leaving group. For example, glycosyl donors carrying a thioester-forming leaving group can be activated with N-iodosuccinimide and triflic acid.

Step (ii) involves deprotection of the compound of formula (4) to form the compound of formula (5). The skilled person will be familiar with appropriate agents for cleaving any particular Pg.

Compounds of formulae (2) and (3) can be prepared by known methods or by analogy with known methods. Exemplary syntheses of such compounds can be found in the Examples section herein.

In formulae (3) (4) and (5), ¾ represents the point of attachment of the saccharide moiety to the remainder of the molecule. The remainder of the molecule may be a protecting group, a protected linker moiety, or a linker moiety attached to a carrier protein. When the remainder of the molecule is a protecing group, the compound of formula (5) can be deprotected to form a tetrasaccharide, which can be reacted with a linker moiety and a carrier protein to give the glycoconjugate of formula (la). Where the remainder of the molecule is a protected linker moiety, the compound of formula (5) can be deprotected and reacted with a carrier protein to give the glycoconjugate of formula (la). Where the remainder of the molecule is a linker moiety attached to a carrier protein, the compound of formula (5) is the glycoconjugate of formula (la).

The skilled person would be able to prepare glyco conjugates of formula (I) by processes analogous to that of scheme 2 described above. Thus, for example, glyconjugates of formula (I) maybe prepared according to scheme 3 :

Step (i)

(SI'X (Sl')s (SI'X

(M2¾— (M2 -Mi

Step (i)

Scheme 3

In scheme 3, Ml, M2, M3 and SI, as well as p, q, r, s and t are as defined herein, and Μ , M2', M3' and SI' represent protected versions of the basic saccharide unit. As for scheme 2,

^ represents the point of attachment of the saccharide moiety to the remainder of the molecule. The remainder of the molecule may be a protecting group, a protected linker moiety, or a linker moiety attached to a carrier protein. Thus, when the remainder of the molecule is a protecting group, removal of the protecting group provides an oligosaccharide of formula (lb):

(lb)

wherein:

Ml represents a mannose unit or a modified mannose unit, which modified mannose unit is as defined above;

Each M2 and SI is as defined above;

- p and q are as defined above;

- r, and s and t are as defined above; M3 represents a modified mannose unit having (a) modification A at one or more of the CI to C4 and C6 positions, the modified C6 position having (R) configuration, and/or (b) modification B at one or more of the C2 to C5 positions; wherein modifications A and B are as defined above.

Preferably, in Formula (lb), Ml represents a mannose unit.

For the avoidance of doubt, preferred embodiments of the oligosaccharides of Formula (lb) are the same as for those in glycoconjugates of Formula (I), except that Ml carries a hydro xyl group at the anomeric carbon atom and is not bound to a linker moiety or a carrier protein.

Treatment of HIV infections and AIDS

The glycoconjugates of the present invention are capable of eliciting an immune response against HIV, typically against HIV- 1.

When administered to a subject as part of a vaccine composition, the glycoconjugates are recognized by the B-cell antigen receptor (BCR) surface immunoglobulin, which signals to the B-cell's interior when the glycoconjugate antigen is bound and delivers the antigen to intracellular cites where it can be degraded. The glycoconjugate may activate the B-cell directly. Alternatively, or additionally, peptide fragments of the carrier protein may be returned to the B-cell surface as part of a peptide:MHC complex for recognition by antigen- specific helper T-cells which make cytokines that cause the B-cell to proliferate and its progeny to differentiate into antibody-secreting cells and memory B-cells.

Antibodies thus secreted are capable of binding to the oligomannose glycans, typically the Dl arm, displayed on the surface of HIV envelope glycoprotein gpl20. The antibody:HIV complex is recognized by Fc receptors on effector cells that bind to Fc portions provided by the antibody. Binding activates the effector cell and triggers neutralization of the HIV virus via phagocytosis and/or granule release.

Vaccine compositions The present invention provides a vaccine composition comprising a glycoconjugate of the invention.

The glycoconjugate is preferably administered together with one or more pharmaceutically acceptable carriers or diluents and optionally one or more other therapeutic ingredients. The carrier (s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free. Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.

For example, a glycoconjugate of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides,

hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen- free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

Other parentally-administrable compositions which are useful include those which comprise the active ingredient in micro crystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Alternatively, the glycoconjugate of the invention may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from

poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.

Once formulated the compositions can be delivered to a subject in vivo using a variety of known routes and techniques. For example, the vaccine composition can be provided as an injectable solution, suspension, emulsion or dry powder and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system, or using a patch. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, sublingual administration, and active or passive transdermal delivery techniques.

The administered compositions will comprise a suitable concentration of the glycoconjugate of the invention which is effective without causing adverse reaction. Typically, the concentration of the glycoconjugate in the composition will be in the range of 0.03 to 200 nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 10 to 150 nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml. The composition or formulations should have a purity of greater than 95% or 98% or a purity of at least 99%.

Dosages for administration will depend upon a number of factors including the nature of the composition, the route of administration and the schedule and timing of the administration regime. Suitable doses may be in the order of up to 15μg, up to 20μg, up to 25μg, up to 30μg, up to 50μg, up to 100μg, up to 500 μg or more per administration. Suitable doses may be less than 15μg, but at least lng, or at least 2ng, or at least 5ng, or at least 50ng, or least lOOng, or at least 500ng, or at least ^g, or at least 10μg. For some molecules, the dose used may be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may be provided in a liquid formulation, at a concentration suitable to allow an appropriate volume for administration by the selected route.

An adjuvant may also be used in combination with the glycocojugate. The adjuvant is preferably administered in an amount which is sufficient to augment the effect of the glycoconjugate or vice versa. The adjuvant or other therapeutic agent may be an agent that potentiates the effects of the glycoconjugate. For example, the other agent may be an immunomodulatory molecule. Non-limiting examples of adjuvants include alum, monophosphoryl lipid, oligonucleotides, cholera toxin, Freund's complete or incomplete adjuvant, Ribi and QS-21.

In one embodiment, the glycoconjugates are used for therapy in combination with one or more other therapeutic agents. The agents may be administered separately, simultaneously or sequentially. They may be administered in the same or different compositions as the glycoconjugate. Accordingly, in a method of the invention, the subject may also be treated with a further therapeutic agent. A composition may therefore be formulated with a glyco conjugate of the invention and also one or more other therapeutic molecules. A vaccine composition of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment.

Examples

Modified monosaccharide synthesis Reference Example 1

D-rhamnose donor synthesis:

60 % 75 %

Reference Example 2

C-5-Me-Mannose donor synthesis:

Reference Example 3

C-3-Me mannose donor synthesis:

77% 98%

Ph-CH(OMe) ₂ CSA, CH ₃ CN

75% 68%

DMSO/Ac ₂ 0 Et

79% 77%

Reference Example 4

C-6-R-Me mannose donor synthesis:

H, H ₂

75% (2 steps) 87%(2 steps)

Reference Example 5 Scheme S5: i) DMSO, C ₂ 0 ₂ C1 ₂ , DCM, -78°C→RT, ii) MeMgBr, THF, 56% over 2 steps, iii) H ₂ , Pd/C, MeOH,

95%.

Scheme S6: i) Ac ₂ 0, Pyridine, 92%, ii) Thiophenol, BF ₃ .OEt ₂ , DCM, 0°C-RT, 80, iii) NaOMe, MeOH then

BnBr, NaH, DMF, 74%.

Scheme S7: i) PhCH(OMe) ₂ , pTSA, DMF, 50 °C, 27%.

Tetrasaccharide synthesis

i) TMSOTf, DCM, 78%, ii) Tf ₂ 0, Me ₂ S ₂ , TTBP, DCM, 4A sieves, -78°C→RT, 68%, iii) NaOMe, MeOH, 95%, iv) DMTST, TTBP, DCM, 4A sieves, -78°C→RT, v) AcOH, H ₂ 0, 50 °C, vi) H ₂ , Pd/C, MeOH.

Reference Example 6 Benzyl (2-0-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-( 3,4,6-tri-0-benzyl- a-D-mannopyranosyl)-(l→3)-2,4-di-0-benzyl-6-0-tei"i-butyld imethylsilyl-a-D- mannopyranoside 61

Benzyl 2,4-di-O-benzyl-6-0-tert-butyldimethylsilyl-a-D-mannopyranos ide (77 mg,

0.14 mmol), ethyl 2-O-acetyl-3,4,6-0-benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-t ri-O- benzyl-thio-a-D-mannopyranoside 16 (160 mg, 0.16 mmol) and 2,6-di-tert-butyl-4- methylpyridine 17 (235 mg, 0.95 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (2 mL) and transferred using a cannula to a flame dried flask containing 4A molecular sieves. The mixture was stirred for 1 h and cooled to -78 °C. DCM (2 mL) was added to a flame dried flask containing 4A molecular sieves and stirred for 1 h then cooled to 0 °C. To this flask was added dimethyldisulfide (73 μί, 0.816 mmol) and trif uoromethylsulfonic anhydride (137 μί, 0.816 mmol). After 2 min, the solution was transferred to the flask containing the flask containing the sugar reagents at -78 °C. The mixture was stirred at -78 °C under an atmosphere of argon. After 1 h, t.l.c (5 : 1 , petrokethyl acetate) indicated formation of a product (R _f 0.5) with complete consumption of the starting materials (R _f 0.6, 0.3). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite. The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6: l , petrohethyl acetate) to afford benzyl 2-O-acetyl- 3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl)-( 1→2)-(3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl)- (l→3)-2,4,di-0-benzyl-6-0-tert-butyldimethylsilyl-a-D-mann opyranoside 61 (136 mg, 68%) as a colourless oil.

Reference Example 7

Benzyl (3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-(3,4,6-tri-0 -benzyl-a-D- mannopyranosyl)-(l→3)-2,4,di-0-benzyl-6-0-tei"i-butyldimet hylsilyl-a-D- mannopyranoside 18A

Benzyl (2-O-acetyl-3,4,6-tri-O-benzyl-a-D-mannopyranosyl)-(l→2)-( 3,4,6-tri-0-benzyl-a- D-mannopyranosyl)-(l→3)-2,4,di-0-benzyl-6-O-tert-butyldime thylsilyl-a-D- mannopyranoside 61 (40 mg, 0.026 mmol) was dissolved in methanol (2 mL) and sodium methoxide (0.2 mL of a 0.1 M solution in methanol) was added. After 24 h, t.l.c. (3 : 1 , petrohethyl acetate) showed formation of a product (R _f 0.3) and complete consumption of the starting material (R _f 0.6). Ammonium chloride (a drop of a saturated aqueous solution) was added followed by sodium hydrogen carbonate (10 mL of a saturated aqueous solution). The mixture was extracted with DCM (3 x 25 mL) and the combined organic layers dried (MgSC^), filtered and concentrated in vacuo to afford benzyl (3,4,6-tri-O-benzyl-a-D- mannopyranosyl)-(l→2)-(3,4,6-tri-0-benzyl-a-D-mannopyranos yl)-(l→3)-2,4,di-O-benzyl- 6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 18A (35 mg, 95 %) as a colourless oil.

Reference Example 8

Benzyl 2-0-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l→2)- 3,4,6-tri-O-benzyl-a- D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-mannopyranos yl-(l→3)-2,4-di-0- nzyl-6-O-teri-butyldimethylsilyl-a-D-mannopyranoside 25A

Benzyl 2,4-di-O-benzyl-6-0-tert-butyldimethylsilyl-a-D-mannopyranos ide 5.3.13 (29 mg, 0.051 mmol), ethyl 2-O-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l→2)- 3,4,6-tri-O- benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-thio-a- D-mannopyranoside 5.3.106 (60 mg, 0.043 mmol) and 2,6-di-tert-butyl-4-methylpyridine (76 mg, 0.30 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (1 mL) and transferred using a cannula to a flame dried flask containing 4 A molecular sieves. The mixture was stirred for 1 h and cooled to -78 °C. DCM (1 mL) was added to a flame dried flask containing 4A molecular sieves and stirred for 1 h then cooled to 0 °C. To this flask was added

dimethyldisulfide (23 μί, 0.26 mmol) and trifluoromethylsulfonic anhydride (44 μΐ,, 0.26 mmol). After 2 min, the solution was transferred to the flask containing the flask containing the sugar reagents at -78 °C. The mixture was stirred at -78 °C under an atmosphere of argon. After 1 h, t.l.c (5 : 1 , petrokethyl acetate) indicated formation of a product (R _f 0.4) with complete consumption of the starting materials (R _f 0.1, 0.3). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite ^® . The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6: l , petrokethyl acetate) to afford benzyl 2-O-acetyl-3,4,6-tri-0-benzyl-a-D-mannopyranosyl- (1→2)- 3,4,6-tri-0-benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-be nzyl-a-D- mannopyranosyl-(l→3)-2,4-di-0-benzyl-6-O-tert-butyldimethy lsilyl-a-D-mannopyranoside 25A (24 mg, 29%) as a colourless oil.

Reference Example 9

Benzyl (2,3,4,6-tetra-0-benzyl-5-C-ethyl-a-D-mannopyranosyl)-(l→2 )- 3,4,6-tri-O- benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-man nopyranosyl-(l→3)-2,4- -O-benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 23b

Benzyl (3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-(3,4,6-tri-O -benzyl-a-D- mannopyranosyl)-(l→3)-2,4,di-O-benzyl-6-O-tert-butyldimeth ylsilyl-a-D-mannopyranoside 18 (59 mg, 0.041 mmol), phenyl 2,3,4,6-tetra-O-benzyl-5-C-ethyl-l-thio- -D- mannopyranoside 20b (31 mg, 0.050 mmol) and 2,4,6-tri-t-butylpyrimidine (85 mg,

0.328 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (1 mL) and transferred using a cannula to a flame dried flask containing 4A molecular sieves. The mixture was stirred for 1 h and cooled to -78 °C. DCM (1 mL) was added to a flame dried flask containing 4 A molecular sieves and stirred for 1 h then cooled to 0 °C. To this flask was added dimethyldisulfide (28 μί, 0.31 mmol) and trifluoromethylsulfomc anhydride (54 μΐ _^ , 0.31 mmol). After 2 min, the solution was transferred to the flask containing the sugar reagents at -78 °C. The mixture was stirred at -78 °C under an atmosphere of argon. After 1 h, t.l.c (5 : 1 , petrokethyl acetate) indicated formation of a product (R _f 0.5) with complete consumption of the starting materials (R _f 0.1 , 0.7). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite ^® . The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6 : 1 , petrohethyl acetate) to afford benzyl (2,3,4,6-tetra-O-benzyl-5-C-ethyl-a-D- mannopyranosyl)-( 1→2)- 3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl-( 1→2)-3 ,4,6-tri-O- benzyl-a-D-mannopyranosyl-(l→3)-2,4-di-O-benzyl-6-0-tert-b utyldimethylsilyl-a-D- mannopyranoside 23b (16 mg, 20%) as a colourless oil.

Reference Example 10

Benzyl (2,3,4,6-tetra-0-benzyl-5-C-methyl-a-D-mannopyranosyl)-(l→ 2)- 3,4,6-tri-O- benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-man nopyranosyl-(l→3)-2,4- di-O-benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 23a

Benzyl (3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-(3,4,6-tri-O -benzyl-a-D- mannopyranosyl)-(l→3)-2,4,di-O-benzyl-6-O-tert-butyldimeth ylsilyl-a-D-mannopyranoside 18 (102 mg, 0.071 mmol), phenyl 2,3,4,6-tetra-0-acetyl-5-C-methyl-l-thio-D- mannopyranoside 20a (55 mg, 0.086 mmol) and 2,4,6-tri-t-butylpyrimidine (92 mg, 0.36 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (1 mL) and transferred using a cannula to a flame dried flask containing 4A molecular sieves. The mixture was stirred for 1 h and cooled to -20 °C. Dimethylthiosulfonium triflate (710 of a 0.4 M solution in DCM was added to the reaction mixuture. After 1 h, t.l.c (5 : 1 , petrohethyl acetate) indicated formation of a product (R _f 0.5) with complete consumption of the starting materials (R _f 0.1 , 0.7). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite ^® . The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6: l , petrohethyl acetate) to afford benzyl (2,3,4,6- tetra-O-benzyl-5-C-methyl-a-D-mannopyranosyl)-(l→2)- 3,4,6-tri-O-benzyl-a-D- mannopyranosyl-( 1→2)-3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl-( 1→3)-2,4-di-O-benzyl-6- O-tert-butyldimethylsilyl-a-D-mannopyranoside 23a (25 mg, 18%) as a colourless oil.

Reference Example 11 a-D-Mannopyranosyl-(l→2)-a-D-mannopyranosyl-(l→2)-a-D-ma nnopyranosyl-(l→3)- D-mannopyranose 3

Sodium methoxide (1 mL of a 0.1 M solution in methanol) was added to a solution of benzyl 2-0-acetyl-3,4,6-tri-O-benzyl-a-D-mannopyranosyl-(l→2)- 3,4,6-tri-O-benzyl-a-D- mannopyranosyl-( 1→2)-3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl-( 1→3)-2,4-di-O-benzyl-6- O-tert-butyldimethylsilyl-a-D-mannopyranoside 25A (24 mg, 0.013 mmol) in methanol (1 mL). After 18 h the reaction mixture was neutralized with acidified DOWEX, filtered and concentrated in vacuo. The residue was suspended in acetic acid (80% in water) and heated at 50°C. After 24 h, t.l.c (2: 1 , petrohethyl acetate) indicated formation of a product (R _f 0.2) with consumption of the starting material (R _f 0.6). The reaction mixture was concentrated in vacuo and coevaporated with toluene (3 x 10 mL). The residue was purified by flash column chromatography (2: 1 , petrol: ethyl acetate). The desilylated product was dissolved in ethanol and palladium (10 mg of 10%> palladium on carbon) was added. Hydrogen gas was bubbled through the solution until saturated. After 48 h under hydrogen, the reaction mixture was filtered through celite ^® , concentrated in vacuo and passed through a silica plug to afford a-D- mannopyranosyl-( 1→2)-a-D-mannopyranosyl-( 1→2)-a-D-mannopyranosyl-( 1→3)-D- mannopyranose 3 (5 mg, 60% over 3 steps) as an amorphous white solid.

Reference Example 12

5-C-Methyl-a-D-mannopyranosyl-(l→2)-a-D-mannopyranosyl- (l→2)-a-D- mannopyranosyl-(l→3)-D-mannopyranose 4a

Benzyl (2,3,4,6-tetra-0-benzyl-5-C-methyl-a-D-mannopyranosyl)-(l→ 2)- 3,4,6-tri-O- benzyl-a-D-mannopyranosyl-( 1→2)-3 ,4,6-tri-0-benzyl-a-D-mannopyranosyl-( 1→3)-2,4-di- O-benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 23a (25 mg, 0.013 mmol) was suspended in acetic acid (80% in water) and heated at 50°C. After 24 h, t.l.c (3 : 1 , petrohethyl acetate) indicated formation of a product (R _f 0.2) with consumption of the starting material (R _f 0.6). The reaction mixture was concentrated in vacuo and coevaporated with toluene (3 x 10 mL). The residue was purified by flash column chromatography (2: 1 , petrohethyl acetate). The desilylated product was dissolved in ethanol and palladium (10 mg of 10%> palladium on carbon) was added. Hydrogen gas was bubbled through the solution until saturated. After 48 h under hydrogen, the reaction mixture was filtered through celite ^® , concentrated in vacuo and passed through a silica plug to afford 5-C-methyl-a-D-mannopyranosyl-(l→2)-a-D- mannopyranosyl-(l→2)-a-D-mannopyranosyl-(l→3)-D-mannopyr anose 4a (6 mg, 70% over 2 steps) as an amorphous white solid; Partial assignment: 5c (500 MHz, D ₂ 0) 1.22 (3H, s, Me), 3.30 (1H, ddd, J 2.0 Hz, J 6.2 Hz, J 8.8 Hz, H-5), 3.44, 3.54 (2H, ABq, J 12.0 Hz, H-6d,

H-6'd), 3.59-4.08 (20 H, m), 4,82 (1H, s, H-la ), 5.06 (1H, d, J 1.9 Hz, H-l), 5.07 (1H, J 1.5 Hz, H-laa), 5.22 (1H, s, H-l), 5.27 (1H, s, H-l); 5 _C (125 MHz, D ₂ 0) 18.3 (q, Me) 60.8 (t, C-6a,b,c), 66.5 (t, C-6d), 66.0, 66.3, 66.8, 67.1, 67.6, 70.1, 70.3, 70.5, 70.9, 72.5, 73.3, 75.9, 77.9, 78.1, 78.2 (16 x d, C-2a,b,c,d, C-3a,b,c,d, 4a,b,c,d, 5a,b,c), 80.4 (s, C-5d), 93.5, 94.0, 100.5, 100.7, 102.9 (5 x d, C-laa, , b, c, d); m/z (ESI ⁺ ) 703 (M+Na ⁺ , 100%); HRMS (ESI ⁺ ) calcd. for C ₂₅ H ₄₄ 0 ₂ iNa (M+Na ⁺ ) 703.2267. Found 703.2265. Reference Example 13

5-C-Ethyl-a-D-mannopyranosyl-(l→2)-a-D-mannopyranosyl-( l→2)-a-D- mannopyranosyl-(l→3)- D-mannopyranose 4b

Benzyl (2,3,4,6-tetra-0-benzyl-5-C-ethyl-a-D-mannopyranosyl)-(l→2 )- 3,4,6-tri-O-benzyl- a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-mannopyran osyl-(l→3)-2,4-di-O- benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 23b (16 mg, 0.008 mmol) was suspended in acetic acid (80% in water) and heated at 50°C. After 24 h, t.l.c (3: 1, petrohethyl acetate) indicated formation of a product (R _f 0.2) with consumption of the starting material (R _f 0.6). The reaction mixture was concentrated in vacuo and coevaporated with toluene (3 x 10 mL). The residue was purified by flash column chromatography (2: 1, petrohethyl acetate). The desilylated product was dissolved in ethanol and palladium (10 mg of 10%> palladium on carbon) was added. Hydrogen gas was bubbled through the solution until saturated. After 48 h under hydrogen, the reaction mixture was filtered through celite ^® , concentrated in vacuo and passed through a silica plug to afford 5-C-ethyl-a-D-mannopyranosyl-(l→2)-a-D- mannopyranosyl-(l→2)-a-D-mannopyranosyl-(l→3)-D-mannopyr anose 4b (4 mg, 78% over 2 steps) as a white amorphous solid; Partial assignment: 5 _H (500 MHz, D ₂ 0) 0.84 (3H, t, J 7.5 Hz, CH ₃ ), 1.57-1.76 (2H, m, CH ₂ ), 3.34 (1H, ddd, J2.5 Hx, J6.5 Hz, J9.5 Hz, H-5), 3.53 (1H, d, J 11.5 Hz, H-6d), 3.60-4.09 (H, m), 4.83 (1H, s, H-la ), 5.04 (1H, d, J 5.5 Hz, H- 1), 5.09 (1H, d, J, ₂ 1.5 Hz, H-laa), 5.23 (1H, s, H-l), 5.30 (1H, s, H-l); m/z (ESI ⁺ ) 717

(M+Na ⁺ , 100%); HRMS (ESI ⁺ ) calcd. for (M+Na ⁺ ) 717.2424. Found 717.2424. Reference Example 14

Benzyl-(2,3,4,6-tetra-0-benzyl-6-S-6-C-methyl-a-D-mannopy ranosyl)-(l→2)-3,4,6-tri-0- benzyl-a-D-mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-man nopyranosyl-(l→3)-2,4- i-O-benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 24A

Benzyl (3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-(3,4,6-tri-O -benzyl-a-D- mannopyranosyl)-(l→3)-2,4,di-O-benzyl-6-O-tert-butyldimeth ylsilyl-a-D-mannopyranoside 18A (95 mg, 0.067 mmol), phenyl 2,3,4,6-tetra-0-benzyl-6-5 ^* -6-C-methyl-l-thio-a-D- mannopyranoside 21 (52 mg, 0.080 mmol) and 2,4,6-tri-t-butylpyrimidine (87 mg, 0.34 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (1 mL) and transferred using a cannula to a flame dried flask containing 4 A molecular sieves. The mixture was stirred for 1 h and cooled to -78 °C. Dimethylthiosulfonium triflate (69 mg, 0.27 mmoL) was added to the reaction mixuture and after 30 min the reaction mixture was allowed to warm to room temperature. After a further 1 h, t.l.c (5 : 1 , petrohethyl acetate) indicated formation of a product (R _f 0.6) with complete consumption of the starting materials (R _f 0.1 , 0.7). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite ^® . The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6: l , petrohethyl acetate) to afford benzyl-(2,3,4,6-tetra-O- benzyl-6-5'-6-C-methyl-a-D-mannopyranosyl)-(l→2)-3,4,6-tri -O-benzyl-a-D- mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-mannopyranosyl -(l→3)-2,4-di-O-benzyl-6- O-tert-butyldimethylsilyl-a-D-mannopyranoside 24A (65 mg, 55%) as a colourless oil.

Reference Example 15

6-C-Methyl-a-D-mannopyranosyl-(l→2)-a-D-mannopyranosyl- (l→2)-a-D- mannopyranosyl-(l→3)-D-mannopyranose 5

Benzyl-(2,3,4,6-tetra-O-benzyl-6-5'-6-C-methyl-a-D-mannopyra nosyl)-(l→2)-3,4,6-tri-O- benzyl-a-D-mannopyranosyl-( 1→2)-3 ,4,6-tri-0-benzyl-a-D-mannopyranosyl-( 1→3)-2,4-di- O-benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 24A (96 mg, 0.049 mmol) was suspended in acetic acid (80% in water) and heated at 50°C. After 48 h, t.l.c (5: 1, petrohethyl acetate) indicated formation of a product (R _f 0.1) with consumption of the starting material (R _f 0.6). The reaction mixture was concentrated in vacuo and coevaporated with toluene (3 x 10 mL). The residue was purified by flash column chromatography (2: 1, petrohethyl acetate). The desilylated product was dissolved in ethanol and palladium (10 mg of 10% palladium on carbon) was added. Hydrogen gas was bubbled through the solution until saturated. After 48 h under hydrogen, the reaction mixture was filtered through celite ^® , concentrated in vacuo and passed through a silica plug to afford 6-C-methyl-a-D-mannopyranosyl-(l→2)-a-D- mannopyranosyl-(l→2)-a-D-mannopyranosyl-(l→3)-D-mannopyr anose 5 (30 mg, 90% over 2 steps) as an amorphous white solid; δ _Η (500 MHz, D ₂ 0) 1.23 (3H, d, J 6.7 Hz, CH ₃ ), 3.41- 4.11 (23H, m, 23 x CH), 4.61 (1H, s, H-la ), 5.01 (1H, s, H-l), 5.06 (1H, s, H-laa), 5.15 (1H, s, H-l), 5.27 (1H, s, H-l); 5 _C (125 MHz, D ₂ 0) 19.0 (q, Me), 60.8, 60.9 (t, C-6a, C-6b, C-6c), 64.7 (d, C-6d), 65.9, 66.2, 66.7, 66.9, 67.0, 69.9, 70.0, 70.1, 70.4, 70.5, 72.5, 73.0, 74.9, 75.9, 77.9, 78.1, 78.8, 80.6 (d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b, C-5b, C-2c, C-3c, C-4c, C-5c, C-2d, C-3d, C-4d, C-5d), 93.5 (d, C-la ), 94.0 (d, C-laa), 100.7 (d, C-1), 100.8(d, C-1), 102.1 (d, C-1); m/z (ESI ⁺ ) 679 (M+H ⁺ , 100%); HRMS (ESI ⁺ ) calcd. for C ₂₅ H ₄₃ 0 ₂ i (M+H ⁺ ) 679.2302. Found 679.2302.

Reference Example 16 Benzyl-(2,3,4-tri-0-benzyl-a-D-rhamnopyranosyl)-(l→2)-3,4, 6-tri-0-benzyl-a-D- mannopyranosyl-(l→2)-3,4,6-tri-0-benzyl-a-D-mannopyranosyl -(l→3)-2,4-di-0- benzyl-6-O-tert-butyldimethylsilyl-a-D-mannopyranoside 22

Benzyl (3,4,6-tri-0-benzyl-a-D-mannopyranosyl)-(l→2)-(3,4,6-tri-O -benzyl-a-D- mannopyranosyl)-(l→3)-2,4,di-O-benzyl-6-O-tert-butyldimeth ylsilyl-a-D-mannopyranoside 18A (62 mg, 0.043 mmol), phenyl 2,3,4-tri-O-benzyl-thio-a-D-rhamnopyranoside 19 (27 mg, 0.052 mmol) and 2,4,6-tri-t-butylpyrimidine (56 mg, 0.22 mmol) were dried in a dessicator overnight. The reagents were dissolved in DCM (1 mL) and transferred using a cannula to a flame dried flask containing 4 A molecular sieves. The mixture was stirred for 1 h and cooled to -78 °C. Dimethylthiosulfonium triflate (44 mg, 0.17 mmoL) was added to the reaction mixuture and after 30 min the reaction mixture was allowed to warm to room temperature. After a further 1 h, t.l.c (5 : 1 , petrohethyl acetate) indicated formation of a product (R _f 0.6) with complete consumption of the starting materials (R _f 0.1 , 0.7). The reaction mixture was quenched with triethylamine (0.5 mL) and filtered through celite ^® . The filtrate was concentrated in vacuo and the residue purified by flash column chromatography (petrol→6: l , petrokethyl acetate) to afford benzyl-(2,3,4-tri-O-benzyl-a-D-rhamnopyranosyl)-(l→2)- 3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl-( 1→2)-3 ,4,6-tri-O-benzyl-a-D-mannopyranosyl- (l→3)-2,4-di-0-benzyl-6-0-tert-butyldimethylsilyl-a-D-mann opyranoside 22 (39 mg, 49%) as a colourless oil.

Reference Example 17 a-D-Rhamnopyranosyl-(l→2)-a-D-mannopyranosyl-(l→2)-a-D-m annopyranosyl- (l→3)-D-mannopyranose 7

Benzyl-(2,3,4-tri-O-benzyl-a-D-rhamnopyranosyl)-(l→2)-3,4, 6-tri-0-benzyl-a-D- mannopyranosyl-( 1→2)-3 ,4,6-tri-0-benzyl-a-D-mannopyranosyl-( 1→3)-2,4-di-0-benzyl-6- O-tert-butyldimethylsilyl-a-D-mannopyranoside 22 (110 mg, 0.060 mmol) was suspended in acetic acid (80% in water) and heated at 50°C. After 48 h, t.l.c (4: 1, petrohethyl acetate) indicated formation of a product (R _f 0.1) with consumption of the starting material (R _f 0.6). The reaction mixture was concentrated in vacuo and co evaporated with toluene (3 x 10 mL). The residue was purified by flash column chromatography (2: 1, petrohethyl acetate). The desilylated product was dissolved in ethanol and palladium (10 mg of 10% palladium on carbon) was added. Hydrogen gas was bubbled through the solution until saturated. After 48 h under hydrogen, the reaction mixture was filtered through celite ^® , concentrated in vacuo and passed through a silica plug to afford a-D-rhamnopyranosyl-(l→2)-a-D- mannopyranosyl-(l→2)-a-D-mannopyranosyl-(l→3)-D-mannopyr anose 7 ( mg, % over 2 steps) as an amorphous white solid; δ _Η (500 MHz, D ₂ 0) 1.09 (3H, d, J 6.0 Hz, Οϊ ₃ -β), 1.19 (3H, d, J6.3 Hz, CH ₃ -a), 3.36 (1H, at, J 9.7 Hz, CH), 3.61-4.03 (21H, m, 21 x CH), 4.82 (1H, s, H-la ), 4.89 (1H, s, H-l), 5.07 (1H, s, H-laa), 5.12 (1H, s, H-l), 5.25 (1H, s, H-l); 5c (125 MHz, D ₂ 0) 16.5 (q, Me), 60.8, 60.9 (2 x t, C-6a, C-6b, C-6c), 66.1, 66.3, 66.7, 66.9, 69.0, 70.0, 70.2, 70.4, 70.9, 72.0, 72.5, 73.3, 75.9, 77.9, 78.1, 78.2, 80.4 (d, C-2a, C-3a, C-4a, C-5a, C-2b, C-3b, C-4b, C-5b, C-2c, C-3c, C-4c, C-5c, C-2d, C-3d, C-4d, C-5d), 93.5 (d, C- lap), 93.9 (d, C-laa), 100.6, 100.8, 102.1 (d, C-lb, C-lc, C-ld); m/z (ESI ⁺ ) 649 (M+H ⁺ , 100%); HRMS (ESI ⁺ ) calcd. for C ₂₄ H ₄ i0 ₂ o (M+H ⁺ ) 649.2197. Found 649.2199.

Tetrasaccharide synthesis with linker: Reference Example 18 5-Azido-pentyl-6-C-6-S-methyl-a-D-mannopyranosyl-(1^2)-a-D-m annopyranosyl- (1— >2)-a-D-mannopyranosyl-(l— >3)-a-D-mannopyranoside 5B

Sodium azide (102mg, 1.57mmol, 150eq) was dissolved in water (3ml) was DCM (3ml). The resulting biphasic mixture was cooled to 0°C and Tf ₂ 0 (132μ1, 0.79mmol, 75eq) was added to the organic layer via syringe. The mixture was stirred at 0°C for 3 hours, after which the aqueous layer was removed and the organic layer washed with water (2 x 3ml), saturated NaHCC"3 solution (3ml) and water (3 x 2ml). To this solution of TfN3 was added NH ₄ HCO3 (2.5mg, 0.031mmol, 3eq), CuCl ₂ (0.2mg, 1.6 x 10 ^"3 mmol, 0.15eq), 5B' (8mg, O.Olmmol, leq), and water (3ml). Methanol (~15ml) was added to yield a monophasic solution. The reaction was monitored using TLC (5 ethanol : 3 NH ₄ OH : 1 water) and HPLC using a Phenomenex Luna NH ₂ column (4.6 x 300mm, 5μιη) and 1 water : 1 acetonitrile as the mobile phase at flow rate of lml/min, with ELS detection of eluants. After 17 hours of reaction, complete consumption of starting material was detected. The organic solvents were removed in vacuo and the aqueous layer washed with ethyl acetate (2 x 15ml). The aqueous layer was lyophilized to remove NH ₄ HCO3. The crude solid was dissolved in water (1ml) and Cu ²⁺ removed by cation exchange through a column of Dowex 50WX8 (H ⁺ form), eluted with water as the mobile phase. Lyophilization yielded the desired compound as a white amorphous solid 5B (7.5mg, 91%).

Protein conjugation: Reference Example 19

QP-alkyne: QP bearing alkyne at surface-exposed lysine residues was prepared by incubating a 10 mg/mL solution of QP with 25 mM of N-(4-Pentynoyloxy) succinimide (35- fold excess with respect to protein subunit) in 0.1 M potassium phosphate buffer (pH 7) with 10% DMSO for 12 hours. The derivatized virus was separated from excess reagent by ultracentrifugation using a 10-40% sucrose gradient.

Starting materials used in the foregoing Examples are commercially available, can be prepared by known methods, or can be prepared by methods analogous to those known in the art.

The glycoconjugates of Examples 1 to 15 can be prepared by methods analogous to the foregoing Examples:

Example la

[D-Rham-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man-(CH ₂ )5-NH-C(S)-NH-CH ₂ ]9-BSA

Example lb

[D-Rham-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man-(CH ₂ ) ₅ -NH-C(S)-NH-CH ₂ ] ₁₃ -BSA Example 2

[D-Rham-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man-(CH ₂ ) ₅ -NH-C(S)-NH-CH ₂ ] ₄ -CRM Example 3

[D-Rham-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man-(CH ₂ ) ₅ -NH-C(S)-NH-CH ₂ ] _1-2 -QP Example 4 {[C6-(S)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Ma n-(CH ₂ ) ₅ -NH-C(S)-NH- CH ₂ } ₇ -BSA

Example 5 {[C6-(S)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Ma n} ₉ -CRM Example 6 {[C6-(S)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Ma n} _! -QP

Tetrasaccharide azide 5B (Reference Example 13, 0.5 mM) was added to QP-alkyne (Reference Example 19, 1 mg/ml) in 0.1 M potassium phosphate buffer pH 7. The following reagents were added sequentially: amino guanidine (AG, 5 mM), mixture of CuS0 ₄ :THPTA [tris(3 -hydro xypropyltrazolylmethyl)amine] in a molar ratio of 1 :5 (0.25 mM CuS0 ₄ , 1.25 mM THPTA), and sodium ascorbate (5 mM). The reaction mixture was incubated at room temperature for 1 firs. Samples of the title compound were analyzed and purified by size- exclusion chromatography (SEC) using a Superose6 column. Example 7

{[C3-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man } ₉ -BSA Example 8

{[C3-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man } ₇ -CRM Example 9 {[C3-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man} ₁ -QP Example 10

{[C5-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man } ₇ -BSA

Example 11

{[C5-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man } ₈ -CRM Example 12

{[C5-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Man } _2-3 -QP Example 13

{[C6-(R)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3 )Man} ₁₃ -BSA Example 14

{[C6-(R)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3 )Man} ₁₀ -CRM Example 15 {[C6-(R)-Methyl]-Man-a-(l→2)-Man-a-(l→2)-Man-a-(l→3)Ma n} ₁ -QP

Example 16 - Enzyme Linked Immunosorbent Assays (ELISA) Four New Zealand white rabbits were immunized with a prime and three boost

immunizations of glycoconjugate (100 μg per injection in case of BSA and CRM conjugates and 50 μg per injection in the case of QP). The prime was given at Week 0 and the boost immunizations were given at Weeks 4, 8 and 12. Pre bleed (Week 0) and post bleed (Week 14) was used for all the ELISA analysis. Serum binding titers were determined against gpl20s absorbed directly onto ELISA wells. Initially gpl20 JRCSF WT was chosen as a candidate and serum from the rabbits was analysed for binding ability. Some serum was subjected to further gpl20s. Correlation between the rabbits immunised and the conjugates used are given in Table la. Binding results are summarized in Table lb.

265 - 268 Example 3

269 - 272 Example lb

273 - 276 Example 4

285 - 288 Example 7

289 - 292 Example 8

293 - 296 Example 9

297 - 300 Example 10

301 - 304 Example 11

305 - 308 Example 12

374-377 Example 13

378-381 Example 14

Table la

294 Med - -

295 Med -

299 Med - -

304* High High Med

308 Weak - Weak

374 Medium - -

379 Weak High Medium

381 Weak High Weak rom rabbit 304 also binds g l2C ) Bal

Table lb

Serum from rabbits 290, 304 and 379 show strong binding with different gpl20s and are shown in Figures 2a, 2b and 2c. Weak, medium and high binding categories in Table lb were assigned based on the serum dilution value when 50% binding was observed. The relevant serum dilution values are shown for rabbits 290, 304 and 379 in Figures 2a, 2b and 2c.

Results show that synthetic glycoconjugates raise antibodies which bind to the ohgomannose glycans displayed in the surface of envelope glycoprotein gpl20.

Example 17 - Binding ELISA Assay (Conjugates/Serum)

IgG from serum samples was tested for binding ability with carrier protein and also with the corresponding glycoconjugates. The serum samples chosen for this assay were from rabbits 304 and 290. Results are shown in Figure 3.

It was found that the CRM conjugates raised high IgG type antibody titers (EC ₅₀ in the range of 1 : 10,000). Serum shows no binding towards the non Histag proteins such as BSA and QP. IgG binds to carrier protein CRM, suggesting that serum IgG contains some anti-carrier properties as well. To verify specific reactivity against non-self Mari ₄ variants, serum binding was further probed using glycoconjugates where the platform changed from CRM to BSA or QP conjugated with non-self sugar variants. In this case also serum IgG binds in the range of 1 : 10,000. This clearly suggests sugar specificity of serum IgG irrespective of its anti-carrier properties. Moreover the binding of serum IgG to different glycoconjugates suggests that the observed reactivity towards the glycoconjugates is not too specific to their arrangement on the chosen carrier protein.

Similar binding ELISA were performed for the QP conjugates with serum from rabbit 267 (weeks 0 and 14), against carrier proteins and glycoconjugates. The results (Figure 4) show that serum IgG binds to the carrier protein and also the corresponding conjugate in the range of 1 : 10,000. When the serum was tested against different carriers with non-self sugar variant, it still shows similar Ab titers (1 : 10,000) and it clearly suggests sugar specificity.

Similarly serum from immunization with BSA based conjugate was tested against the natural glycoconjugate and also with the different platform based conjugates (CRM & QP). In this case serum IgG did not bind to the carrier protein and the observed binding titers were in the range of 1 : 1 ,000 (Figure 5). Thus, the serum IgG is sugar specific.

Binding ELISA results show that serum IgG obtained from immunization of BSA conjugates shows higher titers and is highly sugar specific. QP and CRM conjugates show high sugar specific IgG titer, and also some anti-carrier properties. These results are summarized in Table 2.

Table 2

Example 18 - CFG Glycan array Serum was probed against a wider panel of related oligomannosides on a printed glycan array. Serum was assayed at a 1 : 100 dilution to facilitate detection of lower-affinity interactions while minimizing the nonspecific background binding. Binding of serum IgG (Week 0 and Week 14) from rabbit 304 (CRM-C-5-Me-Dl) is shown in Figure 6. Week 0 bleed showed no binding to the mannose glycosides in printed array. Serum antibodies from week 14 bleed of rabbit 304 shows that CRM-(C-5-Me-Dl)s elicits IgG which recognizes synthetic fragments of high-mannose oligosaccharides in Man a(l→2)Man (glycans 205, 206, 207, 314, 313) and terminating in Man a(l→3)Man motifs (glycans 211, 212). Serum Abs did not bind the corresponding natural, high mannose glycans on a printed covalent array, only binding fragments which were linked by means of a lipid chain linker. From the glycan array list, it is also apparent that the raised antibody contains sugar specific properties.

Example 19 - Neutralization Assay Serum from rabbit and 304 was subjected to neutralization assay using the JRCSF pseudo virus which is sensitive to monoclonal Abs such as bl2, 2G12 and 4E10 (Figure 7). The result shows that serum from rabbit 304 effectively neutralizes the virus. Based on these results similar assays were performed for this serum with different HIV-1 isolates. Neutralization assays were performed with different HIV isolates including HxB2, JRFL, JRFL NB-DBJ and Yu2. In addition to that 2G12 neutralization was also performed as a positive control. In all the cases only serum from rabbit 304 neutralizes the virus (Figure 8). The same assay was performed using pre bleed (week 0) and post bleed (week 14) and the results are shown in Figure 9