WO2005092918A2

WO2005092918A2 - Targeting polypeptide

Info

Publication number: WO2005092918A2
Application number: PCT/GB2005/001084
Authority: WO
Inventors: Benjamin Michael Chain; Sean Paddy Nair
Original assignee: University College London
Priority date: 2004-03-22
Filing date: 2005-03-22
Publication date: 2005-10-06
Also published as: WO2005092918A3

Abstract

A targeting polypeptide is provided that may be used to target a chosen antigen to an antigen presenting cell. Complexes comprising such targeting polypeptide and antigen, nucleic acids and vectors encoding them, and cells comprising the nucleic acids and vectors may be used in methods of immunisation and enhance the immunogenicity of the antigen.

Description

TARGETING POLYPEPTIDE

Field of he Invention The present invention relates to targeting polypeptides and their use in targeting antigens to antigen presenting cells (APCs).

Background of the Invention Staphylococcus aureus is a major causative agent of community and hospital acquired infections worldwide. The organism is an important pathogen due to a combination of invasiveness, toxin production, and antibiotic resistance. S. aureus causes a wide variety of clinical syndromes, ranging from uncomplicated infections of the skin to life-threatening toxic shock syndrome (TSS). The bacterium causes disease by producing large numbers of exoproteins or virulence factors. Among the many -known virulence factors are two families of staphylococcal pyrogenic superantigens, staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST-1). Superantigens bind to major histocompatibility complex (MHC) class-fl on antigen presenting cells (B cells, monocytes, and dendritic cells) outside the classical antigen-binding groove, and activate T cells by binding with the variable region ofthe β chain of T cell receptors (Vβ-TCR). This cross-linking triggers the non-specific activation and proliferation of T cells, induces the production of high levels of a variety of cytokines, and causes toxic shock syndrome characterized by fever, rash, hypotension and multiple organ failure. Staphylococcal enterotoxins are responsible for many cases of food poisoning (intoxication) associated with ingestion of toxin- contaminated food. To date, more than thirteen staphylococcal enterotoxins have been described. As well as these classical superantigens, S. aureus also produces a family of proteins that have sequence homology to the superantigens, these proteins are -known as the staphylococcal exotoxin-like proteins (SETs) and are a family of polymorphic paralogs. They were first identified as a genetic locus encoding at least five exotoxin- like proteins (SETl-5). More recently, data from the sequencing ofthe genomes of several different S. aureus strains has revealed a large number of related (36-67%) set genes clustered on a genomic island. This putative pathogenicity island, which is present in all strains of S. aureus examined to date, codes for between seven and fourteen set genes, which have varying degrees of sequence homology. In addition, there appears to be extensive inter-strain allelic polymorphism for each ofthe set genes. The -International Nomenclature Committee for Staphylococcal Superantigen Nomenclature (INCSS) has recently recommended that the SETs be renamed staphylococcal su erantigen-like exoproteins (SSLs) and numbered from SSL1 to SSL14 in clockwise order from the replication origin ofthe chromosome based on homology to the full complement of genes found in strain MW2. This nomenclature is essentially as described by Fitzgerald et al (Infect. Immun., 2003, 71, 2827-2838) except that the numbering ofthe genes is in the opposite direction. To differentiate between allelic- variants the ssl gene is prefixed by the strain name. The three-dimensional structure of one member ofthe family, SSL5

(previously SET3) has been determined. The crystal structure of this protein shows many ofthe characteristic structures ofthe superantigen superfamily, but significant differences also exist. In addition, SSLs do not show the main properties of classical superantigens, such as polyclonal T cell activation, pyrogenicity, or enhancement of endotoxin shock . The function of SSLs is therefore unknown.

Summary of the Invention The present invention is based on the finding that Staphyloccal superantigen- like exoproteins (SSLs) are able to target themselves to antigen presenting cells (APCs). This targeting to antigen presenting cells and hence the antigen presentation pathway of these cells means that a chosen antigen can also be targeted to the antigen presenting cell facilitating presentation ofthe antigen and hence increasing immunogenicity. The invention may also be used to target antigens to antigen presenting cells in order to induce tolerance. Accordingly, the invention provides for the use of a complex comprising: (a) a targeting polypeptide comprising a staphylococcal superantigen-like protein (SSL), a fragment thereof or a variant of either, where the SSL, fragment or variant has the ability to target the complex to an antigen presenting cell; and

(b) an antigen and/or a nucleic acid molecule encoding an antigen, in the manufacture of a medicament for use in immunization or the induction of tolerance. The invention also provides a complex comprising: (i) a targeting polypeptide as defined in any one ofthe preceding claims; and (ii) an antigen or a nucleic acid encoding an antigen, wherein the antigen or encoded antigen is selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen. The invention also provides a viras comprising a targeting polypeptide ofthe invention In addition, the invention provides: a nucleic acid molecule comprising a polynucleotide sequence encoding a targeting polypeptide and an antigen selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen; - a vector comprising a nucleic acid ofthe invention; and a cell comprising a nucleic acid or a vector ofthe invention or infected with a viras ofthe invention. The invention also provides a method of loading antigen presenting cells comprising contacting an antigen presenting cell with a complex or viras ofthe invention. An antigen presenting cell which has been loaded with a complex or viras is also provided. The invention additionally provides: a pharmaceutical composition comprising a complex ofthe invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus ofthe invention or an antigen presenting cell ofthe invention and a pharmaceutically acceptable carrier or diluent; a vaccine comprising a complex ofthe invention, a nucleic acid encoding the targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a viras ofthe invention or an antigen presenting cell ofthe invention; and a complex ofthe invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus ofthe invention, or an antigen presenting cell ofthe invention for use in a method of treatment ofthe human or animal body by therapy. The invention also provides for the use of a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a viras ofthe invention or an antigen presenting cell ofthe invention in the manufacture of a medicament for use in immunisation. The invention further provides a method of immunising a subject, the method comprising administering an effective amount of a complex ofthe invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a viras ofthe invention or an antigen presenting cell ofthe invention to a subject. The invention also provides an agent for immunising a subject, the agent comprising a complex ofthe invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus ofthe invention or an antigen presenting cell ofthe invention.

Brief Description of the Figures Figure 1 - Panel (a) shows the structure of SSL7, shaded from white (N- terminal) to dark (C-terminal). Panel (b) shows the structure of SSL7 (dark) optimally superposed on that of SSL5 (grey) and the same structure is shown on the right rotated by 90°. Panel (c) shows the structure of SSL7 (dark) optimally superposed on that of SPEC (grey) and the same structure is shown on the right rotated by 90°. Panel (d) shows the SSL7 dimer. Figure 2 - shows residues in the SSL7 dimer interface with residue numbers being given on the x-axis, and buried surface area (A²) on the y-axis. The two forms are show by broken and unbroken lines. Figure 3 - Panel (a) shows the results of FACS analysis of PBMCs (peripheral blood mononuclear cells) incubated with varying concentrations of SSL-7-FITC at 4°C and 37°C. The graph is representative often separate experiments carried out with PBMCs from healthy donors. Panel (b) shows the results of competition experiments between SSL9-FITC (top) and SSL7-FITC (bottom) with various molecules for binding to PBMCs. Results shown are from one representative experiment of three. Figure 4 - shows the results of FACS analysis of PBMC stained with SSL7 (top) or SSL9 (bottom) and various markers. The results shown are representative of five separate experiments. Figure 5 - shows the results of FACS analysis of PBMC-derived dendritic cells stained with SSL9, SSL7 or no stain. The results are representative of a set of three separate experiments on cells from healthy donors. Figure 6 - shows that SSL and SSL9 interact selectively with dendritic cells. FACS results for unpurified Dendritic cells incubated with SSL7-FITC or SSL9-FITC and then stained for CD la are shown. The numbers show the percentage of SSL positive cells that were also CD la positive. Figure 7 - shows that SSLs do not alter Dendritic cell morphology or cell surface phenotype. Panel (A) shows the morphology of Dendritic cells treated with SSL proteins. Results for lipopolysaccharide (LPS) and peptidoglycan (PG) are shown as positive controls. Results are representative of three experiments. Panel (B) shows FACS results for phenotypic analysis of dendritic cells treated with SSL proteins. Data are shown for expression of cell surface molecules on Dendritic cells that have been treated with SSL7 or SSL9. LPS and PG were used as positive controls. Expression ofthe indicated markers is shown by the solid histograms, whereas cells stained with relevant control mAb are indicated by the open line histograms. The numbers on each histogram correspond to the median fluorescence intensity (MFI) of mAb staining. Results shown are from one donor and are representative of similar data obtained from experiments carried out with dendritic cells from four different donors. Figure 8 - shows endocytosis of FITC-Dextran by Dendritic cells exposed to SSL proteins. The results of one of three separate experiments are shown. Figure 9 - shows that the effect of SSL protein on the T cell stimulatory capacity of dendritic cells. Panel (A) shows the effect of SSL7 (black bars) or SSL9 (empty bars) or medium alone (grey bars) on stimulation of autologous T cells in the presence or absence (M) of purified protein derivative (PPD). Data shown are mean + SEM of 3 experiments. Figure 10 - shows autologous T cell responses to Dendritic cells loaded with SSL7 or SSL9 protein. Data are mean + SD of triplicate cultures from individual experiments. Figure 11 - shows the effects of SSL protein on cytokine production. Purified protein derivative is used as control. Figure 12 - shows antibody responses in human sera. Panel (A) show levels of SSL7 or SSL9 measured by ELISA. Results for a polyclonal rabbit antibody raised against purified His-tagged SSL7 are included as a positive control. Data are representative of 3 experiments. Panels (B) and (C) show the results of a competitive ELISA with plates coated with either SSL7 (B) or SSL9 (C) where serum diluted 1:2000 (final dilution) was mixed with varying concentrations of SSL7, SSL9 or Emblp32 as indicated.

Brief Description of the Sequences SEQ ID No: 1 provides the nucleotide sequence of a genomic fragment comprising the pathogenicity island SaPIn2 from S. aureus strain N315 which includes the ssll to ssl5 and ssl7 to sslll genes. SEQ ID Nos: 2 to 11 provide the amino acid sequences of SSL1 to SSL7 and SSL7 to SSL 11 from S. aureus strain N315 and also indicate the location ofthe CDS ofthe genes in SEQ ID No:l. SEQ ID No: 12 provides the nucleotide sequence of a genomic fragment from S. aureus strain N315, which includes the ssll 2 to ssll 4 genes. The sequence indicated is the complement ofthe coding strand. The order ofthe genes in the indicated sequence going from 5' to 3' is ssll 4, ssl 13 and ssll2. The complementary sequence is given by SEQ ID No: 103. SEQ ID Nos: 13 to 15 provide the amino acid sequences of SSL14, SSL 13 and SSL12 respectively from S. aureus strain N315 and also indicate the location of the CDS ofthe genes in SEQ ID No: 12. The start of each coding sequence indicated is the higher nucleotide position listed. SEQ ID No: 16 provides the nucleotide sequence of a genomic fragment comprising the pathogenicity island SaPIn2 from S. aureus strain Mu50, which includes the ssll to 3, ssl5 and ssl7 to sslll genes. SEQ ID Nos: 17 to 25 provide the amino acid sequences of SSL1 to SSL3, SSL5 and SSL 7 to 11 respectively from S. aureus strain Mu50 and also indicate the location ofthe CDS ofthe genes in SEQ ID No: 16. SEQ ID Nos: 26 to 36 provide the amino acid sequences of SSL1 to SSL11 respectively from S. aureus strain MW2 and also indicate the location ofthe CDS in SEQ ID No: 103. SEQ ID No: 37 provides the nucleotide sequence of a genomic fragment from S. aureus strain NCTC8325, and includes the sequences ofthe ssll to sslll genes. The sequence indicated is the complement ofthe coding strand. The order ofthe genes in the indicated sequence going from 5' to 3' is sslll to ssll. The complementary DNA sequence is given by SEQ ID No: 104. SEQ ID Nos: 38 to 48 provide the amino acid sequences of SSL1 to 11 respectively from S. aureus strain NCTC8325 and also indicate the location ofthe CDS ofthe genes in SEQ ID No:37. The start of each coding sequence indicated is the higher nucleotide position listed. SEQ ID No: 49 provides the nucleotide sequence of a genomic fragment from S. aureus strain NCTC8325, and includes the sequences ofthe ssll 2 to ssll4 genes. SEQ ID Nos: 50 to 52 provide the amino acid sequences of SSL12 to 14 respectively from S. aureus strain NCTC8325 and also indicate the location ofthe CDS ofthe genes in SEQ ID No:49. SEQ ID No: 53 provides the nucleotide sequence of a genomic fragment from S. aureus strain EMRSA 16(252), which includes the sequences ofthe ssll to ssl5, ssl7, ssl9 to sslll genes. The sequence indicated is the complement ofthe coding strand. The order ofthe genes in the indicated sequence going from 5' to 3' is sslll to ssl9, ssl7 and ssl5 to ssll. The complementary DNA sequence is given by SEQ ID No:105. SEQ ID Nos: 54 to 62 provide the amino acid sequences of SSL1 to SSL5, SSL7, SSL9 to SSL11 respectively from S. aureus strain EMRSA 16(252) and also indicate the location ofthe CDS ofthe genes in SEQ ID No:53. The start of each coding sequence indicated is the higher nucleotide position listed. SEQ ID No: 63 provides the nucleotide sequence of a genomic fragment from S. aureus strain E-MRSA 16(252) and includes the sequences ofthe ssll 2 to 14 genes. The sequence indicated is the complement ofthe coding strand. The order ofthe genes in the indicated sequence going from 5' to 3' is ssll 4 to ssll 2. The complementary sequence is given by SEQ ID No:106. SEQ ID No: 64 to 66 provide the amino acid sequences of SSL12 to 14 respectively from S. aureus strain EMRSA 16(252) and also indicate the location of the CDS ofthe genes in SEQ ID No:63. The start of each coding sequence indicated is the higher nucleotide position listed. SEQ ID No: 67 provides the nucleotide sequence of a genomic fragment from S. aureus strain MSSA-476 which includes the sslll to sslll genes. SEQ ID No: 68 to 78 provide amino acid sequences of SSL1 to SSL11 respectively from S. aureus strain MSSA-476 and also indicate the location ofthe CDS ofthe genes in SEQ ID No:67. SEQ ?ED No: 79 provides the nucleotide sequence of a genomic fragment from S. aureus strain MSSA-476 which includes the sequences ofthe ssll 2 to ssll 4 genes. SEQ D No: 80 to 82 provides the amino acid sequences of SSL12 to SSL 14 from S. aureus strain MSSA-476 and also indicate the location ofthe CDS ofthe genes in SEQ ID No:79. SEQ ID Nos: 83 and 84 provide the nucleotide and amino acid sequences respectively of sslll from S. aureus strain COL. SEQ ID Nos: 85 and 86 provide the nucleotide and amino acid sequences respectively of ssll 2 from S. aureus strain COL. SEQ ID Nos: 87 and 88 provide the nucleotide and amino acid sequences respectively of ssll 3 from S. aureus strain COL. SEQ ID Nos: 89 and 90 provide the nucleotide and amino acid sequences respectively of ssll4 from S. aureus strain COL. SEQ ID Nos: 91 and 92 provide the nucleotide and amino acid sequences respectively of ssl9 from S. aureus strain COL. SEQ ID Nos: 93 and 94 provide the nucleotide and amino acid sequences respectively of ssll 0 from S. aureus strain COL. SEQ ID Nos: 95 and 96 provide the nucleotide and amino acid sequences respectively of sslll from S. aureus strain COL. SEQ -CD Nos: 97 and 98 provide the nucleotide and amino acid sequences respectively of ssll 2 from S. aureus strain COL. SEQ ID Nos: 99 and 100 provide the nucleotide and amino acid sequences respectively of ssll 3 from S. aureus strain COL. SEQ ID Nos: 101 and 102 provide the nucleotide and amino acid sequences respectively of ssll4 from S. aureus strain COL. SEQ ID No: 103 provides the complementary sequence to SEQ ID No: 12. SEQ ID No: 104 provides the complementary sequence to SEQ ID No: 37. SEQ JD No: 105 provides the complementary sequence to SEQ ID No: 53. SEQ ID No: 106 provides the complementary sequence to SEQ ID No: 63. SEQ -ID No: 107 provides the nucleotide sequence of a genomic fragment from S. aureus strain MW2 which includes the ssll to sslll genes. The amino acid sequences ofthe encoded proteins are provided by SEQ ID Nos: 26 to 36 which indicate the position ofthe coding sequences in SEQ ID NO: 107. Detailed Description of the invention Throughout the present specification and the accompanying claims the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows. In some cases, where specific constituents are recited, the embodiment may, for example, consist essentially of such constituents. The present invention is based on the finding that SSLs are able to target themselves to antigen presenting cells (APCs). The targeting to antigen presenting cells leads to the SSLs entering the antigen presenting pathway ofthe antigen presenting cells and being presented on the surface ofthe antigen presenting cells. This enhances the immunogenicity ofthe SSLs. The invention uses targeting polypeptides employing, or based on, the sequence of SSLs to target a chosen antigen to antigen presenting cells to enhance the immunogenicity ofthe chosen antigen. In effect, the targeting polypeptides are used to deliver a chosen antigen to an antigen presenting cell. In some instances the targeting polypeptides are used to deliver a nucleic acid molecule encoding the antigen, rather than the antigen itself. The nucleic acid molecule will then give rise to expression ofthe antigen in the antigen presenting cell and the subsequent presentation ofthe antigen. The invention employs targeting polypeptides comprising the polypeptide sequence of an SSL, a fragment of an SSL, or a variant sequence based on either, to target a chosen antigen or encoding nucleic acid to an APC. The invention uses complexes comprising the targeting polypeptide and antigen or an encoding nucleic acid to achieve delivery ofthe chosen antigen to an antigen presenting cell.

Targeting polypeptides The targeting polypeptides employed comprise a staphylococcal superantigen- like protein (SSL), a fragment thereof or a variant of either, where the SSL, fragment or variant has the ability to target the targeting polypeptide to an antigen presenting cell (APC). The ability to target a polypeptide to an antigen presenting cell is referred to herein as targeting activity. The targeting polypeptide may comprise the sequence of any naturally occurring SSL polypeptide. Such polypeptides may typically be isolated from Staphylococcus aureus. The amino acid sequences of SSLs 1 to 14 from a variety of strains of Staphylococcus aureus are provided herein and any of these sequences may be present in a targeting polypeptide ofthe invention. The Table below indicates the corresponding SEQ ID Nos for the SSLs.

Table - SSLs

The SSL sequence employed may be one or more of SSLl to SSL 14. In a preferred case where, for example, it is desired to use the sequence of an SSL9, the SSL9 will be have the sequence of one ofthe SSL9 indicated in Table 1 i.e. will be selected from SEQ ID Nos: 9, 23, 34, 46, 60, 76 and 92. In a preferred instance where is desired to employ the sequence of a SSL5, the SSL5 may be selected from the sequences of SEQ ID Nos: 6, 20, 30, 42, 58, and 72. In another preferred case, where it is desired to employ the sequence of a SSL 7, the SSL7 may be selected from the sequences of SEQ ID No: 7, 21, 32, 42, 59 and 74. Thus Table 1 indicates examples of preferred sequences for a particular SSL. In a particularly preferred embodiment the SSL sequence employed may be SSL5, SSL7 and/or SSL9 and may preferably be one ofthe specific SSL 7 and SSL9 molecules whose sequence is provided herein. The SSL sequence may be a fragment of such a sequence. The SSL sequence employed may be a variant ofthe specific SSL 7 and SSL9 molecules whose sequence is provided herein or may be a variant of a fragment of such a sequence. hi some cases, the SSL employed may be an allelic form of an SSL as SSL genes from different Staphylococcus aureus strains vary in sequence. In some instances the SSL may be from one ofthe Staphylococcus aureus strains MW2, NCTC6571, F-RI326 or NCTC8325, N315, Mu50, EMRSA 16(252), MSSA-476 or COL. hi a particularly preferred embodiment the SSL may be from NCTC6571. The targeting polypeptide may comprise a fragment of a naturally occurring SSL where the fragment retains the ability to target the targeting polypeptide to an antigen presenting cell. Such fragments may comprise subregions of any one or more ofthe sequences of any of SSLl to SSL14 and in particular ofthe sequences provided herein of particular SSLl to SSL14 molecules. The targeting polypeptide may, for example, contain a sub-region of an SSL that is from 25 to 200, preferably from 35 to 175, even more preferably from 50 to 150 and even more preferably 75 to 125 amino acids in length. The targeting polypeptide may comprise a sub-region of an SSL that is 220 or less, preferably 180 or less, more preferably 160 or less, still more preferably 140 or less and even more preferably 120 or less amino acids in length. Such fragments may be derived from any SSL and in particular from the amino acid sequences indicated herein for SSLl to SSL14. In some cases, the subregion may comprise at least 30, preferably at least 50, more preferably at least 75 and even more preferably at least 100 amino acids from the SSL. In a preferred case the fragment may be from SSL5, SSL7 and/or SSL9. In a particularly preferred case the fragment may be from SSL9 and/or SSL7. The targeting polypeptide may comprise a variant sequence of an SSL or a fragment thereof. Such variant sequences will be able to target themselves to antigen presenting cells. Any suitable variant polypeptide capable of directing the targeting polypeptide to an antigen presenting cell may be employed. In some cases, the variant will have at least 25%, preferably at least 30%, more preferably at least 35%, still more preferably at least 40% and even more preferably at least 45% amino acid sequence identity to an SSL and in particular to an amino acid sequence of any one or more ofthe specific sequences provided herein for SSL 1 to 14. Thus the variant may have the specified level of sequence identity with the equivalent SSL whose sequence is provided herein. The level of amino acid sequence identity may be at least 50%, more preferably at least 55%, even more preferably at least 60% and still more preferably at least 65% amino acid sequence identity. The variant may, for example, have at least 75%o, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%) sequence identity and even more preferably at least 95%) sequence identity to an SSL and in particular to one or more ofthe specific SSLl to 14 molecules whose sequence is provided herein. some cases the SSL employed may be an allelic variant of a -known SSL gene including an allele of any of SSLs 1 to 14. Such variants may have a high degree of sequence identity to a -known SSL allele and particular to one of those provided herein. Thus in some cases the variant may have at least 85%, preferably at least 95%, more preferably at least 97%, and even more preferably at least 99% sequence identity to any ofthe SSLs mentioned herein. The amino acid sequences of alleles of SSL5, SSL7 and SSL9 are indicated in Table 1 and the targeting polypeptide may comprise one or more of these sequences. In instances where the targeting polypeptide comprises a variant sequence, the variant sequence may have one ofthe levels of sequence identity specified herein to more than one SSL. Thus, for example, the invention encompasses a variant of an SSL or a variant of a fragment of an SSL that has one ofthe specified levels of sequence identity to all ofthe SSLs whose sequence is provided herein or to a particular SSL. The sequence may have one ofthe specified levels sequence identity to all ofthe alleles provided herein for a particular SSL whose sequence is provided herein. hi some cases a variant sequence may have one ofthe specified levels of sequence identity to at least two, preferably at least three, more preferably at least five and even more preferably at least six ofthe sequences provided herein for a particular SSL. In a preferred embodiment it will have one ofthe levels of sequence identity specified to all ofthe sequences provided herein for SSL5, SSL7 and/or SSL9. The variant may in particular have one ofthe specified levels of sequence identity to all of the sequences provided herein for SSL7 and/or SSL9. In a preferred case, the variant may have the specified level of sequence identity to the alleles from strain NCTC6571.1 The targeting polypeptide may comprise a variant sequence of a fragment of an SSL. Thus the fragment may be any ofthe lengths referred to herein. It may have any ofthe degrees of sequence identity referred to herein. In general a variant sequence may have the specified level of sequence identity over the whole ofthe variant, over at least 20, preferably at least 50, more preferably at least 75, and even more preferably over at least 100 amino acids. In the case of fragments, the sequence identity may be over any ofthe lengths specified herein and in particular over the entire fragment or targeting sequence. The variant or fragment has to be able to target the polypeptide to an antigen presentation cell. In cases where the targeting polypeptide comprises sequences, other than those responsible for targeting, the level of sequence identity may be over the region corresponding to the SSL, fragment thereof or variant of either. The specified level of sequence identity may be over the minimum region in the polypeptide necessary for targeting to the antigen presenting cell. The variant may have amino acid substitutions in comparison to the normal sequence ofthe SSL. For example, it may have from 1, 2, 3 or more substitutions such as from 5 to 10, 10 to 20, 20 to 30 or more amino acid substitutions. The variant may have, in addition or alternatively, such numbers of amino acids deleted or inserted into it in comparison to the normal SSL. The amino acid changes may be conservative substitutions, for example according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

The targeting polypeptide may comprise additional sequences to those responsible for targeting to an antigen presenting cell. In some cases the targeting polypeptide will consist essentially ofthe targeting sequences. In cases where there are additional sequences present, they may serve a variety of roles. In a particularly preferred embodiment, the targeting polypeptide will also comprise the antigen it is desired to target to the immune cells. The antigen may be any of those discussed herein. Polypeptide sequences may be present which separate the various regions of the targeting polypeptide. For example, regions may be present to separate the region responsible for targeting from an antigen. Polypeptide sequences may be present which allow for purification ofthe polypeptide such as, for example, a histidine tag or an antibody recognition site. The targeting polypeptide may include an enzymatic cleavage site and in particular a protease recognition site may be present in the targeting polypeptide. It may be desirable to be able to remove sequences from the polypeptide such as those used to purify the polypeptide and cleavage sites may be employed. The targeting polypeptide may lack specific sequences, for example because they have been cleaved after they are no longer of use. Thus in a preferred instance the targeting polypeptide may, for example, lack a Histidine tag and hence lack four, five, six, seven, eight, ten or more consecutive histidine residues typically at the terminus ofthe protein. The targeting polypeptide may lack an antibody recognition sequence used in purification, a reporter sequence and/or sequences such as thioredoxin or Pho A sequences. In some embodiments ofthe invention such sequences will not be present at any stage and will not be encoded by the nucleic acids ofthe invention. The targeting polypeptide may comprise viral sequences. A targeting polypeptide ofthe invention may be used to target a viral particle to an antigen presenting cell, hi such cases, the targeting polypeptide will typically be provided wholly or partially on the surface ofthe virus to allow the targeting sequence to target the virus to an antigen presenting cell. The targeting polypeptide may increase the affinity ofthe viral particle for the antigen presenting cell and may mean that the viral particle is more selective in binding to antigen presenting cells as opposed to other cell types. The viral sequences may be, or comprise sequences from, surface proteins or polypeptide sequences which allow the targeting polypeptide to be displayed on the surface ofthe virus. The targeting polypeptide may comprise trans-membrane sequences to allow the targeting polypeptide to be present in a membrane and in particular in a viral membrane. The targeting sequence may comprise linker sequences to allow different domains in the targeting polypeptide to function. In one case the targeting polypeptide may comprise a proline rich linker and in particular all or part ofthe pro line rich sequence from the 4070A protein from a leukaemia viras surface protein. In cases where the targeting polypeptide is employed to target a viral particle to an antigen presenting cell the targeting sequences may be present in the targeting polypeptide together with the sequences necessary for fusion ofthe membranes ofthe virus and antigen presenting cell to allow entry ofthe virus into the cell. The targeting polypeptide may comprise a protease cleavage site which will allow cleavage ofthe part ofthe targeting polypeptide and in particular a protease site which may be cleaved by a protease present on the surface ofthe antigen presenting cell. In a preferred embodiment, the targeting polypeptide may comprise the sequence of SSL 5, 7, or 9 and in a particularly preferred case the sequence of SSL 7 or 9 may be employed. In some cases the sequence of SSL 7 may be employed, particularly where it is desired to target B cells such as CD19⁺ B cells. In other cases, such as for example where it is not desired to target B cells, other SSLs may be employed and in particular SSL9 may be employed, hi each case fragments of such sequence or variants of either may be employed including any of those referred to herein. In a preferred embodiment of the invention the targeting polypeptide comprises:

(a) a SSL polypeptide having the amino acid sequence of any of SEQ ID Nos:

2 to 11, 13 to 15, 17 to 36, 38 to 48, 50 to 52, 54 to 66, 68 to 78, 80 to 82, 84, 86,88, 90, 92, 94, 96, 98, 100 and 102;

(b) a fragment of any of the sequences of (a), the fragment having the ability to target the complex to an antigen presenting cell; and/or

(c) a variant polypeptide having at least 30% amino acid sequence identity to any ofthe polypeptides of (a) or (b) and the ability to target the complex to an antigen presenting cell. h another the targeting polypeptide comprises: (a) the sequence of SEQ ID No: 7, 21, 32, 44, 59 and/or 74;

(c) a variant polypeptide having at least 70 % amino acid sequence identity to any ofthe polypeptides of (a) or (b) and the ability to target the complex to an antigen presenting cell. In another the targeting polypeptide comprises:

(a) the sequence of SEQ ID No: 9, 23, 34, 46, 60, 76 and/or 92;

(b) a fragment of any of the sequences of (a), the fragment having the ability to target the complex to an antigen presenting cell; and/or (c) a variant polypeptide having at least 70 % amino acid sequence identity to any ofthe polypeptides of (a) or (b) and the ability to target the complex to an antigen presenting cell. In some cases a targeting polypeptide may comprise a plurality of sequences which individually would be able to lead to targeting to an antigen presenting cell. A minimal sequence necessary for targeting to an antigen presenting cell may be referred to as a targeting sequence. Such targeting sequences may be any of those discussed herein which have the ability to target a polypeptide to an antigen presenting cell. Thus a targeting polypeptide may comprise, for example, 1, 2, 3, 5 or more such targeting sequences. The targeting polypeptide may comprise any pair of those targeting sequences referred to herein. In some cases the targeting polypeptide may comprise different targeting sequences with different properties. ?l?n some cases the targeting polypeptide will be employed to target a viral particle to an antigen presenting cell. Thus the targeting polypeptide collectively present on the viras will be able to increase the affinity and/or specificity ofthe viral particle for an antigen presenting cell in comparison to an equivalent viras lacking the targeting polypeptide.

Antigens The targeting polypeptides ofthe invention are used to deliver a chosen antigen to an antigen presenting cell. The antigen may be any suitable antigen and typically will be a peptide or a polypeptide antigen. The antigen may, for example, be an antigen selected from a pathogenic antigen, an auto-antigen, an allergen and a cancer antigen In some cases the invention may be used to deliver a nucleic acid molecule encoding an antigen to an antigen presenting cell. The nucleic acid may then be expressed in the antigen presenting cell to give rise to the antigen and presentation of the antigen. Thus the invention also encompasses the targeting of a nucleic acid molecule encoding any ofthe antigens mentioned herein to an antigen presenting cell. In a preferred instance the antigen, may be an antigen from an infectious organism. The antigen may, for example, be derived from a virus, bacterium, parasite, protozoan, fungus, or prion. The antigen may be a surface antigen expressed on the surface ofthe pathogen or may be an infracellular antigen. The antigen may be from an intracelluar pathogen or alternatively an extracellular one. The antigen may, for example, be from a bacterium. It may be from a gram positive or a gram negative bacterium. The antigen may, for example, be: an antigen from Mycobacterium (for example from Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansaii, o Mycobacterium gordonae); Pseudomonas; Yersinia; Salmonella (for example from Salmonella typhimurium); Helicobacter (for example from Helicobacter pylori); Borelia (for example from Borelia burgdorferi); Bordetella (for example from Bordtella pertussis or Bordetella parapertussis); Legionella (for example from Legionella pneumophilia); Staphylococcus (for example from Staphylococcus aureus); Neisseria (for example from Neisseria gonorrhoeae or Neisseria meningitides); Listeria (for example from Listeria monocytogenes); or Streptococcus (for example from Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, or Streptococcus pneumoniae). In some cases the antigen may be from: a Campylobacter; Enterococcus; Haemophilus (for example from Haemophilus influenzae); Bacillus (for example from Bacillus antracis); Corynebacterium (for example from Corynebacterium diphtheriae); Erysipelothrix (for example from Erysipelothrix rhusiopathiae); Clostridium (for example from Clostridium perfringers, or Clostridium tetani); Vibrio (for example Vibrio cholerae); Enterobacter (for example from Enterobacter aerogenes); Klebsiella (for example from Klebsiella pneumoniae); Pasturella (for example from Pasturella multocida); Bacteroides; Fusobacterium (for example from Fusobacterium nucleatum); Streptobacillus (for example from Streptobacillus moniliformis); Shigella; Escherichia (for example from Escherichia coli); Rickettsia; Treponema (for example from Treponema palladium); Lactococcus; Lactobacillus; Brucella; Aeromona;, Franciesella; Citrobactor; Rhodococcus; Leishmania; or Strongylus (for example from Strongylus vulgaris). Examples of preferred bacterial antigens include: the Shigella sonnei form 1 antigen; the FI antigen of Yersinia pestis; antigens from Neisseria meningititidis and in particular those encoded by the GNA33, GNA2001, GNA1220 and GNA1946 genes; the O-antigen of V. cholerae Inaba sfrain 569B; protective antigens of enterotoxigenic E.coli, such as fimbrial antigens including colonisation factor antigens, in particular CFA/I, CFA/II, and CFAJTV and the nontoxic B-subunit ofthe heat-labile toxin; pertactin of Bordetella pertussis, adenylate cyclase-hemolysin of E. pertussis; fragment C of tetanus toxin of Clostridium tetani and the LT (heat labile enterotoxin) and ST (heat stable toxin) antigens. In some instances the antigen may be a viral antigen. The antigen may, for example, be a viral coat protein, glycoprotein or other proteins expressed on the surface of a viras. The antigen may be from a Picornaviridae (for example from a polio viras, a hepatitis viras, an enteroviras, a coxsackie virus, a rhinovirus, or an echoviras); Calciviridae ; Togaviridae (for example from a equine encephalitis virus or a rubella viras); Flaviridae (for example from a dengue viras, an encephalitis viras, or a yellow fever viras); Coronaviridae (for example from a coronaviras); -Rhabdo viridae (for example from a vesicular stomatitis virus, or a rabies viras); Filoviridae (for example from an ebola virus); Paramyxoviridae (for example from a parainfluenza virus, mumps virus, measles viras, or a respiratory syncytial viras); Orthomyxoviridae (for example from an influenza viras such as influenza types A, B and C); Bungaviridae (for example from a Hanta viras, bunga viras, phleboviras or a Nairo virus); Arena viridae (for example from a hemorrhagic fever viras) ; Reoviridae (for example a rotaviras); Birnaviridae; Hepadnaviridae (for example a Hepatitis B viras); Parvoviridae; Papovaviridae (for example from a papilloma viras, or polyoma virus); Adenoviridae; Herpesviridae (for example from herpes simplex virus (HSV) 1 or 2, varicella zoster virus, cytomegaloviras or a herpes viruses); Poxviridae (for example from a variola viras, vaccinia virus, or a pox viras); or an Irido viridae (for example from African swine fever virus). hi a preferred case the antigen may be from a Retroviradae (e. g., HTLV-I ;

HTLV-11; or HIV-1 (also known as HTLV-111, LAV, ARV, hTLR, etc.)). In a particularly preferred case the antigen may be one derived from HIV and in particular the isolates HlVIllb, HIVSF2, HTVLAV, HIVLAI, HIVMN ; HIV-1CM235, HIV-1; or HIV-2. In a particularly preferred embodiment, the antigen may be a human immunodeficiency viras (HIV) antigen. Examples of preferred HIV antigens include, for example, gpl20, gap 160 gp41, gag antigens such as p24gag and p55gag, as well as proteins derived from the pol, env, tat, vif, rev, nef, vpr, vpu or LTR regions of HIV. In a particularly preferred case the antigen may be HIV gp 120 or a portion of HIN gpl20. The antigen may be from an immunodeficiency viras, and may, for example, be from S?TV or a feline immunodeficiency viras. In a preferred case the viral antigen may be one from a hepatitis viras such as an antigen from hepatitis A viras (HAV), hepatitis B viras (HBV), hepatitis C viras (HCN), the delta hepatitis virus (HDN), hepatitis E viras (HEN) or hepatitis G viras (HGV). In a particularly preferred embodiment the antigen may be from hepatitis B virus (HBV) and may preferably be a surface or core antigen from ItBN. In another preferred case the antigen may be from a herpesvirus family. Particular antigens include those from herpes simplex viras (HSN) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH; antigens from varicella zoster viras (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) including CMV gB and gH; and antigens from other human herpesvirases such as HHV6 and HHV7. The antigen maybe a fungal antigen, such as one from Candida oτAspergilus. m particular, it may be from Candida albicans or Aspergillus fumigatus. The antigen may be from Sporothrix (e.g from Sporothrix schenckii), Histoplasma (e.g. from Histoplasma capsulatum) Cryptococcus (e.g. from Cryptococcus neoformans) or Pneumocystis (e.g. from Pneumocystis carinii). The antigen may be from a parasitic pathogen and may, in particular, be from Taenia, Flukes, Roundworms, A ebiasis, Giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii, Trichomoniasis and Trichinosis. In some cases the antigen may be an antigen from a prion. In particular, the antigen may be one from the causative agent of kura, Creutzfeldt- Jakob disease (CJD), scrapie, transmissible mink encephalopathy and chronic wasting diseases, or from a prion associated with a spongiform encephalopathy, particularly BSE. The antigen may be from the prion responsible for familial fatal insomnia. In some cases the antigen may be from a parasitic pathogens including, for example, one from the genera Plasmodium, Chtamydia, Trypanosome, Giardia, Boophilus, Babesia, Entamoeba, Eimeria, Leishmania, Schistosome, Brugia, Fascida, Dirofilaria, Wuchereria and Onchocerea. Examples of preferred antigens from parasitic pathogens to be expressed as the heterologous antigen include the circumsporozoite antigens of Plasmodium species, such as the circumsporozoite antigen of P. bergerii or the circumsporozoite antigen of P. falciparum; the merozoite surface antigen of Plasmodium species; the galactose specific lectin of Entamoeba histolytica; gp63 of Leishmania species; paramyosin of Brugia malayi; the triose- phosphate isomerase of Schistosoma mansoni; the secreted globin-like protein of

Trichostrongylus colubriformis; the glutathione-S-transferases of Frasciola hepatica, Schistosoma bovis and S.japonicum; and ?KXH of Schistosoma bovis and S. japonicum. In some cases the antigen may be a cancer antigen and in particular a tumour antigen. Examples of particular cancers that the antigen may be derived include those from cancers ofthe lung, prostate, breast, colon, ovary, melanoma, a lymphoma and leukaemia. Examples of particular tumour antigens include MART-1, Melan-A, tyrosinase, p97, beta-HCG,GaINAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3, ?MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen,K19, Tyrl, Tyr2, members ofthe pMel 17 gene family, c-Met, PSA (prostate antigen), PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretary protein, alpha-fetoprotein, CA125, CA19.9, TAG-72, BRCA-1 and BRCA-2 antigens. The antigen may be an auto-antigen, i particular, the antigen may an antigen associated with an autoimmune disease. Auto-antigens include those associated with autoimmune diseases such as multiple sclerosis, insulin-dependent type 1 diabetes mellitus, systemic lupus erythematosus (SLE) and rheumatoid arthritis. The antigen may be one associated with, Sjorgrens syndrome, myotis, scleroderma or Raynaud's syndrome. Further examples of auto-immune disorders that the antigen maybe associated with include ulcerative colitis, Crohns' disease, inflammatory bowel disorder, autoimmune liver disease, or autoimmune thyroiditis. Examples of specific autoantigens include insulin, glutamate decarboxylase 65 (GAD65), heat shock protein 60 (HSP60), myelin basic protein (MBP), myelin ohgodendrocyte protein (MOG), proteolipid protein (PLP), and collagen type II. In cases where the antigen is an autoantigen the antigen will typically be administered in order to promote tolerance to the auto-antigen. Although in some cases models ofthe diseases may be produced using the invention to be produce an immune response. hi some cases the antigen may be an allergen. The allergenic antigen may be any suitable antigen from an antigen. For example, the allergen may be from Ambrosia artemisiifolia, Ambrosia trifida, Artemisia vulgaris, Helianthus annuus, Mercurialis annua, Chenopodium album, Salsola kali, Parietariajudaica, Parietaria ojficinalis, Cynodon dactylon, Dactylis glomerata, Festuca pratensis, Holcus lanatus, Lolium perenne, Phalaris aquatica, Phleum pratense, Poa pratensis or Sorghum halepense. The allergen antigen may be from a tree, such as, for example, from Phoenix dactylifera, Betula verrucosa, Carpinus betulus, Castanea sativa, Corylus avellana, Quercus alba, Fraxinus excelsior, Ligustrum vulgare, Olea europea, Syringa vulgaris, Plantago lanceolata, Cryptomeriajaponica, Cupressus arizonica, Juniperus oxycedrus, Juniperus virginiana, or Juniperus sabinoides. hi some cases the antigen may be from an antigen from a mite such as, for example, from Acarus siro, Blomia tropicalis, Dermatophagoides farinae, Dermatophagoides microceras, Dermatophagoides pteronyssinus, Euroglyphus maynei, Glycyphagus domesticus, Lepidoglyphus destructor or Tyrophagus putrescentiae. The allergen antigen may be from an animal such as, for example, from a domestic or agricultural animal. Examples of allergens from animals include those from cattle, horses, dogs, cats and rodents (e.g from rat, mouse, hamster, or guinea pig), h some cases the antigen may be from a food allergen and in others it may be from insect. The antigen may be one involved in transplant rejection. The invention may be use to induce or promote tolerance to such an antigen in order to ameliorate or prevent transplant rejection. Homologues of antigens may also be employed. Protein antigens employed may have homology and/or sequence identity with naturally occurring antigens, such as any ofthe antigens mentioned herein. They may have any ofthe levels of sequence identity or sequence changes specified herein. The antigen may be a model antigen. The antigen may be one commonly used in experiments to assess immune responses. For example the antigen may be a lysozyme and in particular chicken egg lysozyme. The antigen may be ovalbumin and in particular chicken ovalbumin. Such model antigens may have the advantage that antigens, T cells and other reagents may be readily available for assessing antigen presentation by the targeted antigen presenting cell.

Complexes The targeting polypeptides ofthe invention may be used to deliver a chosen antigen to antigen presenting cells (APCs). The targeting polypeptide and antigen are combined in the form of a complex. Thus a complex comprising a targeting polypeptide and an antigen is provided. In particular, the complex may comprise an antigen selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen In some cases a nucleic acid molecule encoding the antigen may be delivered to antigen presenting cells using the targeting polypeptides ofthe invention. Thus the invention also provides a complex comprising a targeting polypeptide and a nucleic acid molecule encoding an antigen. The nucleic acid molecule will be delivered to the antigen presenting cell and result in expression ofthe antigen in the antigen presenting cell. Thus reference herein to an antigen includes a nucleic acid molecule encoding such an antigen. A complex of the invention may comprise: - a targeting polypeptide; and - an antigen and/or a nucleic acid molecule encoding an antigen. The targeting polypeptide and antigen may be joined together by any suitable means that ensures that the antigen is also targeted to the antigen presenting cell. Preferably, the targeting polypeptide and antigen may be present in the same polypeptide. Thus in some cases the antigen and targeting polypeptide may be directly fused to each other in a single polypeptide. In others the two may be present in the same polypeptide, but separated by an intervening sequence. For example, they may be separated by from 1 to 50, preferably from 5 to 25, more preferably from 10 to 20 amino acids. In cases where the targeting polypeptide and fusion polypeptide are present in the same polypeptide, the targeting polypeptide may be separated by a sequence designed to be cleaved by a proteases in the antigen presenting cell in order to allow the two to be separated. In some cases where the targeting polypeptide and antigen are present in the same polypeptide, there may be a plurality of antigens in the polypeptide. For example, the same antigenic sequence may be repeated several times in the polypeptide such as from 1 to 50, preferably from 2 to 25, and more preferably from 5 to 10 times. In some cases the polypeptide may therefore comprise repeats of the same epitope or a group of epitopes from a particular antigen, hi other cases different antigens may be present in the polypeptide. For example, two, three, four, five or more of any ofthe antigens mentioned herein may be present in the same polypeptide. The antigens may be from the same source or different source, and may, for example, be from different organisms. The targeting polypeptide and antigen may not be part ofthe same polypeptide. For example, they may be joined together by other covalent means. The targeting polypeptide and antigen may be joined together by a covalent bond, such as a covalent bond between side chains, for example by disulphide bridges. The two may be joined by a linker or other bridging molecule. In some cases the targeting polypeptide and antigen may be provided or coated on a moiety and the complex including the targeting polypeptide, antigen and moiety may be targeted to antigen presenting cells by virtue ofthe presence ofthe targeting polypeptide. The complex may comprise a plurality of antigens and/or targeting polypeptides. For example, a plurality of antigens may be present and may be linked to a single targeting polypeptide or alternatively multiple antigens may be present in a polypeptide with a single targeting sequence. The complex, may for example comprise one, two, three, five, ten or more antigens. The complex may comprise antigens from different sources such as antigens from different organisms. The complex may comprise antigens from different strains ofthe same organism, such as from different strains ofthe same pathogen, hi some cases the complex may comprise different allelic or mutant forms ofthe same antigen. For example, the antigens may be different forms of an antigen that display diversity that leads to strains of that pathogen with differing pathogenicity. The complex may comprise a plurality of copies of a single epitope where the epitope sequence is either present in the same polypeptide as the targeting polypeptide or is joined to it by one of the other means discussed herein. The complex may comprise a plurality of targeting polypeptides. For example, the complex may comprise 1, 2, 3 or more targeting polypeptides, such as for example from 5 to 50, more preferably from 10 to 25 or even more preferably from 15 to 20 targeting polypeptides. The ratio of targeting polypeptides to antigen and or epitope maybe for example 1:1, 1:2, 1:5, 1:10 or 1:25, in some cases the ratio maybe from 1 : 1 to 1 :75, preferably from 1 :2 to 1 : 50 and more preferably from 1 :5 to 1 : 25. The ratio of targeting sequences to antigen and/or epitope may have such ratios. In a particularly preferred embodiment ofthe invention the targeting polypeptides in the complex may be present in a dimeric form. This may be the case, for example, where the targeting sequence is, or is a fragment thereof or a variant of either of SSL7 and/or 7 and preferably of SSL 7. In a preferred case, where a fragment or variant is employed as well as having targeting activity it may be able to form a dimer. The complex may be chemically modified, e.g. one or more, or indeed all, of the polypeptide types in the complex may be post-translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. The polypeptides in the complexes and targeting polypeptides ofthe invention may comprise amino acid analogs, h some cases, one or more peptides in the complex may have been generated synthetically. In some cases libraries of different complexes maybe generated. The libraries may comprise complexes with different antigens, for example, from different pathogens. In some cases, the library may comprise complexes with antigens from the same source, such as from the same organism including any of those mentioned herein. The libraries may be encoded by libraries of nucleic acids and/or vectors of the invention. Libraries may be generated and then screened to identify those complexes showing advantageous properties. In some cases the complex may comprise a nucleic acid encoding the antigen rather than the antigen itself, hi particular, the complex may comprise a nucleic acid molecule capable of expressing the antigen. Any ofthe sequences discussed herein for expressing polypeptides may be employed to express the antgen. The invention therefore provides a viral particle which comprises a targeting polypeptide ofthe invention. In a particularly preferred instance the complex may comprise a viral particle which comprises: (i) a targeting polypeptide; (ii) a nucleic acid molecule encoding an antigen. The targeting polypeptide will preferably be wholly or partially exposed on the surface ofthe viral particle to allow the viras to be targeted to an antigen presenting cell. The complex may comprise the nucleic acid in any suitable manner to allow expression ofthe antigen in the antigen presenting cell. The complex may comprise a liposome with the targeting polypeptide present wholly or partially on the surface of the liposome to allow targeting to antigen presenting cells.

Nucleic acids The present invention also provides a nucleic acid molecule comprising a polynucleotide sequence encoding a targeting polypeptide and an antigen and in particular an antigen selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen. The targeting polypeptide and antigen may be encoded by separate open reading frames (ORFs) or alternatively the nucleic acid may comprise an open reading frame encoding both the polypeptide and the antigen. In a preferred case, the nucleotide sequence encoding the targeting polypeptide and the antigen are present in a single open reading frame. In a particularly preferred embodiment the nucleic acid will be able to express the targeting polypeptide and antigen in the form of a polypeptide comprising both. As discussed elsewhere herein the targeting polypeptide and antigen may be directly fused or alternatively may be separated by intervening sequences which are also encoded by the nucleic acid. The nucleic acid sequence may therefore encode any ofthe targeting polypeptides referred to herein. Thus the nucleic acid may comprise a sequence that encodes any ofthe SSLs, fragments thereof, or variants of either discussed herein. The nucleic acids may also encode any ofthe additional polypeptide sequences referred to herein. In a preferred case the nucleic acid molecule may comprise one or more ofthe polynucleotides sequences of SEQ ID Nos 1, 12, 16, 37, 49, 53, 63, 67, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 and/or 107. In particular, the nucleic acid may comprise one or more ofthe regions of SEQ ID Nos 1, 12, 16, 37, 49, 53, 63, 67, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 and/or 107 indicated herein to represent the CDS of a particular SSL. Thus any ofthe nucleic acid sequences provided herein which encode one or more of SSLl to SSL14 may be used. The sequences used from the sequences provided herein may be restricted to the coding regions indicated or may also employ other regions from the gene and in particular the whole gene. The nucleic acid may comprise the polynucleotide sequence of one or more of the coding sequences provided herein for SSL 1 to SSLl 4, a fragment of one or more of those sequence or a variant sequence of such a sequence or fragment, where the sequence encodes a targeting sequence able to target the encoded polypeptide to an antigen presenting cell. The sequence of an SSL may be modified by nucleotide substitutions, insertions or deletions. In particular, the sequences provided herein which encode a SSL may be altered in such a way. The nucleic acid sequence may, for example, comprise from 1, 2, 5, 10 or 20 such substitutions, insertions and/or deletions as long as the encoded polypeptide has targeting activity. The variant sequence may comprise from 1 to 50, preferably from 5 to 25, more preferably from 10 to 15 amino acid insertions, deletions or substitutions as long as the encoded polypeptide displays targeting activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as shown in the Table above. The nucleic acid may comprise a sequence that has at least 25%), preferably at least 30%), more preferably at least 35% and even more preferably at least 40% sequence identity to any one or more of SEQ ID Nos 1, 12, 16, 37, 49, 53, 63, 67, 79, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101 and/or 107. In particular, they may have such a level of sequence identity to the region encoding the SSL and/or over the whole gene. In some cases the nucleic acid may have at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80% sequence identity to such sequences, hi other cases the level of sequence identity may be higher, because, for example, the sequence is a natural allelic variant or an engineered variant. Thus in some instances the sequence may have at least 85%, preferably at least 90%), more preferably at least 95%>, even more preferably at least 97% and still more preferably at least 99% sequence identity to any of the sequences provided herein which encode SSLl to SSL 14. In one case, the nucleic acid molecule will have one ofthe specified levels of nucleotide sequence identity to all of, three of, or two ofthe sequences encoding SSL5, SSL7 and/or SSL9 provided herein. The levels of sequence identity specified may be over the entire sequence encoding the targeting polypeptide or targeting sequence. They may, for example, be over from 25 to 900 nucleotides, preferably over 50 to 700 nucleotides, more preferably over 75 to 350 nucleotides and even more preferably over 100 to 250 nucleotides. Thus in some cases the level of sequence identity specified may be over a region of at least 50, preferably at least 75, for instance at least 100, at least 150, more preferably at least 200 contiguous nucleotides or most preferably over the full length ofthe nucleic acid encoding the targeting sequence or polypeptide. The nucleic acid may comprise the nucleotide sequence of one or more of the sequence SSL5, SSL7 and/or SSL9 genes whose sequence is provided herein, a fragment of any ofthe sequences or a variant ofthe preceding sequences where the encoded polypeptide displays targeting activity. In a preferred instance, the nucleic acid will comprise the nucleotide sequence of one or more of the sequences provided herein which encode SSL5, SSL7 and/or SSL9. In a prefened case the nucleic acid may comprise the nucleotide sequence of SEQ ID Nos: SSL7 and/or SSL9, a fragment of either or a variant of any such sequence, where the fragment or variant retains targeting ability. In a particularly preferred case the nucleic acid will comprise the polynucleotide sequence of a sequence provided herein encoding SSL7 and/or SSL9. Sequence identity and comparisons may be performed in a number of ways. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux βt al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al (1990) J. Mol. Biol. 215:403-10. Software for performing BLAST analyses is publicly available through the

National Centre for Biotechnology -Information (http://www.ncbi.nlm.nih. gov/ . This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word ofthe same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm performs a statistical analysis ofthe similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison ofthe first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Any combination ofthe above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides ofthe invention, with the more stringent combinations (i.e. higher sequence identity over longer lengths) being prefened. Thus, for example, a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect ofthe invention, as does a polynucleotide which has at least 95% sequence identity over 40 nucleotides. The nucleic acids ofthe invention may comprise a number of another sequences in addition to that encoding the targeting polypeptide and antigen. For example, the nucleic acid may comprise sequences involved in the expression ofthe polypeptide it encodes such as those discussed below on the section in vectors. The nucleic acids may comprise primer sites, restriction sites, multiple cloning sites and other sequences to facilitate manipulation. The nucleic acid may comprise enhancer sequences to faciliate gene expression. The nucleic acid may comprise sequences allowing for the secretion ofthe encoded polypeptide or its targeting to a particular cellular compartment. In some cases the nucleic acids may also comprise a reporter gene or sequences, in other cases they may not. The nucleic acids ofthe invention may be used in the production of targeting polypeptides, antigens and/or complexes ofthe invention. Such production may take place in vitro, in vivo or ex vivo. The polynucleotides may be used in recombinant protein synthesis or indeed as therapeutic agents in their own right. Polynucleotides encoding a targeting polypeptide and not an antigen or alternatively those encoding an antigen, but not a targeting polypeptide may be used to produce targeting polypeptide and/or antigen which can then be utilised to form complexes ofthe invention.

Vectors The present invention provides vectors comprising a nucleic acid ofthe invention. Thus in one instance the invention provides a vector comprising a nucleic acid sequence that encode a targeting polypeptide ofthe invention and an antigen.

The antigen may be one selected from any of those mentioned herein and in particular may be selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen. The vector may comprise any ofthe nucleic acid sequences mentioned herein, hi a prefened instance, the vector may encode the targeting polypeptide and antigen via the same open reading frame (ORF). The invention also provides vectors which comprise a nucleic acid which encodes the chosen antigen where the nuleic acid will be targeted to an antigen presenting cell using a targeting polypeptide ofthe invention. Such vectors will preferably be viral vectors. The vector may, for example, be a cloning, expression and/or viral vector. The vector may, for instance, be a plasmid vector. The vector may be a viral vector. The vector may be a shuttle vector. The vector may comprise a selectable marker, for instance an antibiotic resistance selectable marker. hi particular, expression vectors are provided which are capable of expressing a targeting polypeptide ofthe invention and an antigen. In a preferred instance, the vector will express a targeting polypeptide ofthe invention as a fusion protein with the chosen antigen. Thus the vector may be capable of expressing a complex ofthe invention. Alternatively the targeting polypeptide and antigen may be produced separately and then linked to form a complex or the targeting polypeptide and antigen may be expressed as a single polypeptide and then further processing is carried out to produce a particular complex. For example, individual polypeptides may be linked together or sequences may be cleaved from the expressed polypeptides. In some case sequences used in purification may be removed by cleavage. Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the conect orientation, in order to allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al., 1989. The expression vector may be a prokaryotic or a eukaryotic expression vector. In some cases the expression vector may be used to produce the encoded protein in vitro, hi other cases, the expression vector will be intended to generate in vivo expression ofthe encoded protein and may be used, for example, in a method of therapy. Once coding sequences for desired proteins have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are -known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences may be performed using standard procedures, -known in the art. The vector may be, for example, plasmid, viras or phage vectors provided with a origin of replication, optionally a promoter for the expression ofthe polynucleotide and optionally a regulator ofthe promoter. The vectors may contain one or more selectable marker genes. Expression ofthe targeting polypeptide and/or antigen will typically be driven by a promoter. The promoter will usually be chosen on the basis ofthe cell the expression vector is to be used in. Thus for prokaryotic expression a prokaryotic promoter will typically be used, whilst for eukaryotic expression a eukaryotic or viral promoter will typically be employed. The promoter employed may be a viral or non- viral promoter. The promoter may be a mammalian promoter, such as a cell or tissue specific promoter or alternatively a promoter expressed in a wide range of cells. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences. Mammalian promoters, such as β-actin promoters, may be used. Tissue- specific promoters are especially prefened. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma viras (RSV) LTR promoter, the S V40 promoter, the human cytomegalovirus (CMV) ?IE promoter, adenoviras, HS V promoters (such as the HS V IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art. In one prefened case the promoter employed may be one that is capable of being expressed in an antigen presenting cell. Thus a vector comprising a promoter capable of giving rise to expression of the antigen in an antigen presenting cell is provided. A promoter which is specific for antigen presenting cells may be employed. Additional sequences may also be present in the open reading frame encoding the targeting polypeptide as well as optionally those encoding the antigen. In some cases, sequences directing secretion or release from the cell ofthe targeting polypeptide may be included. Peptide sequences allowing purification ofthe targeting polypeptide may be included. Preferably sequences allowing cleavage may be included and, for example, may be used to release the targeting polypeptide from the sequence used to purify the expressed polypeptide. Cells The invention provides cells comprising a polynucleotide or vector ofthe invention. Thus the invention provides for a cell comprising a polynucleotide and/or vector encoding a targeting polypeptide and an antigen. The targeting polypeptide and/or antigen may be any of those mentioned herein. Preferably, the cell will express the targeting polypeptide and antigen and in particular will express polypeptides comprising both. The cell may therefore be able to produce complexes ofthe invention and in one case may secrete them. The invention also provides for cells that can produce viruses ofthe invention which have a targeting polypeptide provided on their surface either wholly or partially to allow for targeting ofthe viral particle to an antigen presenting cell. The invention provides a helper cell line that is capable of expressing the targeting polypeptide in such a way that it is incorporated in a viral particle. Such cells may also comprise the nucleic acid to be incorporated into the viral particles. The invention also provides cells infected with a virus ofthe invention. Cells ofthe invention include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, produced using, for example, a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells include mammalian cells such as HEK293T, CHO, HeLa and COS cells. The cells may be human cells. The cells may be, for instance, from any ofthe species and/or subjects mentioned herein. Preferably the cell line selected may be one which is not only stable, but also allows for normal post-translation modifications, particularly so that the antigen or epitope is in the form it would be naturally expected to be encountered as. The cell may, for example, allow normal glycosylation. A polypeptide ofthe invention may be expressed in cells of a transgenic non- human animal. A transgenic non-human animal expressing a polypeptide ofthe invention is included within the scope ofthe invention. Such an animal may for example be a rodent (e.g. a mouse or rat). Prefened polypeptides ofthe invention may also be expressed in Xenopus laevis oocytes. The sequences encoding the targeting polypeptide ofthe invention and/or the antigen may be introduced into a chosen cell by any suitable technique and may be generally refened to without limitation as "transformation". For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other viras, e.g. vaccinia or, for insect cells, baculovirus. For example, the calcium phosphate precipitation method of Graham and van der Eb, Virology 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No. 4,399,216. For various tec-hniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527 537 (1990) and Mansour et al, Nature 336:348-352 (1988). Nucleic acids and vectors ofthe invention may be introduced into target cells both in vitro and in vivo. In particular, viral based systems may be used to introduce the nucleic acids and/or vectors ofthe invention into cells, particularly in vivo.

Antigen Presenting cells (APCs) The invention allows a chosen antigen to be delivered to an antigen presenting cell. The targeting polypeptide present in the complexes direct the complex to an antigen presenting cell. The complex is taken up by the antigen presenting cell. This means that the antigen may then be presented by the antigen presenting cell. The invention also provides for the delivery of a nucleic acid molecule encoding an antigen through the use of a targeting polypeptide ofthe invention. The targeting polypeptide will preferably be on the surface of a viras. -Infection ofthe antigen presenting cell by the viras results in the production of antigen which may then be presented by the cell. The antigen presenting cell may be any suitable antigen presenting cell. In particular, it may be a professional antigen processing cell and will typically express MHC molecules. The antigen processing cell will typically express MHC II molecules and the complex will allow the chosen antigen, or peptides derived from it, to be presented via MHC II molecules. In some cases the antigen, or peptides derived from it, may be presented via MHC I. The antigen may be presented by both MHC I and II, typically presentation will be via -MΗCII. Examples of -MHC I and II molecules which may be involved in presentation include HLA-A2, HLA-B62, HLA-Bw62,

HLA-B35, HLA-DRB1, HLA-DRB2, HLA-DRB3, HLA-DRB5, HLA-DRB7, HLA- A25, HLA-B8, HLA-B52, HLA-DQB1, HLA-A3, HLA-A11 or HLA-B27. Examples of antigen presenting cells that the invention may be used to target antigens to include dendritic cells, monocytes, and/or or a B cells. In particular, monocytes and/or dendritic cells may be targeted and in a prefened instance the antigen presenting cell is a dendritic cell. The B cells may typically be CD19⁺ B cells. The monocytes may, for example, be CD14⁺ cells and such cells may also be CD2¹⁰ cells. In cases where the antigen presenting cell is a dendritic cell it may express cell surface markers known to be characteristic of dendritic ells, hi particular the dendritic cell may express CD1 lc, CD209 (also known as DC sign) and/or CD13. The cell may be CD14¹⁰. In some cases the cell may express all of CD lie, CD209 and CD 13 and may also be CD 14^l0. In a prefened instance the antigen presenting cell may be a dendritic cell. Any type of dendritic cell may be targeted. Examples of dendritic cells include a monocyte derived dendritic cell, a plasmacytoid derived dendritic cell or an intersititial dendritic cell. The dendritic cell may be a langerhans cell. The dendritic cell may be one present in an organ or tissue. The dendritic cell may be one from, or present in, a mucosal surface. The dendritic cell may be an intestinal dendritic cell such as one obtained from Peyers patches. The dendritic cell may be one present in, or obtained, from an immune tissue such as from a secondary lymph node. The dendritic cell may be present in, or obtained from the spleen or a lymph node. The term antigen presenting cell covers any cell that can present an antigen targeted to it via a complex ofthe invention, hi some cases antigen presenting cells may have different stages in their development during which they, for example, predominately take up antigen, rather than present it. For example, it is thought that dendritic cells may have immature stages characterised by the uptake of large amounts of potential antigens and more mature stages characterised by lower amounts of antigen uptake, but increased amounts of antigen presentation ofthe antigens they acquired earlier. The dendritic cell may, for example, be present in the periphery and effectively collect antigen and then move to areas such as secondary lymphoid tissues to present antigens, hi one case the invention encompasses targeting of an antigen to an immature antigen presenting cell subsequently capable of presenting the antigen it has taken up, such as to an immature dendritic cell. In some cases it may be desirable to target immature dendritic cells in instances where it is desired to induce tolerance. In particular, delivery of antigen by employing a targeting polypeptide ofthe invention in the absence of a stimuli which induces or promotes dendritic cell maturation can result in tolerance. To achieve tolerance the targeting polypeptide may preferably be used to deliver the chosen antigen or nucleic acid encoding the antigen in the absence of an adjuvant. An advantage of employing the targeting polypeptide ofthe invention to induce tolerance is that unlike many methods for inducing tolerance a large dose of antigen is not required. Steinman et al (2003) Ann. Rev. Immunol., 21:685-711 discusses the induction of tolerance via dendritic cells and the methods and assays discussed therein may be employed when inducing tolerance using the methods ofthe invention. In situations where it is desired to induce tolerance peripheral dendritic cells may preferably be targeted. Conversely, in situations where it is desired to promote an immune response against the antigen, the targeting polypeptide may preferably be employed with an adjuvant and in particular one which induces or promotes stem cell maturation such as aluminium hydroxide. In some cases the antigen cells may be manipulated in vitro and this may allow control of whether the cells are exposed to stimuli which promote dendritic cell maturation. Thus by ensuring that the cells are not exposed to stimuli responsible for inducing maturation the resultant cells may be used to induce tolerance. In other cases the cells will be exposed to stimuli which promote dendritic cell maturation and hence the cells can be used to promote an immune response when they are transfened to a subject. Antigen presenting cells may be isolated from any suitable source including any of those mentioned herein. For example, such cells may be isolated from the white cells ofthe blood. Methods based on cell density such as LYMPHOP?REP™ and centrifugation may be employed. The cells may be isolated using a variety of techniques including antibody based techniques. The cells may be isolated using negative and positive selection techniques based on surface markers which present and/or those that are not present on antigen presenting cells. In some cases, antigen presenting cells may be obtained by exposing other cells, such as precursor cells, to appropriate stimuli. Dendritic cells may be obtained by treating monocytes with appropriate stimuli such as GM-CSF and/or IL-4. For example, cells may be culture in the presence of 10 to 500 ng/ml, preferably from 25 to 200 ng/ml, more preferably from 50 to 150 ng/ml and even more preferably in the presence of 100 ng/ml GM-CSF. hi particular, human and preferably recombinant human GMCSF may be employed. The cells may be cultured in the presence of 10 to 250 ng/ml, preferably from 25 to 150 ng/ml, more preferably from 40 to 70 ng/ml and even more preferably in the presence of 50 ng/ml IL-4. T and B lymphocytes maybe removed by appropriate selection such as on the basis ofthe markers CD3, CD2 and/or CD19. In other cases plasmacytoid cells may be induced to differentiate into dendritic cells by exposure to IL-3. In some instances immature antigen presenting cells may be induced to mature using appropriate stimuli. Such treatments will typically be performed in vitro. The antigen presenting cells may be treated ex vivo. Thus the cells may be recovered from a subject loaded with antigen using the methods ofthe invention and then used therapeutically. The invention provides loaded antigen presenting cells. The invention provides antigen presenting cells which have been infected by a viral particle ofthe invention.

Assays The targeting polypeptides ofthe invention are utilised to target antigens to antigen presenting cells. This targeting ability and the downstream effects of targeting can be assessed in a number of ways. The ability of a polypeptide to target itself, and hence a complex, to an antigen presenting cell can be assessed using any suitable technique, hi one case in vitro assays may be performed to monitor binding ofthe targeting polypeptide to an antigen presenting cell. The targeting polypeptide may be labelled and the binding to the antigen presenting cell followed. The targeting polypeptide may be labelled with a fluorochrome and its binding to an antigen presenting cell assessed using techniques such as flow cytometry. Binding at 4°C and 37 °C may be compared to help demonstrate that the binding is specific. The ability ofthe polypeptide under study to target to an antigen presenting cell may be compared with a polypeptide known to have targeting ability. For example, where the test targeting polypeptide is based on the sequence of a particular SSL the ability to compete against that SSL may be compared using two or more different labels. The ability of a targeting polypeptide to compete against varied concentrations of a polypeptide with known binding activity may be measured. In this way polypeptides with binding activity may be identified and the level of targeting ability that they display quantified. Such assays will typically be performed in vitro but the ability of antigen cells in vivo or ex vivo conditions to take up test polypeptides and/or complexes may also be measured. Again, labelling may be used to study delivery to antigen presenting cells. The ability of a targeting polypeptide to enter the antigen presentation pathway may also be assessed. The presence ofthe targeting polypeptide in a subcelluar compartment associated with antigen presentation may be measured. Labelling may be used to achieve this. Confocal microscopy can be used to confirm that the label, and hence the targeting polypeptide, has entered the cell. Stains for subcellular compartments associated with antigen presentation may be employed. For example, Texas red dexfran staining may be employed to identify such compartments and co-staining may be used to confirm the presence ofthe targeting polypeptide in such compartments, h addition, the association ofthe complex, or part of it, in the same regions as ?MHC molecules, particularly MHC II may be examined. The ability of a particular targeting polypeptide to lead to antigen presentation may be studied. Thus the presence of peptide sequences from the antigen being presented on the cell surface by ?MHC may be studied. In particular, presentation by MHCD? may be examined. Techniques may be used to elute peptides bound to MHC and identify those originating from the chosen antigen. The degree of peptide presented when the antigen is provided on its own and when it is provided as part of a complex may be compared. The ability of particular polypeptides to lead to antigen presentation may be compared including using control polypeptides with -known targeting ability. Thus the ability of two polypeptides to lead to presentation ofthe same antigen may be compared. hi some assays the downstream effects of antigen presentation may be measured. Thus assays may involve an antigen presentation cell being loaded using test polypeptides and then antigen presentation to a second cell may be measured. The ability of different targeting polypeptides and complexes to bring about the downstream effects of antigen presentation may be measured including controls with -known activity. Such assays may be performed in vitro and, for example, they may be performed using an antigen presenting cell and a T cell -known to have a receptor specific for an epitope present in the antigen. The T cell may be any suitable T cell. It may be a CD4⁺ or CD8⁺ T cell and in particular may be a CD4⁺ T cell. The downstream effects of antigen presentation may also be measured in vivo Downstream effects of antigen presentation include activation ofthe T cell. Various signal transduction effects associated with activation ofthe T cell may be measured. The activation may include the differentiation and/or proliferation ofthe T cell. Thus the number and proliferation ofthe T cells may be measured, using, for example, suitable labelling techniques or by measuring cell number. The expansion of particular subsets of T cells may be measured. For example, by flow cytometry. Assays involving autologous and/or allogenic T cells may, for instance be employed, including any of those mentioned herein. The activation ofthe T cells may lead to the release of cytokines. For example, the activation may lead to the release of interleukins (e.g. 11-2, IL-4 , IL-5, IL-6 and/or IL-10) , IFNγ and/or T?NF-β. -In some cases antigen presentation may lead to a Thl type response. Thus the cytokines released may be, or predominately be, IL-2, IFNγ and/or T-NF-β. In other cases the response may be a Th2 type response. The cytokines released may be, or predominately be, IL-3, IL-4, IL-5, IL-6 and/or IL-10. The T cells may release factors that stimulate their own proliferation such as IL-2. and/or IL-4. Particular complexes and substances ofthe invention may be designed to give particular responses such as any of those mentioned herein. The release of such factors can be studied both in vitro and in vivo using techniques such as ELISA to measure levels of such compounds, hi some cases, the cytokine levels measured may be one or more, or indeed all, of IFNγ, IL- 10 and IL- 13. The presence and/or number of effector T cells and memory T cells may be assessed. In addition various downstream effects may be measured. The number and activity of CD4 and/or CD8⁺ T cells may be measured and in particular those specific for the chosen antigen. Antibody responses may be assessed in terms ofthe amount and types of antibody produced. In one prefened instance, the delivery ofthe chosen antigen using the targeting sequences ofthe invention leads to an antibody response against the chosen antigen. Any ofthe assays discussed herein may, for instance, be used to detect such an antibody response. In some cases tolerance may be induced and an antibody response may not be seen and in particular subsequent challenge with the antigen may result in a lower response or no response in comparison to the response seen without the initial induction of tolerance. The effects on other immune cells may be measured such as on macrophages and/or granulocytes. The skilled person will be aware of appropriate assays for assessing the immune response and immunogenicity. In some embodiments ofthe invention a nucleic acid encoding an antigen, rather than the antigen itself, is targeted to the antigen presenting cell. In a particularly prefened embodiment the viral particles comprising targeting polypeptides may be employed in the invention. The ability ofthe viruses to selectively bind to antigen presenting cells may be measured. Non-antigen presenting cells may be employed as controls to assess the specificity ofthe viruses. Standard techniques may be used to monitor the expression ofthe nucleic acids targeted to antigen presenting cells. For example, test experiments may be done using reported genes in place of an antigen encoding gene. Techniques such as RT- PCR, Northern blotting, Western Blotting and cell staining may be used to monitor expression in antigen presenting cells. Presentation ofthe antigen may be evaluated. The above techniques may be used to identify effective targeting sequences. They may also be used to assess the efficacy ofthe invention in promoting an immune response against a chosen antigen. The techniques may also be used to assess the efficacy of the substances of the invention in the treatment or prevention of any of the conditions refened to herein. Typically suitable controls will be employed. In some cases standard test antigens and/or targeting polypeptides may be employed and compared to the substance under test.

Loading antigen presenting cells The invention provides for the loading of antigen presenting cells. Thus the invention provides a method of loading an antigen presenting cell, comprising contacting an antigen presenting cell with a complex ofthe invention. The targeting polypeptide present in the complex directs the complex to the antigen presenting cells. The complex is taken up by the antigen presenting cell and the antigen is presented by the antigen presenting cell as discussed herein. The targeting ofthe complex to the antigen presenting cell is termed loading ofthe antigen presenting cell. The invention also encompasses the loading of antigen presenting cells using a targeting polypeptide ofthe invention to deliver a nucleic acid encoding an antigen to antigen presenting cells. In particular viral particles comprising the targeting polypeptide may be employed. The invention therefore provides a method of loading an antigen presenting cell comprising using a targeting polypeptide ofthe invention to deliver a nucleic acid molecule encoding an antigen to the antigen presenting cell. The invention provides for the infection of an antigen presenting cell with a viras ofthe invention. The loading of antigen presenting cells or their precursors may be performed in vitro, ex vivo or in vivo, h the case of in vitro loading the antigen presenting cell may simply be contacted with a complex ofthe invention. The cell may be cultured in the presence ofthe complex under suitable conditions. The cell and complex may, for example, be contacted for between five minutes and ten days, preferably from an hour to five days, more preferably from five hours to two days and even more preferably from twelve hours to one day. Ex vivo loading may,for instance, be carried out in the same manner once the cells to be loaded have been obtained. Loading of antigen presenting cells or their precursors may be performed in vivo. Thus the invention provides an in vivo method of loading antigen presenting cells comprising administering to a subject and effective amount of a complex ofthe invention. Administration may be via any ofthe routes discussed herein. Loading in vivo may also be achieved by administering a nucleic acid, vector, cell, viras, vaccine or pharmaceutical composition ofthe invention. The administration of such products results in complexes ofthe invention coming into contact with antigen presenting cells. Thus the nucleic, vector, virus or cells may lead to the production of a complex ofthe invention which then loads an antigen presentation cell present in the subject. hi one case antigen presenting cells recovered from a subject are loaded in vitro with a complex ofthe invention. The invention therefore provides an ex vivo method of loading antigen presenting cells. The loaded cells may then be returned to a subject and in particular to promote an immune response against the antigen that the cells have been loaded with. The invention also provides antigen presenting cells which have been loaded using the complexes ofthe invention. Such antigen presenting cells will typically comprise the complex or breakdown products ofthe complex and in particular the antigen to be presented. The loaded cells may comprise epitopes ofthe antigen. The cell may also comprise the targeting polypeptide and/or breakdown products thereof. The invention provides such cells in an isolated form. The loaded cells may comprise a nucleic acid ofthe invention and in particular may comprise a viral vector ofthe invention. Preferably, the viral vector will be replication deficient. Generally the antigen presenting cell ofthe invention carries peptides, and in particular an antigenic epitope, derived from the chosen antigen on its surface in conjunction with an ?MHC class I or class II molecule and in particular in conjunction with an -MHC π molecule, -hi one embodiment the antigen presenting cell has at least 100, preferably at least 200, for example at least or about 500 or 1000, class I and/or class II molecules on its surface loaded with the product and in particular class II molecules, hi some cases, the cells may carry a label or be labelled, such as, for example, with thymidine or radioactive chromium. The invention also provides T cells that have been activated by a loaded antigen presentation cell ofthe invention. In some cases antigen presenting cells may be recovered from a subject, loaded in vitro and then returned to the same subject. In other cases, it may be that T cells are recovered from a subject, exposed to loaded antigen presenting cells ofthe invention in vitro and then returned to the subject. h one case the invention may provide a composition comprising T cells, antigen presenting cells and a complex ofthe invention. The T cells or antigen presenting cells may be any ofthe cells mentioned above. In particular, the antigen presenting cells may be dendritic cells. The T celhantigen presenting cell ratio may be typically from 500:1 to 1:500. Typically at least 10³, such as (e.g. at least or about) 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ cells are present per millilitre ofthe composition. The composition typically also comprises a culture medium capable of supporting the T cells or antigen presenting cells, such as ?RPMI medium. The medium may also comprise cytokines, such as IL-2, IL-4, IL-7 or T?NF-α. The T cells and antigen presenting cells may be from the same individual. The cells employed in the invention, particular the antigen presenting cells and/or T cells, may be autologous cells, or cells which have been partially or fully matched with the subject for -MHC class I HLA-A or HLA-B; and/or for MHC class II type, hi a prefened case, the cells employed in the invention may be recovered from a subject and utilised ex vivo and subsequently returned to the same subject.

Delivery of nucleic acid molecules encoding antigens to antigen presenting cells The targeting polypeptides ofthe invention may be used to deliver a nucleic acid molecule which encodes an antigen to an antigen presenting cell. The nucleic acid molecule gives rise to expression ofthe antigen in the antigen presenting cell and to the subsequent presentation ofthe antigen by the cell. The nucleic acid molecule will preferably comprise a promoter which is operably linked to the sequences encoding the antigen and which is active in antigen presenting cells or which can be induced in antigen presenting cells. hi a particularly prefened embodiment the nucleic acid encoding the antigen may be delivered to the antigen presenting cell via a viral particle which comprises a targeting polypeptide ofthe invention. The targeting polypeptide will typically be provided wholly or partly on the surface ofthe virus in order for the polypeptide to be able to target the viras to an antigen presenting cell. Any suitable viras may be used in such embodiments. The viras may, for example, be a retrovirus, a lentiviras, an adenovirus, an adeno-associated virus , a vaccinia virus or a herpes simplex viras. lh a particularly prefened embodiment the viras may be a lentiviras. The lentiviras may be a modified HIV viras suitable for use in delivering genes. The lentiviras may be a SIN, F1V, or equine infectious anemia viras (EQ-LA) based vector. The virus may be a moloney murine leukaemia virus (MMLV). The targeting polypeptide may comprise sequences from the viras. For example, the targeting polypeptide may comprise sequences from a viral surface protein. In particular, the targeting sequences may be at the Ν terminus ofthe targeting polypeptide and be fused or linked to surface protein sequences. The targeting polypeptide may also comprise a transmembrane domain so that is can be provided in the viral membrane. hi a particularly prefened embodiment the targeting polypeptide may include sequences from a surface protein ofthe viras. Ih particular the targeting polypeptide may comprise sequences from a surface protein that is involved in the normal binding ofthe viras to its target cell. The binding ofthe targeting polypeptide to the antigen presenting cell may lead to conformational changes allowing the viral surface protein sequences to bind to their target on the cell surface. This may lead to fusion ofthe viral and cell membranes allowing entry ofthe virus into the antigen presenting cell. Thus the targeting sequences and surface protein domains may show receptor cooperation to facilitate the entry ofthe viras specifically into the antigen presenting cells. The targeting polypeptide may include linker sequences which facilitate the cooperation and in particular proline rich sequences present in the viral surface protein may be employed. The proline rich linkers discussed in Martin et al (2003) Journal of Virology 77(4): 2753-2756 and Valsesia-Wittmann et al (1997) EMBO Journal, 16(6): 1214-1223 maybe employed. The invention provides a viras comprising a targeting polypeptide as well as a cell infected by such a viras. The virus will typically also comprise a nucleic acid molecule encoding the chosen antigen. The virus may, for instance, be any of those mentioned herein. The nucleic acid molecule may also encode other sequences, for example the nucleic acid sequences may comprise sequences which express proteins which boost the immune response to the antigen. The nucleic acid may encode a cytokine, including any of those mentioned herein and in particular IL-1, IL2 and/or IL12. The nucleic acid may also encode a costimulatory molecule such as a surface polypeptide which enhances the immune response. The nucleic acid may encode, for example, CD80 and/or CD86. The viruses ofthe invention are preferably replication deficient. In some cases the nucleic acid sequences encoding the targeting polypeptide will not be included in the viral vector. Thus the invention also provides helper cells which express the targeting polypeptide in such a way that the targeting polypeptide is incorporated into the viral particles. The invention also provides nucleic acid vectors that encode a targeting polypeptide ofthe invention which comprises viral sequences.

Medicaments, methods and therapeutic use The complexes ofthe invention and various related aspects ofthe invention may be used be used in a method of therapy ofthe human or animal body. Thus the invention provides for the use of a targeting polypeptide, a complex, a nucleic acid, a vector, a cell, a viras, or an antigen presenting cell ofthe invention in a method of treatment of the human or animal body by therapy. The invention provides for the use of a complex ofthe invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex ofthe invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus ofthe invention or an antigen presenting cell ofthe invention in the manufacture of a medicament for use in immunisation. h a prefened case the invention provides for the use of a complex comprising: (a) a targeting polypeptide comprising a staphylococcal superantigen-like protein (SSL), a fragment thereof or a variant of either, where the SSL, fragment or variant has the ability to target the complex to an antigen presenting cell; and (b) an antigen or a nucleic acid encoding an antigen, in the manufacture of a medicament for use in immunisation. hi some instances the antigen comprises a polypeptide which is present in the complex as a fusion polypeptide with the targeting polypeptide. In others the antigen and targeting polypeptides are not part ofthe same polypeptide, but are covalently joined to each other or are joined through a linker. In a prefened case the antigen is a pathogenic antigen, an auto-antigen, an allergen and/or a cancer antigen. In another, the targeting polypeptide is present as a dimer. Immunisation may result in promoting an immune response against the chosen antigen. Any ofthe effects resulting from targeting antigen presenting cells mentioned herein may be promoted or achieved. In particular the level of presentation ofthe chosen antigen will be increased. An increase in presentation via MHC I and/or MHC II molecules and in particular via ?MHC II molecules may be seen. In a prefened case the level of antigen presentation achieved may be such that when the same antigen is encountered again an increased immune response is seen in comparison to if the initial immunisation had not taken case, h particular a therapeutic and/or protective immune response may be raised. The invention may therefore ensure that a higher level of immune response is seen when the antigen is next encountered. The invention may be used to enhance the level of antigen presentation or of any ofthe downstream effects thereof, such as any of those mentioned herein, in comparison to administration of an equivalent amount of antigen in the absence of a targeting polypeptide. The increase may be double, treble, or more fold, in some cases it may be at least ten-fold, preferably at least twenty-fold and even more preferably at least 100 fold, or 1000 fold or more. It may be that a protective, or therapeutic response, is seen whereas in the absence ofthe use of a targeting polypeptide it is not. The invention also provides for an agent for immunising a subject, the agent comprising a complex of the invention, a nucleic acid encoding a targeting polypeptide and antigen of a complex of the invention, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, or an antigen presenting cell ofthe invention. The various diseases and conditions to be prevented or treated may be any of those mentioned herein or associated with an antigen mentioned herein. In particular, the disease may be one associated with a pathogen, such as a bacterium, viras, bacterium, parasite, protozoan, fungus, and/or prion. In some cases the disease may be a cancer such as any of those mentioned herein. In some instances the invention will be used to induce tolerance to a particular antigen and in particular to an allergen or an auto-antigen hi such cases typically the method will involve the delivery ofthe desired antigen to an antigen presenting cell in the absence of a stimulus which promotes antigen presenting cell maturation. In particular, the antigen may be delivered in the absence of an adjuvant such as aluminium hydroxide. The immunisation methods and vaccines ofthe invention may be used to induce tolerane to a selected antigen. The substances ofthe invention may be administered to any suitable subject.

The subject on which the method ofthe invention is performed is generally a vertebrate subject. By "vertebrate subject" is meant any member ofthe subphylum cordata, particularly mammals, including, without limitation, humans and other primates, as well as rodents, such as mice and rats. The subject may be a non-human animal. The non-human animal may be a domestic animal or an agriculturally important animal. The animal may be a domestic pet. The animal may be a monkey such as a non-human primate. The term subject does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered, h one embodiment the subject is susceptible to or at risk from the relevant disease. For example, the subject may have been exposed, or will be in a region where there is a risk of exposure, to a particular antigen and in particular a pathogen. The invention also covers the use ofthe complexes ofthe invention to promote antigen presentation of a chosen antigen, hi some cases the invention may be used to bring about antigen presentation in an animal model, for example to study whether or not a particular immune response can be raised. The efficacy ofthe invention may be assessed in such non-human animal models. In some cases the invention may be used to help generate an immune response in a non-human animal in order to obtain antibodies against a chosen antigen that can then be recovered. Thus the invention may be used in a method of antibody production. The invention may be used in combination with other means of, and substances for, immunisation, h some cases the complexes ofthe invention may be administered simultaneously, sequentially or separately with antigen which is not part of a complex ofthe invention. Thus complexes may be administered with the same antigen in a form not linked to a targeting polypeptide. The substances ofthe invention may be used in combination with existing vaccines for a particular antigen and may, for example, be simply mixed with such vaccines. Thus the invention may be used to increase the efficacy of existing vaccines including, for example, peptide, polypeptide, nucleic acid, viral and/or bacterial based antigens.

Pharmaceutical compositions, vaccines and administration The invention additionally provides pharmaceutical compositions comprising a complex, nucleic acid, vector and/or cell ofthe invention and a pharmaceutically acceptable carrier or diluent. The present invention also provides a vaccine composition comprising a complex, nucleic acid, vector and/or cell ofthe invention. The vaccines and compositions may comprise any ofthe substances mentioned herein and in particular the complexes, nucleic acid molecules, vectors, viruses and cells of the invention. The invention provides a method of vaccination comprising administering to a subject an effective amount of a vaccine composition ofthe invention. The various compositions, vaccines and other substances ofthe invention may be fonnulated using any suitable method. Formulation with standard pharmaceutically acceptable carriers and/or excipients may be carried out using routine methods in the pharmaceutical art. For example, an active substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, 19^th Edition, Mack Publishing Company, Eastern Pennsylvania, USA, the disclosure of which is included herein of its entirety by way of reference. The substances may be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, infra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes. The substances may in some cases be administered to sites characterised by the presence of antigen presenting cells, hi cases where loaded antigen presenting cells are administered they may be administered, for example, to sites of antigen presentation such as secondary lymph nodes. Vaccines may be prepared from one or more ofthe complexes, nucleic acids, vectors, and/ or cells ofthe invention together with a physiologically acceptable carrier or diluent. Typically, such vaccines are prepared as mjectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the encapsulated in a liposome, particularly in the case of nucleic acids and vectors of the invention. The active ingredient may be mixed with an excipient which is pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, ofthe like and combinations thereof. In addition, if desired, the vaccine and/or pharmaceutical compositions ofthe invention may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance effectiveness. The complexes ofthe invention enhance the immunogenicity of a chosen antigen. They may therefore act as adjuvants for a chosen antigen and may be used as adjuvants. In some cases other adjuvants may be present in the various formulations ofthe invention or be administered simultaneously, separately or sequentially with them. Suitable adjuvants include, for example, any substance that enhances the immune response ofthe subject to the antigen (including when delivered by the polynucleotide ofthe invention). They may enhance the immune response by affecting any number of pathways, for example, by stabilizing the antigen/MHC complex, by causing more antigen/MHC complex to be present on the cell surface, by enhancing maturation of APCs, or by prolonging the life of APCs (e. g., inhibiting apoptosis). Examples of adjuvants that may be employed include cytokines. Certain cytokines, for example TRANCE, flt-3L, and CD40L, enhance the immunostimulatory capacity of antigen presenting cells and may be employed. Non- limiting examples of cytokines which may be used alone or in combination include, interleu?kin-2 (IL-2), stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 a), interleukin- 11 (IL-11), MlP-la, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO), CD40 ligand (CD40L), tumor necrosis factor-related activation-induced cytokine (TRANCE) and flt3 ligand (flt-3L). Further examples of adjuvants which may be effective include but are not limited to: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr- MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, refened to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'- dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835 A, refened to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion. In cases where the invention is used to target a nucleic acid which encodes an antigen to an antigen presenting cell, the nucleic acid may also encode molecules capable of acting as an adjuvant. Thus the nucleic acid may lead to the production of any of the adjuvants mentioned herein and in particular a cytokine or costimulatory molecule. The cytokine may, for example be, IL-1, IL2, and/or IL-12 which will preferably be secreted from the antigen presenting cell. The costimulatory molecule may, for example, be CD80 or CD86 which will be preferably expressed on the cell surface ofthe antigen presenting cell. The substances ofthe invention, and in particular, the vaccines, are typically administered parentally, by injection, for example, either subcutaneously or intramuscularly. Additional possible formulations include suppositories, oral formulations and formulations for transdermal administration. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%), preferably 1% to 2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10% to 95% of active ingredient, preferably 25% to 70%>. Where the substance is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer. Capsules, tablets and pills for oral administration to a patient may be provided with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose. Substances of the invention and in particular nucleic acids and vectors ofthe invention may be administered by needleless injection, for example, transdermally, may also be used. The substances ofthe invention may be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include the acid addition salt (formed with free amino groups ofthe peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric and maleic. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine and procaine. The substances are administered in a manner compatible with the dosage formulation and in such amount will be prophylactically and/or therapeutically effective. The quantity to be administered depends on the subject to be treated, capacity ofthe subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgement ofthe practitioner and may be peculiar to each subject. A substance ofthe invention may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple does schedule is one in which a primary course of administration maybe 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example at 1 to 4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgement of the practitioner. Examples of dosages of complex will may be administered include from 5 μg to 100 mg, preferably from 50μg to 50 mg, more preferably from 250μg to 10 mg. In some cases the administered substances may comprise cells. The cells may, for example, be those comprising nucleic acids or vectors ofthe invention. In other cases the cells may be loaded antigen presenting cells or may be T cells that have had antigen presented to them by loaded antigen presenting cells ofthe invention. Any suitable number of cells maybe administered to a subject. For example, at least, or about, 10 , 10 , 10 , 10 , 10 cells maybe administered. As a guide the number of cells ofthe invention to be administered may be from 10⁵ to 10¹³, preferably from 10⁷ to 10¹¹. In such cases where cells are administered or present, culture medium may be present to facilitate the survival ofthe cells, hi some cases the cells ofthe invention may be provided in frozen aliquots and substances such as DMSO may be present to facilitate survival during freezing. Such frozen cells will typically be thawed and then placed in a buffer or medium either for maintenance or for administration. The nucleotide sequences ofthe invention and vectors can also be used administered as outlined above. Preferably, the nucleic acid, such as ?RNA or DNA, in particular DNA, is provided in the form of an expression vector, which may be expressed in the cells ofthe individual to be treated. The vaccines may comprise naked nucleotide sequences or be in combination with cationic lipids, polymers or targeting systems. The vaccines may be delivered by any available technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration. Uptake of nucleic acid constructs may be enhanced by several -known transfection techniques, for example those including the use of fransfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dexfran and lipofectants, for example, lipofectam and transfectam. The dosage ofthe nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of lpg to lmg, preferably to lpg to lOμg nucleic acid for particle mediated gene delivery and lOμg to lmg for other routes.

The following Examples illustrate the invention.

Examples

Example 1

Methods

Recombinant protein expression and purification Recombinant N-terminal histidine tagged SSL7 and SSL9 proteins from S. aureus sfrain NCTC6571 were produced in E. coli using the expression vector pQΕ30 containing the genes NCTC6571-s-s 7 and NCTC6571-s-s^,/P respectively as previously described (Williams,R.J. et al, Infect. Immun. 68, 4407-4415 (2000)). Embp32 from S. epidermidis was also expressed as a recombinant N-terminal histidine tag fusion protein in E. coli and purified as previously described (Williams,R.J. et al, Infect. Immun. 70, 6805-6810 (2002)).

Crystallisation Crystals of SSL7 were obtained by the hanging drop vapour diffusion technique, at room temperature. Crystals were obtained in two different conditions. For the first condition (form I), the well buffer contained 25-30% (w/v) PEG-MME 2K, 0.2 M ammonium sulphate and 0.1 M MES, pH 6.5. Drops consisted of 1 μl recombinant SSL7 at 10 mg/ml and 1 μl well buffer. Crystals had a flat plate morphology with dimensions up to 0.3x0.2x0.01mm³. The second condition (form II) had the same protein concentration and drop size, but the well buffer in this case consisted of 28% (w/v) PEG 2K and 0.1 M Li₂SO₄ buffered with 0.1M Tricine at pH 8.5. h this case the crystals were rod-shaped with approximate dimensions 0.3x0.05x0.05mm³.

X-ray Data Collection Data were collected from cryocooled crystals following immersion in mother liquor containing 30% (v/v) glycerol (as described in Garmen,E. & Sc?hneider,T. Macromolecular Cryocrystallography. J. Appl. Cryst. 30, 211-237 (1997)). Form I The data were collected to 2.75A resolution on line BM14 at the European Synchrotron Radiation Facility (ESRF; Grenoble, France), on a Mar Research ccd detector. The data were indexed and integrated with Mosflm (Leslie, Joint CCP4 and ESF-EAMCB Newsletter on Protein Crystallography 26, (1992)), and scaled and merged using Scala (Evans et al, 97-103. 1997. CCLRC, Daresbury Laboratory. Ref Type: Conference Proceeding) from the CCP4 suite (Acta Cryst. D50, 760-763 (1994)). Subsequent analysis was carried out using programs from the CCP4 suite, unless otherwise stated. Data collection statistics are provided in Table 1.

Table 1: Data Collection and Refinement

Res. Rmerge Completeness Redundancy

(A) N_ref (%) I/σl (%) (%) Meas Uni. All High² All High All High All High

Form ] I (P4₃2ι2)

45.5- 59980 12607 9.4 46.0 14.6 3.1 98.7 99.2 4.5 4.3 2.75

Form II (P2ι2ι2

30.0- 99169 11049 14.0 48.0 4.6 1.5 99.9 99.9 4.5 3.9 2.7

Protein Water R-factor³ (%) rmsd

N ⁴B_ave(A²) N I B_ave(A²) Working⁵ I Free⁶ Bonds(A) Angles (°)

Form I (P4₃2_ϊ2)

3127 43.6 18 30.9 23.5 27.5 0.008 1.35

Form II (P2ι2ι2ι) 42.6 24 42.5 23.4 29.9 0.011 1.3

R-_merge ⁼ /_ ι ^~~ ^M \/ _j^_{M >} where Ii is the observed intensity of a reflection and I_M is the mean intensity of all related reflections.

² High: highest resolution shell. Form I: 2.9-2.75A; Form II: 2.85-2.70 A.

³ R- factor =

.

⁴ Mean B-factor

⁵ For the 95.1 % of data included in the refinement.

⁶ For the 4.9% of data randomly selected and excluded from refinment. Autoindexing indicated that the crystals belonged to pointgroup P422, with cell dimensions a=b=81.66 A, c=148.04 A and a Matthews coefficient of 3.1 (conesponding to a solvent content of 60% v/v) for 2 molecules in the asymmetric unit. There was a significant peak in the native Patterson map at fractional coordinates: 0.000, 0.372, 0.500 and no peaks attributable to non-crystallographic symmetry in the self-rotation function, indicating the presence of two molecules related by a 2-fold rotation parallel to the a-axis in the asymmetric unit.

Form II The data were collected to 2.7 A resolution on line ID 14-1 at the ESRF, on an

ADSC Quantum 4R ccd detector. Data indexing, integration, scaling and analysis was carried out as for form I. Autoindexing and analysis of systematic absences indicated spacegroup J?2 2_\2_\, with a=51.65 A, b=71.59 A, c=103.47 A, and a Matthews' coefficient of 2.00, conesponding to a solvent content of 40%. There was a peak on the self-rotation function at κ=l 80°, confirming the presence of two molecules in the asymmetric unit. Data collection statistics are provided in Table 1.

Molecular Replacement The co-ordinates for one monomer of SSL9 (pdb-id: 1M4V; Arcus et al, J. Biol. Chem. 277, 32274-32281 (2002)) were used for molecular replacement, which was carried out with Molrep (Vagin & Teplyakov, J. Appl. Cryst. 30, 1022-1025 (1997)).

Eorm / There was a single clear peak in the rotation function (I/σI=5.3, next peak

I/σI=3.58). Since very few systematic absences were recorded during data collection, molecular replacement was carried out in all nine possible spacegroups to unambiguously identify screw axes. The best solution was for spacegroup P4₃2ι2, for which two molecules could be placed in the asymmetric unit, related by the appropriate translation, with a conelation coefficient of 32.6% (for comparison, the best solution in P4A2 had a conelation coefficient of 26.4%). Sidechains that differed between the SSL5 and SSL7 proteins were replaced by alanines in the conectly positioned model, and rigid-body refinement was carried out using CNS version 1.1 (Brunger et al, Acta Cryst. D54, 905-921 (1998)). At this point, the R- factor was 52.2% and the Rg-ee, 54.6%. A round of simulated annealing reduced the R- and R_free-factors to 44.6 and 48.9% respectively.

Form II Molecular replacement was performed as described for form I. The best solution had a conelation coefficient of 37.4% for two molecules in the asymmetric unit. Following rigid-body and simulated annealing refinement in CNS, the R-factor and R_free were 34.16 and 42.06%> respectively.

Density Modification For both crystal forms, cross-crystal averaging, non-crystallographic averaging and phase improvement were carried out using Dmmulti (Cowtan, CCP4 Newsletter on Protein Crystallography (1994)) in CCP4 (Acta Cryst. D50, 760-763 (1994)), prior to calculation of maps for manual rebuilding ofthe model.

Model Building and Refinement In both cases, manual rebuilding was perfonned using O (Jones & Kjelgaard, Methods in Enzymology. Charles W.Carter . & Sweet,R.M. (eds.), pp. 173-208

(Academic Press, 1997), and in later refinement rounds XtalView (McRee, Practical Protein Crystallography. Academic Press, San Diego, CA (1993)). Refinement was carried out with CNS. Alanine residues in the initial model were exchanged for the conect sidechains where positive Fourier difference density could be seen. At a number of positions the sequence alignment was inconect and additional rebuilding ofthe chain was required. For the refinement, an overall anisotropic B-factor conection and bulk solvent scaling with k=0.36, B=26.9 A², for form I, and k=0.59, B=101 A² for form II were applied. Noncrystallographic symmetry restraints were applied throughout refinement except in the later stages where there was clear evidence of a difference between the chains. After all protein residues had been included in refinement, a number of tightly bound waters were added, where there was a 3 rms peak in the difference Fourier, and a 1 rms peak in the 2F₀-F_C map, and appropriate protein- water hydrogen bonds. At the end of refinement, the R- factor was 23.5% and R_free, 27.5%, for form I and R-23.4% and R_free=29.9% for form II. A homology model of SSL9 was created using the program MODELLER (Lawkowski et al, J. Appl. Cryst 26, 283-291 (1993)) and both SSL7 and SSL5 as template structures. Structural illustrations were drawn with Bobscript, ((Esnouf, Acta Cryst. D55, 938-940 (1999)) a modification of molscript (Kraulis, J Appl Cryst. 24, 946-950 (1991) and rendered with Raster 3D (Merritt & Bacon, Methods in Enzymology 277, 505-524 (1997) and Bacon & Anderson, Journal of Molecular Graphics 6, 219-220 (1988)).

FITC labelling of SSLs SSL7 and SSL9 were dialysed against labelling buffer (0.2 M NaHCO₃, pH 9.0) overnight at room temperature (RT). 50 μl of 1 mg/ml fluorescein isothiocyanate (FITC, Sigma) in dimethyl sulfoxide (DMSO) was added to 1 ml of a 2 mg/ml protein solution. After 4 hours incubation at room temperature in the dark, unbound FITC was removed by size exclusion chromatography using a PD-10 (Sephadex™) column. The concentration of labelled protein, and the FITC:protein ratio were determined by spectrophotometry. All preparations gave FIT C:protein ratios of between 1 : 1 and 2:1.

Antibodies The following monoclonal antibodies (MAbs) were used: CD2 (mouse MAb MAS 593, IgG_2b; Harlan), CD3 (supernatant mouse MAb UCHT1, Igd; obtained from P. C. L. Beverley [Edward Jenner -Institute for Vaccine Research, Compton, UK]), CD14 (supernatant mouse MAb HB246, IgG₂ ; gift from P. C. L. Beverley), and CD19 (supernatant mouse MAbBU12, IgGi; gift firomD. Hardie [Birmingham University, Birmingham, UK]).

Cell culture Human PBMC-derived dendritic cells (DC) were generated from fresh whole blood samples obtained from healthy volunteers (Alderman et al, Cardiovasc. Res. 55, 806-819 (2002) and Newton et al, Clin. Exp. Immunol. 133, 50-58 (2003)). Mononuclear cells separated on Lymphoprep ™ (Nycomed Pharma) by centrifugation at 400 g for 30 minutes were incubated in six- well tissue culture plates for 2 h at 37°C in 5% CO₂ in complete medium (CM)(RPMI 1640 medium (Gibco) supplemented with 10%) fetal calf serum (FCS; PAA Laboratories), 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Clare Hall Laboratories, Imperial Cancer Research Fund)). The adherent cells were cultured in fresh complete medium with 100 ng/ml human recombinant granulocyte-macrophage colony- stimulating factor (GM-CSF) and 50 ng/ml interleukin (IL)-4 (Schering-Plough Research Institute) . On day four of incubation, loosely adherent cells were collected, and contaminating T and B lymphocytes were removed by incubation with CD3, CD2, and CD 19 MAbs, followed by anti-mouse IgG-coated immunomagnetic Dynabeads ™ (Dynal). The supernatant, containing highly purified DC was cultured for another three days in fresh complete medium with GM-CSF and IL-4. Human PBMC-derived macrophages were obtained using the same procedure for dendritic cell culture, except that 10%) human serum was used and no cytokines were added (Swetman et al, Eur. J. Immunol. 32, 2074-2083 (2002)).

Binding and uptake of FITC labelled SSLs by human cells Binding assays were performed by incubating 10⁶ cells/well in complete medium with various concentrations of SSL-FITC (0.05-1.25 μM) for 1 hour at 4°C or 37°C. hi some experiments, 8 μM of unlabelled SSL was added to the cells together with the labelled protein. After incubation, cells were washed three times by centrifugation, and examined by flow cytometry. In some experiments, cells were additionally stained for various surface markers after SSL uptake. Cells were incubated with the relevant MAb for 30 min at 4°C, washed, and then incubated in 1 : 25-diluted phycoerythrein-conjugated goat anti-mouse immunoglobulin (PE, Jackson -l-mmunoResearch) for 30 min at 4°C. Cells were washed, fixed in 2% formaldehyde and examined using a FACScan flow cytometer (Becton Dickinson). Confocal microscopy 10⁵ cells were seeded on 32mm coverslips coated (for dendritic cells only) overnight at 4°C with lOμg/ml fibronectin (FN, Sigma) in HBSS (Gibco). After 2 hours at 37°C in complete medium, cells were incubated with SSL-FITC (1.25 μM) and or Texas Red-dextran (lmg/ml, Molecular Probes) for 1 hour at 37°C in complete medium. The coverslips were then washed three times in cold HBSS and fixed in 2% paraformaldehyde. The slides were examined on a Bio-Rad Confocal Microscope. Images were acquired from 0.5-μm optical sections of individual cells.

Results

Structure determination of SSL Recombinant SSL7, consisting of residues 36-231 ofthe sequence with accession number AF094826 in GenBank, was crystallised in two different conditions each of which gave rise to a different crystal form.

Form / The final, 2.75A resolution, model built into the electron density map contained two SSL7 molecules, representing residues 18-213 ofthe recombinant SSL7. In addition, eighteen water molecules were included at stereochemically sensible locations. Though the His-Tag and N-terminal tail were disordered and omitted from the density, the majority ofthe residues had well-defined electron density. The final R-factor and R_fr_ee were 23.5%> and 27.5% respectively, and refinement statistics are given in Table 1. The final model had good stereochemistry, with 98.6% of residues in the most favoured and additionally allowed regions ofthe Ramachandran plot, and no residues in disallowed regions. The geometry was better than expected for the average 2.75 A structure according to PROCHΕCK analyses (Lawkowski et al, J. Appl Cryst 26, 283-291 (1993)).

Form II This model again contained two SSL7 molecules: in this case residues 21-213 or 23-213 ofthe construct in chains A and B respectively. Twenty-four water molecules were added in this form. Once again, with the exception ofthe His-tag and N-terminal tail, the majority ofthe molecule had well-defined electron density. The final R- and R_ffee-factors were 23.4 and 29.9% respectively. The final model had good stereochemistry with 96.5% ofthe residues in the two most favoured regions of the Ramachandran plot. The geometry was better than expected for the average 2.7 A structure according to PROCHECK. Refinement statistics for both crystal forms are provided in Table 1.

The SSL7 structure The structure ofthe SSL7 monomer is shown in Figure 1(a). As predicted from sequence comparisons, the fold is similar to that ofthe bacterial superantigens, and consists of two domains. The N-terminal domain (residues 18-110) is an OB- fold, a variety of β-barrel associated with oligosaccharide and DNA binding, while the C-terminal domain (111-213) forms a β-grasp domain: a series of β-strands wrapped around a helix. In total, the structure of four copies ofthe SSL7 monomer was obtained (two from each crystal form), and they are all very similar, as can be seen from the Cα- atom root mean square deviations (rmsds; Table 2).

Table 2: Root Mean Square deviations (A) between the different copies ofthe SSL7 monomer.

Form I Form II A B A B

Form I A - 0.228 0.701 0.703 B - - 0.669 0.642

Form II A - - - 0.450 B - - - -

The only differences between the structures arise from differences in the conformations of flexible loops, and the linker between the N- and C-terminal domain (residues 106-112). The relative orientation ofthe two domains to one another, and the orientations ofthe individual secondary structural elements remain unchanged in the different copies ofthe monomer. For this reason, unless explicitly stated, one example monomer, chain A from form I, will be used in the comparisons and discussions that follow.

SSL7 and other proteins When the SSL7 monomer is superposed on the structure of SSL7 (SET3) (the stracture of SSL5 is provided by Arcus et al, J. Biol Chem. 277, 32274-32281 (2002)), the other member ofthe family for wliich a three dimensional stracture is available, the two are seen to share the same fold (Figure 1 (b)), as might be expected for proteins sharing 40%> sequence identity. However, when optimally superposed, the rmsd, which is 1.33 A over 157 spatially equivalent Cα atoms, is surprisingly high for two such highly related proteins (a value of about 1.1 A over the whole structure would be anticipated from sequence identity (Chothia & Lesk, EMBO J. 5, 823-6 (1986)). The high rmsd can be largely accounted for by changes in two regions ofthe structure. Firstly, there is a change in the twist ofthe β-sheet in the C-terminal β- grasp domain (Figure 1, (b) right hand structure-indicated by arrow). The change in the twist results in shifts in individual residue positions as large as 6.65 A (for the Cα atom of G125 in the C-terminal domain). Secondly, there are changes to the conformations ofthe loops on the external face ofthe N-terminal OB-fold (Figure 1 (b), left hand stracture- indicated by anow), these loops are associated with a generic low affinity MHCII binding site in superantigens (Jardetzky et al, Nature 368, 711-8 (1994) and Kim et al, Science 266, 1870-1874 (1994)), and changes in them may indicate differences in function between the two proteins. These large movements account for the low contrast and large R-factor for the initial molecular replacement solution, prior to the simulated annealing which successfully realigned the sheet strands, and some ofthe loop residues. The SSL7 stracture was also compared to a homology model of SSL9, based on both SSL7 and SSL5 as template structures (not shown). The sequence of SSL9 has a much greater homology to SSL7 (sequence identity 49%) than SSL5 (sequence identity 35%_>). This is reflected in the model of SSL9: when the structures are optimally superposed SSL9 has a Cα atom rmsd of 0.6 A over 188 spatially equivalent atoms from SSL7, and 1.4 A over 177 spatially equivalent atoms from SSL5. When SSL7, and the superantigen of known stracture with which it shares the highest sequence identity (streptococcal pyrogenic exotoxin, SPEC; 29% - Roussel., Nat. Struct. Biol. 4, 635-43 (1997)) are optimally superposed (Figure 3), it is seen that once again the overall fold is conserved. SPEC has some extended loops, but the structures are otherwise very similar. Interestingly, despite the SPEC and SSL7 sequences being far more divergent than SSL7 and SSL5, the two structures superpose nearly as well. The Cα atom rmsd for optimally superposed SSL7 and SPEC is 1.48 A over 134 structurally equivalent atoms. The difference between the structures on this occasion being both a slight change in the orientation ofthe β- grasp domain (Figure 1, (c) right hand structure, indicated by anow), but also large differences in the orientations ofthe strands ofthe OB-fold (Figure 1, (c), left hand structure-indicated by anow) the very large changes in conformation in this domain are not unexpected since the SSL proteins do not bind -MHC π, and this is the region primarily involved in this interaction in the superantigens.

Dimerisation In both crystal form I and II there are two molecules in the crystallographic asymmetric unit; these two molecules are related by a proper two-fold, resulting in the formation of an intimate dimer (Figure 1(d)). The dimer is virtually identical in both crystal forms, as can be seen from a comparison ofthe residues buried in the dimer interface (Figure 2), and the fact that the form I and form II dimers can be superposed with an all Cα atom rmsd of 0.881 A or 0.902 A depending on orientation. The remainder ofthe crystal pac-king is entirely different for each ofthe forms, since the crystals grew in different conditions and at markedly different pH; it is unlikely that the dimers formed solely as a result of crystal pac-king forces. As can be seen from Figure 1(d) the dimer interface is the result ofthe two β- grasp domains interacting to create an intermolecular β-sandwich. In the process, 1122 A² ofthe monomer surface in form one and 1146 A² of this surface in form II are buried: this is in the range seen in biologically relevant dimers (Jones & Thornton, Progress in Biophysics and Molecular Biology 63, 31-59 (1995)) . The dimer formation results in the burial of a number of hydrophobic residues (including FI 19, L128, 1132) and a number of neutral polar residues contribute to a hydrogen bonding network between the two monomers, however, no charge-charge interactions are created. It has been shown that protein-protein interaction surfaces differ little from the 'normal' exterior surface of proteins, but that they tend to contain additional neutral polar residues and fewer charged ones (Lo Conte & Janin, J. Mol Biol. 285, 2177-98 (1999)): the SSL7 dimer interface is entirely consistent with this. Figure 1 (d) also indicated with an anow the loop with the largest difference between SSL7 and SSL5, the movement of which prevents steric clashes between the two SSL7 molecules in the dimer.

Cellular tropism ofSSL7 and SSL9 Peripheral blood mononuclear cells (PBMC) were incubated with various concentrations of SSL7-FITC or SSL9-FITC for 1 hour, and cell-associated fluorescence measured by flow cytometry. Both SSL7 (Figure 3(a)) and SSL9 (not shown) stained a small proportion of PBMC at 37°C, but not at 4 °C. The level of fluorescence was dose-dependent up to a maximum at 1.25 μM protein. The mean percentage of cells stained with SSL7 (9.8+1.8, range 7.1-12.2, n=7) and SSL9

(10.9+1.1, range 9.4-12.6, n=5) was very similar. Mean fluorescence also increased with time between five minutes and a hundred and twenty minutes (not shown) suggesting progressive uptake of SSL protein by the cells. hi order to determine whether the interaction between SSL protein and PBMC was specific, competitive inhibition of SSL-FITC cell labelling by unlabelled SSL was investigated (Figure 3(b)). Excess unlabelled SSL7 was able to completely block uptake of SSL7-FITC. hi contrast, neither SSL9, nor an unrelated bacterial protein also canying a polyhistidine tag (Embp32) had any effect on the SSL7-FITC signal. Conversely, only unlabelled SSL9, but not SSL7 or Embp32, blocked uptake of SSL9-FITC. Interaction between SSL proteins and the PBMC therefore occurs via a saturable specific receptor, and is not mediated by the histidine tag on these proteins. Furthermore, SSL7 and SSL9 use different receptors, or different sites within one receptor. The PBMC sub-populations which are the targets for SSL7 and SSL9 were further characterised by immunophenotyping, using monoclonal antibodies to the major surface markers CD2, CD3, CD14, and CD19 (Figure 4). Both SSL7 and SSL9 were taken up by all CD 14 positive cells, and by a population of CD2-low cells, a phenotype consistent with that of peripheral blood monocytes (Crawford,K. et al, J. Immunol. 163, 5920-5928 (1999)). Neither SSL7 nor SSL9 showed any interaction with CD3 positive T cells. Interestingly, SSL7-FITC but not SSL9-FITC stained a subpopulation of CD 19 B cells, providing further evidence that the receptor for these two SSLs is distinct.

Uptake of SSLs by dendritic cells Peripheral blood monocytes were cultured in vitro in the presence of GM- CSF and IL-4, in order to drive their differentiation into myeloid dendritic cells

((Sallusto & Lanzavecchia, J. Exp. Med. 179, 1109-1118 (1994)). After depletion of residual lymphocytes, the population obtained after seven days culture consisted of >90% CDla+ HLA-DR high CD14 low dendritic cells (data not shown). These cells were incubated for sixty minutes at 37°C with either SSL7-FITC or SSL9-FITC and examined by flow cytomefry Figure 5 and confocal microscopy (data not shown). Dendritic cells stained uniformly strongly positive for both SSL7 and SSL9. Confocal microscopy confirmed that fluorescence was predominantly due to infracellular uptake of SSL, rather than surface staining. Both SSL7 and SSL9 were concentrated in small vesicular structures, localised particularly to the perinuclear region ofthe cell. In order to characterise the nature of these vesicles further, dendritic cells were cultured in the presence of SSL7 or SSL9-FITC and Texas Red dexfran (data not shown), which is avidly taken up by dendritic cells via mannose receptors (Sallusto et al, J. Exp. Med. 182, 389-400 (1995)). Texas Red dexfran strongly labelled a large number of infracellular vesicles throughout the dendritic cell cytoplasm. SSL distribution and dexfran distribution partially overlapped, with some infracellular vesicles clearly containing both markers. However, SSL positive dexfran negative vesicles were also observed. In a small proportion of cells, vesicles containing SSL9 appeared to aggregate, to generate very large vesicles, which contained high concentrations of both SSL and dexfran. The very large vesicles observed (which were never seen in the presence of dexfran alone) presumably resulted from fusion of many SSL containing vesicles, and may have been driven by intramolecular interactions between SSL molecules (as observed during dimerisation in crystal structure). Similar vesicle distortion is observed in the presence of excess invariant chain, again driven by multiple interactions between invariant chain molecules (Romagnoli et al, J. Exp. Med. 177, 583-596 (1993). ?In order to determine whether uptake of SSLs was a generalised feature of endocytic cells, peripheral blood monocytes were differentiated into macrophages, via culture in human serum, without added cytokines. Under these culture conditions, the cells develop a completely different phenotype (CDla-,HLA-DR-,CD14 high) and morphology (lack of dendrite formation) (Swetman et al, Eur. J. Immunol 32, 2074-2083 (2002). Macrophages, like dendritic cells efficiently endocytosed Texas Red dexfran, but showed no uptake of either SSL7 or SSL9 (data not shown).

Discussion One ofthe most exciting results from the structural studies of SSL7 was the identification of an identical SSL7 homodimer in the asymmetric unit of two otherwise very different crystal forms, grown from very different solution conditions. The dimer has a number of characteristics seen in functionally relevant dimers, and which indicates that the dimer is not purely an artefact of crystallization. Interestingly, the structure of SSL5 did not reveal any such dimer formation (Arcus et al, J. Biol. Chem. 277, 32274-32281 (2002) and the residues making up the interface are not conserved across the different SSL proteins. The change in the orientation ofthe β -grasp domain in SSL7 relative to SSL5 is necessary to allow the dimer to form: if a similar dimer is created from SSL5 monomers, clashes occur between residues 110-114 in one monomer and 197-200 in the other, and more seriously between residues 118-125 and 161-165. These clashes are alleviated by the change in the orientation ofthe β-grasp domain β-strands in SSL7, further suggesting that SSL7 does not form a dimer in the same way. Since no crystal stracture of SSL9 is yet available, a preliminary comparison with SSL7 was carried out using a homology model. As for SSL5, the residues involved in the dimer interface in SSL7 are not conserved between SSL7 and SSL9. However the sequence changes do not create steric or electrostatic clashes between the two monomers; rather they are such that hydrogen-bonding between the two molecules is maintained, and in some cases, new hydrogen-bonds are formed. SSL9 may therefore form a dimer in the same manner as SSL7. SSL7 and SSL5 show differences in the region ofthe N-terminal domain that are implicated in a general low-affinity MHCII binding site in superantigens. The homology model reveals that the residues in these loops are in general highly conserved between the SSL7 and SSL9 sequences. However there are some important differences, including the change of P93 in SSL7 to threonine in SSL9. This proline is part of a well ordered β-turn in SSL7, while in SSL5 there is no ordered secondary structural element present here: in fact this in SSL5 loop is rather disordered. The pattern of sequence conservation in the N-terminal loops indicates that the structures of SSL7 and SSL9 are more related to one another than to SSL5 and this may also be reflected in the functional properties ofthe molecules. However, the small number non-conservative sequence changes between SSL7 and SSL9 are also entirely consistent with differences in their putative receptor binding, as discussed further below. Studies ofthe superantigens have shown that their interactions with -MHCII and T cell receptor molecules are diverse, encompassing a number of different interaction surfaces and stoichiometries. This includes the formation of functionally important superantigen dimers for some superantigens, for example the Zn²⁺- dependent dimers formed by staphylococcal exotoxin D (Sundstrom et al, EMBO J. 15, 6832-40 (1996)), which form via the C-terminal β-grasp domain, in a manner reminiscent ofthe homodimers seen of SSL7. It also includes the formation of heterodimers using the same surface ofthe N-terminal OB-fold but different surfaces of -MHC molecules (as in the complexes of HLA-DR1 with SEB and TSST-1 respectively - Jardetzky et al, Nature 368, 711-8 (1994) and Kim et al., Science 266, 1870-1874 (1994)). The functional and structural studies described here show that SSL7 and SSL7, may be functionally active in different quaternary states. The most significant differences between the structures of SSL7, SSL5 and other superantigens suggest that these molecules may interact with different binding partners, and this is supported by the studies of cellular tropism. The characteristic features ofthe interaction between SSL7 and SSL9, and PBMC are specificity, temperature dependence and cell selectivity. Specificity, indicative that the interaction is mediated by a cell surface receptor, is shown by the demonstration that unlabelled SSL blocks uptake of SSL-FITC. This competition is observed for both SSL7 and SSL9, ruling out the hypothesis that the results are due to significant differences in affinity of binding between the two. The lack of reciprocal inhibition between SSL7 and SSL9 indicates that these two molecules have different binding partners on the cell surface, although the possibility that they bind to different sites on the same molecule cannot be ruled out. Although the binding sites of SSL9 and 5 on the cell surface are distinct, both are able to self-target to APCs. Since it was impossible to measure binding in the absence of uptake, true measurements of affinity could not be obtained. The concentrations required to obtain measurable uptake, however, were in the order of 0.1 micromolar, suggesting that the affinity of interaction with any putative receptor is relatively low. This is a characteristic of many classical superantigens (Labrecque et al, Semin. Immunol. 5, 23-32 (1993)). The temperature and time dependence of SSL interaction are suggestive of receptor mediated uptake rather than simple binding to the cell surface, and this was confirmed by the confocal microscopy studies discussed further below. However, a small amount of surface binding can be detected at 37°C, but not 4°, using indirect labelling of intact cells with an antibody against the histidine tag (not shown). The interaction of SSL with the receptor, as well as its subsequent uptake, is therefore temperature-dependent. The third characteristic of SSLs observed in these cellular studies with PBMC was the highly selective nature ofthe target population with which interaction could be detected. In ex vivo PBMC, the major target population is the monocyte, characterised by high expression of CD 14. Essentially all monocytes were found to interact with both SSL7 and SSL9. In contrast, neither SSL7 nor SSL9 interacted with T cells, identified by expression of CD3 and high levels of CD2. Interestingly, SSL7, but not SSL9 also bound to a proportion of B cells (in the order of 30% although this varied significantly between individuals), providing further evidence that the receptor for these two molecules is distinct. Since a very significant proportion of T cells, and all human B cells also express class II ?MHC (e.g. HLA- DR) this result rules out a direct binding of SSL7 or SSL9 to these molecules, thus clearly distinguishing them from classical superantigens. Monocytes express both class I and class II MHC molecules, and can act as antigen presenting cells for the activation of CD4 or CD8 T cells. However, the prototype antigen presenting cell, and the only cell type which can activate naive T cells, is the dendritic cell. It was therefore of interest that both SSL7 and SSL9 were taken up efficiently by monocyte-derived dendritic cell and hence both molecules can self target to this important class of antigen presenting cells. This cell type, which can be obtained by culture of PB monocytes in appropriate cytokines, provides a widely used model for myeloid dendritic cells. In contrast, neither SSL7 nor SSL9 showed any tropism for macrophages, a cell type also produced by in vitro culture of monocytes, but which has no antigen presenting capabilities. Studies indicate that antigen presenting cell activity remains intact in the presence of SSLs. Conversely, self-targeting to antigen presenting cells results in enhancing the immunogenicity of these proteins. The uptake of SSL7 and SSL9 into an endosomal compartment w-hich intersects with the dexfran uptake pathway indicates that the SSL7 are successfully targeted to the antigen presentation pathway. This is because uptake via the mannose receptor efficiently targets antigens to the Class II MHC antigen processing pathway (Sallusto et al, J. Exp. Med. 182, 389- 400 (1995)). Although enhancing immunogenicity would, at first sight, appear to be paradoxical, the generation of an antibody response to a secreted protein is unlikely to confer any advantage in bacterial clearance by the host. On the contrary, the interaction between secreted toxin and specific antibody in the microenvironment of the bacterium may activate complement and hence contribute to the breakdown of the physical barriers that restricts the invasiveness of these bacteria. SSLs therefore appear to provide S. aureus with an alternative molecular strategy with which to distract the protective adaptive immune response ofthe host, and contribute to bacterial pathogenicity. Specifically, SSLs achieve this through their ability to target antigen presenting cells.

Example 2

The work described in Example 1 shows SSLs (staphylococcal superantigen like proteins) interact selectively with antigen presenting cells, including dendritic cells. The functional consequences ofthis interaction are now examined further. We show that SSL uptake does not adversely effect any ofthe parameters of antigen presenting cell function examined using dendritic cells. SSL7 and 9 were found to have no effect on viability or morphology of dendritic cells. The proteins did not induce dendritic cell maturation, as measured by cell surface phenotype. Exposure to SSL did not alter the ability of dendritic cells to take up FITC-dextran. In addition, exposure to SSLs did not impair the ability ofthe dendritic cells to stimulate allogeneic or antigen specific T-cell responses. The ability of antigen presenting cells to present SSLs was also examined.

Dendritic cells loaded with SSL7 or 9 were able to stimulate a T-cell proliferative response in three out of eight healthy individuals tested. Sera from nine out often individuals tested contained antibodies against both SSL7 and SSL9, and the response to each SSL was specific and not cross-reactive. The results obtained demonstrate that SSLs can be used to specifically target antigen presenting cells and gain access to the antigen presentation pathway of these cells. SSLs may therefore be utilised to specifically deliver chosen antigens to antigen presenting cells in order to elicit an immune response against the chosen antigen.

Methods Recombinant protein expression and purification Recombinant N-terminal histidine tagged SSL7 and SSL9 proteins from S. aureus strain NCTC6571 and Embp32 from S. epidermidis were produced as described above in Example 1. SSL proteins without histidine tag behaved identically to the tagged version in terms of cell binding and uptake (Al Shangiti AM et al, Meet. Immun., 72:4261-70 (2004).

Antibodies The following monoclonal antibodies (MAbs) were used: CD2 (mouse MAb MAS 593, IgG₂ ; Harlan), CD3 (supernatant mouse MAb UCHTl , IgGi ; gift from P. C. L. Beverley [Edward Jenner Institute for Vaccine Research, Compton, UK]), CD14 (supernatant mouse MAb HB246, IgG_2b; gift from P. C. L. Beverley), and CD19 (supernatant mouse MAb BU12, IgGi; gift from D. Hardie [Birmingham University, Birmingham, UK]), HLA-DR (supernatant mouse MAb L243, IgG_2a; gift from P. C. L. Beverley), HLA-ABC (W6/32; Serotec), CD86 (supernatant mouse

MAb BU63, IgGl; gift from D. Hardie), and CD54 (Mouse IgG MEM-I ll, gift from Prof. Horejsi, Academy of Science, Prague, Czech Republic). Fluorescein isothiocyanate (FITC) conjugated anti-mouse rabbit polyclonal antibody was purchased from Dako. PE-conjugated anti-CD la (Monoclonal Mouse IgGl, clone BL6, Immunotech, Marseille, France). The rabbit polyclonal antibody to His-SSL7 was produced under contract by Eurogentec Ltd (Southampton, UK) and validated by Western blot and ELISA.

Dendritic Cell Preparation Monocyte-derived human dendritic cells (MDDC) were generated from fresh whole blood samples obtained from healthy volunteers as described previously (Al Shangiti et al, (2004), supra). Briefly, mononuclear cells separated on Lymphoprep™ (Nycomed Pharma) by centrifugation at 400g for 30 mins were incubated in 6-well tissue culture plates at 37°C in 5% CO₂ in complete medium (CM)(RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS; PAA Laboratories), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (Clare Hall Laboratories, Imperial Cancer Research Fund), 100 ng/ml human recombinant granulocyte-marcrophage colony-stimulating factor (GM-CSF) and 50 ng/ml interleukin (IL)-4 (Schering-Plough Research Institute). On day four of incubation, loosely adherent cells were collected, and contaminating T and B lymphocytes were removed by incubation with CD3, CD2, and CD 19 MAbs, followed by anti-mouse IgG-coated immunomagnetic Dynabeads™ (Dynal). The non-adherent fraction, containing highly purified dendritic cells (less than 5% CD3, CD 19 or CD 14) was cultured for another three days in fresh culture medium with GM-CSF and IL-4.

Cell viability 10⁵ dendritic cells/group were cultured with SSL7 or SSL9 (4.16 μM) in a 96-well plate, and the plate was incubated at 37°C in 5% CO₂ overnight. Cell viability was assessed by trypan blue exclusion assay. 10 μl of cells suspension were diluted in an equal volume of trypan blue solution. 10 μl ofthis mix were loaded on a haemocytometer counting chamber placed under the microscope and white live cells (dead cells turn blue) were counted with in a 4 x 4 square grid.

Cell surface phenotype expression Dendritic cell surface staining was performed by using a panel of monoclonal antibodies (MAbs) directed against surface antigens expressed by dendritic cells and the appropriate specific isotype controls. Briefly, 10⁵ cells were pre-incubated for 24 hours in culture medium with 4.16 μM of SSL7 or SSL9 or with PG (5 μg/ml, from S. aureus, Sigma) or purified LPS (100 ng/ml; Salmonella Minnesota, Sigma) in 96 well U-bottomed plates at 37°C. Cells were resuspended in 100 μl of staining buffer (-HBSS, 1% FBS, 0.1% sodium azide), and incubated first with the relevant MAbs for 30 mins at 4°C. Cells were washed, and secondary immunolabeling was performed using FIT C-conjugated rabbit anti-mouse immunoglobulin (30 min, 4°C). Cells were washed three times and fixed in 3.8%> paraformaldehyde and examined within 5 days on a FACScan flow cytometer (Becton Dickinson). Data were analysed using CellQuest software. Endocytosis assay Dendritic cells (10⁵) were incubated in culture medium with or with 4.16 μM of SSL7 or SSL9 for various times (1 or 18 hours) in 96 well U-bottomed plates at 37°C. Different concentrations (1, 3, 10 and 30 μg/ml) of FITC-dextram (40,000 MW) were incubated with the cells. After 1 hour of incubation at 37°C, cells were washed in ice cold HBSS containing 0.1 % azide to stop further endocytosis, fixed with 3.7% formaldehyde, and analysed by flow cytometry. The uptake of dexfran is expressed as mean fluorescent intensity. For each sample at least 5000 events gated on dendritic cells were analysed.

T-cell Proliferation Assays Autologous T-cells were obtained from non-adherent population of peripheral blood mononuclear cell fraction from eight healthy volunteers (age range 20-50, median approximately 30) and Cryopreserved in FCS containing 10% DMSO (Sigma Aldrich) at -70°C. Cells were thawed rapidly (37°C), and B cells, monocytes, and macrophages were depleted by incubation with CD 19, HLA-DR and CD 14 MoAb for 45 minutes on ice. Cells were washed and then mixed with magnetic microbeads and separated on magnetic columns. T-cells (greater than 90% purity) were used immediately after purification. Allogeneic T-cells used in mixed leucocyte reactions (MLR) were prepared from HLA-mismatched donors in same procedure. Purified dendritic cells (10 ), either untreated or treated for 18 hours to different concentration of SSL proteins (4.16, 1.25 and 0.42 μM), were incubated at 37°C/5%> CO₂ with autologous T-cells (2 x 10⁵ cells/well) in the presence of purified protein derivative (PPD) or with allogeneic T-cells in flat-bottomed 96-well microtiter plates. The dendritic cell autologous and allogeneic T-cell cocultures were incubated 6 days. Both assays were then pulsed with 1 μCi of [³H]thymidine (ICN Biomedical, High Wycombe, United Kingdom) for the final 18 hours of culture. Cells were harvested, and T-cell proliferation was measured by liquid scintillation counting (Microbeta Systems). All assays were performed in triplicate. Results were express as cpm. Enor bars represent the standard deviation (SD). Cytokines assays Autologous T and dendritic cells were incubated with different concentrations of SSL proteins (4.16, 1.25 and 0.42 μM) at 37°C/5% CO₂ in 24-well plates. After 4 days, cell culture supernatants were centrifuged for the removal of cells and stored at -70°C. Cytokine detection was done by enzyme-linked imnxunosorbent assay (ELISA) for interleukin- 10 (IL-10, Pharmingen, UK), gamma interferon (DFN-γ, Pharmingen, UK) and IL-13 (ImmunoTools, Germany). Purified protein derivative (PPD; 500 U/ml) was used as positive control.

Antibody detection Human serum was collected from heparinised blood often normal individuals (three females, seven males, age range 20-50, median 30). ELISA miscroassay plates were coated for 24 hours with 0.04 μM of SSL proteins dissolved in sodium carbonate buffer 0.1 M, pH = 9.5 at 4°C (100 μl/well). After three successive washes with HBSS containing 0.1 %> Tween 20™, blocking was performed with 1 % skimmed milk in ?HBSS for 1 hour at 37°C. The plates were washed again three times, as before, and the tested sera diluted at 1 :2000 in HBSS with 0.1% Tween™ were added to the SSL coated plates for 1 hour at 37°C (preliminary studies showed that this diluation of antisera gave no background staining for any serum tested in control wells). After three additional washes, the remaining bound antibodies were incubated for 1 hour at 37°C with alkaline phosphates - conjugated human antibodies diluated at 1:1000 in -HBSS with 0.1% Tween 20™. Excess conjugate was removed by washing as above and a colorimetric reaction was carried out by addition ofthe chromogen OPD (o-Phenylenediamine dihydrochloride) for 15-20 minutes. The plates were read (405 nm) to detect the optical density (OD) readings. A control well containing no serum was used to detect the background count. A polyclonal rabbit antibody raised against purified His-tagged SSL was included as a positive control (anti-SSL7). Competitive ELISA was performed by mixing the sera with differing concentrations of SSL or a control bacterial protein Embp32 (0.08, 0.17, 0.33 and 0.42 μM), and then testing binding on SSL-coated plates as above. Statistical analysis The means of paired groups were analysed by a 2-tailed Student's t test. The level of significance was O.05.

Results

TJie effects of SSL protein on DC viability, morphology and surface phenotype Monocyte-derived dendritic cells (MDDC) were derived from peripheral blood monocytes. They showed the characteristic phenotype with low CD 14, and high HLA-DR and high CD la. As shown in Figure 1, fluoresceinated SSL7 and SSL9 labelled CD la expressing cells dendritic cells in unpurified dendritic cell cultures, while residual contaminating cells (predominantly lymphocytes) did not show any interaction with either protein. Nevertheless, in order to exclude the possibility of indirect effects mediated on dendritic cells via some other cell type all further experiments were performed on purified dendritic cell cultures, containing less than 5% non-dendritic cells. Dendritic cells were incubated with SSL7 or SSL9 (4.16 μM) for either 1 or 18 hours at 37°C and cell viability was assessed by trypan blue exclusion. Microscopic analysis revealed that SSL proteins were not cytotoxic as more than 95% of cells appeared viable. Untreated cells were predominantly non-adherent with few dendritic cell processes, typical of immature dendritic cells (Figure 7A, top panel). Neither SSL7 or SSL9 induced any noticeable morphological changes over the time period tested (Figure 7A, middle panels). In contrast dendritic cells treated with the TLR4 bacterial ligand LPS (100 ng/ml) or peptidoglycan (PG) 5 μg/ml) became adherent and extended multiple, long dendritic processes (Figure 7A, bottom panels). The cell-surface expression of a panel of characteristic dendritic cell surface markers was analyzed by flow cytometry. Immature dendritic cells were incubated with 4 μM SSL7 or SSL9, or LPS and PG and the surface phenotype of these dendritic cells was analysed after 18 hours of culture. Neither SSL7 or SSL9 (Figure 7B) induced significant changes in any ofthe surface molecules measured. In contrast, dendritic cells incubated with either LPS or PG up-regulated surface expression of HLA-DR, HLA-ABC, CD86 and CD54. Thus, in summary, exposure of dendritic cells to the SSLs protein did not induce dendritic cell maturation, nor indeed any obvious changes in dendritic cell surface phenotype, viability or morphology.

The influence of SSLs on endocytosis Fluorescein isothiocyanate-labelled dexfran (FITC-Dx) is rapidly taken up by dendritic cells via the mannose receptor (Sallusto F., et al, J. Exp. Med., 182:389- 400 (1995)). To determine whether SSL protein altered antigen uptake function, dendritic cells were treated with 4.16 μM of SSL7 or SSL9 and incubated for 1 or 18 hours at 37°C. Different concentrations of FITC-Dx (1, 3, 10 and 30μg/ml) were added to the cell and incubated for a further hour at 37°C, and FITC-Dx update by the dendritic cell was then measured by flow cytometry. Figure 8 shows the results obtained with the total cell associated dexfran being measured by flow cytometry and expressed as mean fluorescent intensity for a minimum of 5000 dendritic cells. As shown in Figure 8, SSL treated dendritic cell showed rapid update of FITC-Dx, and neither protein had any effect on endocytic activity.

Txe influence of SSLs on the T-cell stimulatory capacity of dendritic cells To evaluate the effect of SSL proteins on the stimulatory capacity of dendritic cells in T-cell proliferation, day 6 dendritic cells (10⁴) were incubated for 18 hours with different concentrations of SSL proteins (0.42, 1.25, and 4.16 μM). Residual T- cells were depleted and the functional assays performed using fresh viable purified autologous or allogeneic T-cells (2 x 10⁵). The ability to induce secondary immune responses was unchanged. Figure 9A shows representative experiments eliciting recall responses to tuberculin (PPD - 500 U/ml)). There was no statistical difference in the proliferative responses observed between any ofthe pre-incubated dendritic cell groups with SSL 7 or SSL9 (37182 ± 2036 cpm and 36458 ± 3000 cpm, respectively) and the control (36458 + 6151 cpm) after the 6 days co-culture period (E>0.05). The capacity of SSL-treated dendritic cells (10⁴) to elicit primary T-cell proliferation also was tested in an allogeneic mixed lymphocyte reaction (MLR). The same concentrations of SSL 7 and 9 and number of T cells used in the autologous assay were emplyed with the allogenic T cells. A similar result was observed (Figure 9B), as proliferation response against allogeneic T-cells (25710 + 1140 cpm) was unaffected by the treatment of SSL7 or SSL9 (26149 ± 3674 and 25816 ± 3159 cpm, respectively (E>0.05)). Therefore, SSL proteins have no effect on the ability to induce proliferation of allogeneic T-cells. In general, the results of these experiments demonstrate that the antigen presentation capacity of dendritic cells remains intact in the presence of these secreted proteins.

T-cell responses to SSLs in the normal human population To investigate the ability of SSLs to stimulate a recall T-cell response in healthy volunteers, dendritic cells (10⁴) were incubated with autologous T-cells (2 x 10⁵) from normal donors in the presence of different concentrations of SSL proteins (0.42, 1.25, and 4.16 μM) for 6 days (Figure 10). Purified protein derivative (PPD) was used as a positive confrol. A recall response against SSL7 was documented in 2/8 individuals and a response to SSL9 in 3/8 individuals. All volunteers showed a good recall response against PPD (70889 + 3146 rpm). The supernatants of tlrree dendritic cell/T-cell/SSL co-cultures (individuals 1, 2 and 8 from Figure 10) were tested for IFN-γ (TH1), IL-13 (TH2) and IL-10 (Treg) after 4 days. All cytokine levels were low (IFNγ < 700pg/ml n=3); IL-13 < 50 pg/ml, n=3) or undetectable (IL-10). The results for the individual with maximum response (individual 1 in Figure 10) at different SSL concentrations are shown in Figure 11.

Antibodies responses to SSLs in the normal human population In order to see if the presence of a T-cell response conelated with antibody production, sera from ten individuals (including those tested for T-cell responses as shown above) were tested by ΕLISA against immobilized SSL7 and SSL9 (Figure 12A). Sera was diluted 1 :2000 and tested for binding to SSL7 or SSL9 by ΕLISA as described in the materials and methods section. Nine out of time individuals tested showed antibody responses to both SSL7 and SSL9 at this dilution. Interestingly, competitive ΕLISA (Figure 12B and C) showed that the antibody response was highly specific for individual SSL isotypes. Increasing concentrations of SSL7 (Figure 12B) were able to completely block the interaction between SSL7 protein and SSL7 sera. In contrast, neither SSL9, nor an unrelated bacterial protein (Embρ32) had any effect on the SSL7 antibody binding. Conversely, only SSL9, but not SSL7 or Embp32, were able to block SSL9-antibody (Figure 12C). Therefore, interaction between SSL proteins and SSL-antibodies were specific and do not cross- react.

Discussion

Dendritic cells as professional antigen presenting cells have a key role in the initiation ofthe immune response against microbial infections; therefore, many microbial strategies have been described which interfere with dendritic cell function (Moll H., Cell Microbiol., 5:493-500 (2003)). One possibility was that SSLs might interfere with normal function of dendritic cells and therefore impair the protective immune response to S. aureus. Such functions have recently been proposed for the anthrax lethal toxin (Agrawal A, et al, Nature, 424:329-34 (2003)) and E. coli heat labile toxin (Petrovska L, et al, Vaccine, 21 : 1445-54 (2003)). The possibility of SSLs inhibiting dendritic cell function was therefore ruled out in the present study. SSL7 or SSL9 were shown to be non-toxic to antigen presenting cells and did not alter the characteristic morphology of these cells (cf the effect of Clostridium difficile toxin B, (Swetman CA et al, Eur. J. Immunol., 32:2074-83 (2002)). Conversely, SSL7 and SSL9 did not induce process extension, or up-regulation of cell surface co-stimulatory and HLA molecules on the dendritic cell, two characteristic signs of activation/maturation responses induced by whole S. aureus (Tourkova IL, et al, Immunol. Lett., 78:75-82 (2001)) or bacterial surface components such as peptidoglycan (PG) (Michelsen KS, et al, J. Biol. Chem., 276:25680-6 (2001)). Thus, although SSLs bind to and are taken up by dendritic cells (Figure 6) this interaction does not appear to engage activating receptors on the dendritic cell surface. Previous studies (Williams RJ, et al, hifect. Immun.,

68:4407-15 (2000)) indicating that SSLs could activate high levels of inflammatory cytokine release from peripheral blood cells could not be repeated using the highly purified protein preparations used for this study (data not shown) and may have resulted from trace amounts of contaminating LPS. In addition to their specialized dendritic morphology and cell surface phenotype, dendritic cells are characterized by extremely rapid endocytosis by both fluid phase and receptor mediated update (Swanson JA, et al, Trends Cell Biol., 5:424-8 (1995) and Levine TP, et al, Adv. Exp. Med. Biol. 329:11-5 (1993)). The uptake of FITC-Dextran, which is believed to be mediated via mannose receptors on the cell surface (Sallusto F, et al, J. Exp. Med., 182:389-400 (1995)) is frequently used to measure the latter. Dendritic cells did indeed show efficient internalization of FITC-Dextran (albeit slightly less well after overnight culture) and this uptake was not altered by exposure to SSL7 or SSL9. Finally, since dendritic cells are distinguished by being the most potent stimulators of both primary and secondary T- cell responses, we tested the effects of SSL7 and SSL9 exposure on dendritic cell function directly. Although dendritic cells stimulated powerful proliferative responses to both PPD (a classical recall secondary response to BCG vaccination) and allogeneic purified T-cells (predominantly a primary response) neither SSL7 nor SSL9 altered the antigen presentation activity of dendritic cells. Taken together, therefore, these data do not provide any evidence that SSL proteins inhibit or modify dendritic cell function. Further experiments demonstrated that SSL targeted to antigen presenting cells, in particular dendritic cells, actually are delivered to the antigen presentation pathway. This can hence enhance an immune response to these proteins and can also be used to deliver chosen antigens to the same pathway. The immune response to SSLs was analysed in a small panel of healthy human volunteers. Although none of the individuals tested had any -known history of clinical S. aureus infection, the organism is extremely prevalent in the environment and approximately 30-40%> individuals are persistently colonized by S. aureus, usually in the nasal mucosa (Nair SP, Williams RJ, Henderson B, Advances in our understanding ofthe bone and joint pathology caused by Staphylococcus aureus infection, -Rheumatology, (Oxford), 2000; 39:821-34). Indeed, in eight volunteers tested, three (37%) showed a dose dependent T-cell response to dendritic cells loaded with SSL9 and two to SSL7. The response was detectable with relatively large numbers of T-cells/well (2 x 10⁵) and induced the release of a very low level of either TH1 (IFN-γ) or TH2 (IL-13) cytokines, suggesting precursor frequency of T-cell specific for SSL was likely to be low. Despite a low T-cell precursor frequency, the humoral response to SSL7 and SSL9 was robust. Using solid phase ELISA nine out often sera tested showed specific antibody binding to both SSLs. The response measured was IgG (using an anti-IgG detection antibody) suggesting that class switching had occuned and further implicating the activity of SSL-specific T-cells. Interestingly, some individuals (e.g. individuals 2 and 7 in Figures 10 and 11) show antibody responses, but no detectable T-cells responses, perhaps because precursor T-cell frequency has fall below detectable levels in these individuals. One individual showed a T-cell response to SSL9, but no antibody response to either SSL tested, although we cannot rale out that some antibody might be detectable at a lower dilutions. Interestingly, the antibody response to each SSL was highly specific with minimal evidence of cross- SSL reactivity. Tliis data is consistent with the sequence diversity between SSL paralogs, despite a highly conserved three dimension structure (Al Shangiti et al (2004) Supra and Arcus VL, et al, J. Biol. Chem., 277:32274-81 (2002)). The presence of SSL specific immunity in so many individuals, and the existence of so many SSL paralogs in the S. aureus genome, is suggestive of a strong evolutionary interaction between host immunity and this bacterial family of proteins. In conclusion, this study describes a number of functional consequences of the interaction between SSL and dendritic cells. In contrast to some other bacterial exotoxins (Agrawal et al, (2003), Supra and Pefrovska et al, (2003) Supra) SSLs do not appear to damage dendritic cells, but rather can be taken up by them, and thus stimulate T-cell response in healthy individuals. This means that SSLs may be employed to selectively deliver chosen antigens to antigen presenting cells, in particular to dendritic cells. SSLs may therefore be used to help induce an immune response or tolerance to a selected antigen.

Claims

1. Use of a complex comprising: (a) a targeting polypeptide comprising a staphylococcal superantigen-like protein (SSL), a fragment thereof or a variant of either, where the SSL, fragment or variant has the ability to target the complex to an antigen presenting cell; and

(b) an antigen and/or a nucleic acid molecule encoding an antigen, in the manufacture of a medicament for use in immunization or the induction of tolerance.

2. Use according to claim 1, wherein the antigen comprises a polypeptide which is present in the complex as a fusion polypeptide with the targeting polypeptide.

3. Use according to Claim 1 or 2, wherein the antigen and targeting polypeptides are not part ofthe same polypeptide, but are covalently joined to each other or are joined through a linker.

4. Use according to any one ofthe preceding claims, wherein the antigen is a pathogenic antigen, an auto-antigen, an allergen and/or a cancer antigen.

5. Use according to any one ofthe preceding claims, wherein the targeting polypeptide is present as a dimer.

6. Use according to any one ofthe preceding claims, wherein the targeting polypeptide comprises: (a) a polypeptide having the amino acid sequence of any of SEQ ID Nos 6, 7, 9, 20, 21, 23, 30, 32, 34, 40, 44, 42, 58, 59, 60 72, 74, and/or76; (b) a fragment of any ofthe sequences of (a), the fragment having the ability to target the complex to an antigen presenting cell; and/or

(c) a variant polypeptide having at least 30% amino acid sequence identity to any ofthe polypeptides of (a) or (b) and the ability to target the complex to an antigen presenting cell.

7. Use according to claim 6, wherein the targeting polypeptide comprises: (a) the sequence of SEQ ID No: 7, 9, 21, 23, 32, 34, 40, 42, 59, 60, 74, 76 and/or 92;

(c) a variant polypeptide having at least 70 % amino acid sequence identity to any ofthe polypeptides of (a) or (b) and the ability to target the complex to an antigen presenting cell.

8. Use according to any one ofthe preceding claims wherein the medicament is for the induction of tolerance and is to be administered without an adjuvant.

9 A complex comprising:

(i) a targeting polypeptide as defined in any one ofthe preceding claims; and (ii) an antigen or a nucleic acid encoding an antigen, wherein the antigen or encoded antigen is selected from a pathogenic antigen, auto-antigen, an allergen and a cancer antigen.

10. A complex according to claim 9, wherein the targeting polypeptide is present as a dimer.

11 A virus comprising a targeting polypeptide as defined in any one of claims 1 to 8.

12. A nucleic acid molecule comprising a polynucleotide sequence encoding a targeting polypeptide and antigen, wherein the targeting polypeptide and antigen are as defined in claim 9.

13. A nucleic acid according to claim 12, wherein the nucleotide sequence encoding the targeting polypeptide and the antigen are present in a single open reading frame.

14. A vector comprising a nucleic acid according to claim 12 or 13.

15. A vector according to claim 14, comprising a promoter capable of giving rise to expression of both the targeting polypeptide and the antigen in an antigen presenting cell.

16. A cell comprising a nucleic acid according to claim 12 or 13 or a vector according to claim 14 or 15 or infected with a virus according to claim 11.

17. A method of loading antigen presenting cells comprising contacting an antigen presenting cell or a precursor thereof with a complex as defined in any one of claims 1 to 10 or a virus according to claim 11

18. A method according to claim 17, which is an in vitro method.

19. An antigen presenting cell which has been loaded with a complex as defined in any one of claims 1 to 10 or a virus according to claim 11.

20. A pharmaceutical composition comprising a complex as defined in any one of claims 1 to 10, a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 10, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a viras according to claim 11 or an antigen presenting cell according to claim 19 and a pharmaceutically acceptable carrier or diluent.

21. A vaccine comprising a complex as a complex as defined in any one of claims 1 to 10, a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 10, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus according to claim 11 or an antigen presenting cell according to claim 19.

22. A complex as defined in any one of claims 1 to 10, a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 10, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a viras according to claim 11, or an antigen presenting cell according to claim 19 for use in a method of treatment ofthe human or animal body by therapy.

23. Use of a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 10, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus according to claim 11 or an antigen presenting cell according to claim 19 in the manufacture of a medicament for use in immunisation.

24. A method of immunising a subject, the method comprising administering an effective amount of a complex as defined in any one of claims 1 to 10, a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 10, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, a virus according to claim 11 or an antigen presenting cell according to claim 18 to a subject.

25. An agent for immunising a subject, the agent comprising a complex as defined in any one of claims 1 to 9, a nucleic acid encoding the targeting polypeptide and antigen of a complex as defined in any one of claims 1 to 9, a vector comprising such a nucleic acid, a cell comprising such a nucleic acid or vector, or an antigen presenting cell according to claim 17.