Field of the Invention

The present invention relates to the identification of therapeutic agents and their use in improving immuno-therapies, improving nucleic acid vaccination (for instance DNA and RNA vaccination), and for improving transplantation. Furthermore, the inventive agents allow manipulation of the immune system, with therapeutic applications to autoimmunity and infection. Background of the Invention

Major histocompatibility complex class II (MHCII) molecules are a family of molecules involved in the presentation of antigens to CD4 T cells, and as such play an essential role in the adaptive immune response. The production, trafficking, loading with antigenic peptide, and subsequent trafficking of MHCII molecules is a complicated process. Class II a and β chains assemble in the endoplasmic reticulum (ER) together with the invariant chain (Ii) to form nonomeric complexes (αβΙϊ) ₃ , which exit the ER and follow the secretory pathway to the trans-Golgi network (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). Ii contains dileucine based sorting signals that are responsible for targeting into endosomal-lysosomal compartments; this can be direct from the trans-Golgi network or indirectly via the cell surface (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). It is thought that a significant proportion of (αβΙΐ) ₃ traffics via the plasma membrane (Lapaque et al, PNAS, Vol 106 (33) _> P14052-14057. 18 August 2009). In the endosomal-lysosomal system the MHCII molecule is loaded with an antigenic peptide, after a sequence of events involving the degradation of Ii and exchange of a remaining fragment (CLIP). The peptide-MHCII complex (p MHCII) when on the cell surface is capable of interacting with and stimulating the T cell receptors (TCRs) of CD4 T cells. pMHCII can also be found in intracellular pools in the late endosomal antigen processing compartments. Traditionally MHCII expression is described as being restricted to professional antigen presenting cells (dendritic cells, macrophages, B cells, monocytes, and thymic epithelial cells). However, MHCII expression can also be induced upon cells such as fibroblasts, epithelial cells, keratinocytes, myocytes, and also activated T cells (MHCII is found on a least human, bovine, equine, and rat T cells) (Holling et al, Human Immunology, 2004, 65, P282-290). The primary role of pMHCII is to stimulate the adaptive immune system by the direct activation of CD4 T cells. However, pMHCII has also been shown in many contexts to stimulate the suppressive side of the immune system, and has also been demonstrated to have direct effects upon the cells that express it. An example of pMHCII in a suppressive context is direct expression on tolerogenic dendritic cells. In addition, Foxp3+ Regulatory T cells are restricted to recognition of peptide presented by MHCII.

The expression of MHCII upon activated T cells has been shown to have immune suppressive effects. Antigen presentation by T cells through MHCII has been indicated to cause anergy and apoptosis (Kambayashi and Laufer, Nature Rev Immuno, Vol 14, Nov 2012, P719-730; Tsang et al, Blood, 2003, 101 (7)). In addition, MHCII expression on T cells may directly mediate apoptosis of the MHCII bearing activated T cells (Holling et al, Human Immunology, 2004, 65, P282-290; Haabeth OA et al, Front Immunol. 2014; 5: 174). pMHCII can also be found on the surface of cells that do not directly express pMHCII. Cells can acquire pMHCII from other cell types by a process known as trogocytosis or "nibbling". T cells have been demonstrated to acquire pMHCII in this manner.

Due to the fundamental role of pMHCII on a range of cells involved in the immune system, and the range of activating and suppressive effects, the manipulation of cell surface levels of pMHCII is a powerful tool. It is known that Salmonella typhimurium is able to reduce cell surface expression of pMHCII specifically (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). However, the process by which this effect is achieved has never been identified. While deficiency in a particular Salmonella protein was demonstrated to prevent Salmonella from reducing pMHCII surface levels (SsaV-deficiency), this protein is an essential component of the SPI-2 type III secretion apparatus. The SPI-2 type III secretion is activated after Salmonella has been internalised into acidified vacuoles of host cells and it translocates over 30 different bacterial effector proteins into host cell membranes and cytoplasm (Figueira and Holden, Microbiology, Vol 158 (5), 15 March 2012). These effectors have different functions, and therefore the failure of a strain lacking SsaV to reduce pMHCII surface levels does not provide information on the identity of the effector that reduces pMHCII surface levels (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009).

The inventors have now identified that the SaZmoneZZa-derived protein SteD is capable of reducing the surface level of pMHCII. SteD was previously known only to be a SPI-2 type III secretion effector important for Salmonella virulence, not for a specific function (Supplemental Table 4: Niemann et al, J. Bacteriol, (2013), Vi95(io), 2119- 2125; Neimann et ah, Infect. Immun., (2011), V79(i), 33-43). Furthermore the inventors have demonstrated that the reduction in surface pMHCII has a physiological effect on immune function, suggesting the practical application of SteD as a therapeutic agent for manipulating the immune system.

The inventors also provide data indicating the mechanism behind the function of SteD, demonstrating the broad applicability of the disclosed agents for immune

manipulation.

The identification of the protein in Salmonella that is responsible for the reduction in surface levels of pMHCII, advantageously provides a substantial number of

applications that provide significant therapeutic possibilities.

The invention is applicable to adoptive cell transfer therapy (ACT). ACT is a type of immuno-therapy that involves the administration, to a subject to be treated, of immune cells capable of activity against a chosen target. A great deal of research into this type of therapy has addressed the transfer of anti-tumour T cells into a cancer-bearing host. However, this technique is also applicable to other types of target, such as chronic viral infections, and to the transfer of other types of immune cell.

Key issues with all types of adoptive transfer therapies are difficulties related to achieving the desired strength of immune activity, duration of immune activity, or the required persistence of the transferred cells. The expression of pMHCII on the surface of activated T cells has been associated with a reduced immune response.

The invention is also applicable to techniques involving nucleic acid (RNA, DNA) and vector-mediated transgene expression in a context where expression of a transgene leads to therapeutic benefits, for instance vaccination. Nucleic acid vaccination is a technique whereby a nucleic acid encoding a target antigen is introduced into a host either alone or mediated by a vector (for instance viral or bacterial) for the induction of an immune response. The target antigen is expressed by the host cells and can result in the generation of an immunological response and residual immunological memory. Nucleic acid vaccination is believed to be applicable to protecting both humans and animals from viral, bacterial, and parasitic pathogens. Nucleic acid vaccination is also a potential method for vaccination against tumour antigens, either prophylactically or therapeutically. The encoded transgene can be expressed and presented directly by antigen presenting cells (APCs), or the target antigen can be expressed by non-APC host cells and then acquired by APCs for presentation, for instance by engulfment of apoptotic target antigen expressing cells. An example of a host cell suitable for inoculation with a nucleic acid vaccine is muscle cells. Nucleic vaccination is applicable to many target antigens, for instance target antigens associated with ΗΓν.

Another technique to which the invention is applicable is gene therapy. Gene therapy can involve the replacement or removal of an endogenous gene, or the introduction of a new transgene into a host for a therapeutic benefit. This type of treatment is applicable to the treatment of diseases such as those caused by inherited mutations, and also has broader applications such as the treatment of cancer and viral infections. A limitation of nucleic acid vaccination and gene therapy is that the host cells, or introduced cells, expressing the introduced nucleic acid, such as a target antigen, can be precociously removed, thereby reducing the duration, intensity, or efficacy of the desired immunological response to the target antigen. Presentation of antigen by MHCII expressed on the host cells expressing the target antigen may contribute to this precocious removal.

Another limitation of nucleic acid vaccination is that antigen presentation by cells not providing additional co-stimulatory signals can result in tolerance induction to the target antigen. This could promote the opposite effect to that desired. As the host cells expressing the target antigen may not be professional APCs, direct antigen presentation by MHCII by the host cells, rather than expression and subsequent uptake by professional APCs, may therefore reduce the efficacy of the vaccine.

Transplantation is a well-established therapeutic technique that is applicable to a wide range of tissues, organs, and cells. Transplantation is applicable to whole organs, such as the heart, kidneys, liver, lungs, pancreas, intestine, and thymus. Transplantation is also applicable to tissues such as bones, tendons, cornea, skin, heart valves, tissues of the nervous system, and tissues of the circulatory system. Tissues consisting of individual cells such as bone marrow can also be transplanted. A limitation of transplantation is that an immune response to the transplant can cause transplant rejection. This problem is relevant to all types of transplant. While the use of immunosuppressant drugs and matching tissue types can reduce the risk, there remains a need for more specific agents to reduce the immune response to

transplantation. Summary of the Invention

The ability to manipulate the levels of pMHCII on cell surfaces therefore would be of significant benefit in many therapeutic and prophylaxic contexts. The present inventors have now identified a protein translocated into host cells by Salmonella which is sufficient to reduce surface levels of pMHCII.

Therefore, according to a first aspect of the invention, there is provided an isolated polypeptide comprising an amino acid sequence as set out in any one of SEQ ID NOs: l to 29, or a fragment or variant thereof, for use as a medicament. The polypeptide or protein of the invention has been designated herein as SteD. Embodiments of the invention also include fragments and variants of SEQ ID NOS: 1 to 29 that retain the ability to reduce cell surface levels of pMHCII.

According to another aspect, there is provided a nucleic acid sequence encoding a polypeptide of the invention, for use as a medicament.

In another aspect, there is provided a vector comprising said nucleic acid sequence of the invention, for use as a medicament.

The nucleic acid of the invention may be transformed into a cell and accordingly the invention is also directed in another aspect to a cell comprising a vector of the invention, for use as a medicament.

Another aspect provided by the present invention is a pharmaceutical composition comprising any of the polypeptide, nucleic acid, vector, or cell of the invention together with a pharmaceutically acceptable excipient. Further aspects provide the use of said pharmaceutical composition as a medicament, optionally for use with methods of treatment as disclosed herein.

In a further aspect, there is provided a vaccine composition comprising any of i) a polypeptide of the invention, ii) a nucleic acid of the invention, iii) a vector of the invention, iv) a cell of the invention, or v) a composition of the invention, and a target antigen.

The target antigen may be present as a nucleic acid molecule encoding said target antigen. The nucleic acid molecule may be an RNA molecule, for instance a self- amplifying RNA molecule. The vaccine composition may further comprise a synthetic lipid nanoparticle delivery system.

A medicament of the invention can be for use in immuno-therapy, the treatment of a medical condition (such as allergy, autoimmunity, cancers, infections, graft versus host disease, infertility), for use with adoptive cell transfer therapy, for use with nucleic acid (RNA/DNA) or vector (viral, bacterial) mediated vaccination, or with a transplant related disorder. In particular embodiments, the polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions of the invention can be used in conjunction with adoptive cell transfer therapy, wherein the cell transfer therapy involves the adoptive transfer of leukocytes, lymphocytes, T cells, CAR T cells, TCR transgenic T cells, transgenic T cells, or tumour infiltrating lymphocytes.

In another aspect, the polypeptides, nucleic acids, vectors, cells, or pharmaceutical compositions of the invention can be used in nucleic acid vaccination. The nucleic acid vaccine may comprise DNA and/or RNA. In an embodiment the nucleic acid vaccine comprises a self-amplifying RNA molecule. The inventive agents and/or the nucleic acid vaccine may be delivered by a synthetic lipid nanoparticle delivery system. In a particular embodiment the inventive agents are used in combination with self- amplifying RNA vaccine delivered by a lipid nanoparticle delivery system. The nucleic acid vaccination maybe for the treatment or prevention of diseases or conditions, for example an HIV infection, such as influenza.

In another aspect, the polypeptides, nucleic acids, vectors, cells, pharmaceutical compositions, or medicaments of the invention can be for use with a transplant related disorder or for use with gene therapy for the treatment or prevention of diseases or conditions. In another aspect the invention is directed to a method of screening for molecules for use in immuno-therapy, wherein the molecules are capable of reducing the cell surface levels of pMHCII, wherein the method comprises: l) contacting a test molecule with one or more cells expressing surface pMHCII, 2) measuring the change in surface levels of pMHCII, 3) selecting molecules capable of reducing cell surface levels of pMHCII. The invention is also directed to inhibitors identified by the above method for use as a medicament.

According to another aspect, the invention is directed to a method of preventing, treating, or improving the efficacy of treatment of a medical condition by reduction of cell surface levels of pMHCII within a subject to be treated by administering to a subject in need thereof, any of a polypeptide, nucleic acid, vector or pharmaceutical composition of the invention. In particular embodiments, specific cells maybe targeted for the reduction in pMHCII levels.

In a particular embodiment, the medical condition to be treated may be an immune mediated disorder. In other embodiments, the method of treatment may be immunotherapy. The method of treatment may be directed to a medical condition selected from the group consisting of: an allergy, an autoimmune disease, graft versus host disease, infertility, or infection. In other embodiments the inventive method may be directed to the improvement of adoptive cell transfer therapy, which may involve the adoptive transfer of leukocytes, lymphocytes, T cells, CAR T cells, TCR transgenic T cells, transgenic T cells, or tumour infiltrating lymphocytes. In other embodiments, the inventive method is for the improvement of nucleic acid vaccination, such as DNA and/ or RNA vaccination. The inventive method may be for the improvement of vaccination with a self-amplifying RNA molecule. The compositions may be delivered, at least in part, by a synthetic lipid nanoparticle delivery system. In an embodiment the vaccination may be for the treatment or prevention of HIV. In an alternative embodiment, the inventive method is for use with a transplant related disorder or for use with gene therapy.

In another aspect, the invention is directed to a kit for use in the prevention, treatment, or amelioration of a medical condition, said kit comprising a polypeptide, nucleic acid, vector, or composition of the invention, and means for delivering said composition. In an aspect, the invention is directed to a method for improving the suitability of cells for adoptive cell transfer therapy, whereby a composition comprising any of i) a polypeptide of the invention, ii) a nucleic acid of the invention, iii) a vector of the invention, iv) a cell of the invention, or v) a pharmaceutical composition of the invention, is provided to the cells. In an embodiment, the cells to be adoptively transferred are CAR T cells.

Brief Description of Drawings

Figure 1 illustrates the results of experiments illustrating that Salmonella SPI-2 effector SteD is required and sufficient to reduce surface levels of peptide-loaded MHCII (pMHCII) molecules.

Figure 2 illustrates the results of experiments illustrating that SteD is an integral membrane protein that localizes to the Golgi network after translocation from Salmonella.

Figure 3 illustrates the results of experiments illustrating that SteD increases internalization and ubiquitination of pMHCII.

Figure 4 illustrates the results of experiments showing interactions between SteD, MARCH8 and pMHCII.

Figure 5 illustrates the results of experiments illustrating that reduction of pMHCII surface levels by SteD suppresses T cell proliferation.

Figure 6 illustrates the results of experiments illustrating comparative genomic analysis, showing SteD homologues in several Salmonella serovars.

Figure 7 illustrates the results of experiments identifying regions within SteD that are important for functional activity.

Detailed Description

As described in detail in the Examples, the inventors have now demonstrated that the SaZmoneZZa-derived protein, SteD, reduces cell surface levels of pMHCII. The inventors have shown herein the association of SteD with at least the endogenous ubiquitin ligase MARCH8 and pMHCII, and demonstrate that SteD increases the ubiquitination of residue K225 on DRT3, resulting in the reduction of cell surface levels of pMHCII. The detailed information provided in the Examples also demonstrates that the reduction of pMHCII surface levels has an effect on the immune response both in vitro and in vivo. The sequences SEQ ID NOS: 1 to 29 are sequences of functional SteD polypeptides (Table 1). In another aspect, the invention is directed to a polypeptide exhibiting at least 70% similarity/identity with the sequences of any of SEQ ID NOS: 1 to 29, for use as a medicament. According to another aspect of the invention, there is provided an isolated polypeptide comprising an amino acid sequence at least 70% identical to the sequence according to any one SEQ ID NOS: 1 to 29, for use as a medicament.

Therefore polypeptides of the invention include polypeptides comprising a sequence substantially similar to SteD. In one embodiment, the polypeptide of the invention comprises a sequence that exhibits at least 75%, 80%, 85%, 90%, 95%, or 100% identity/similarity to any of SEQ ID NOS: 1 to 29, for use as a medicament. Two polypeptides are substantially similar when the amino acid sequences are typically at least about 70-75%, more typically at least about 80-85%, and more typically greater than about 90% or more similar or identical.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/polynucleotide/polypeptide sequence", "functional variant" and "functional fragment", can be a sequence that has at least 40% sequence identity with the amino acid/ polynucleotide/ polypeptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleic acids identified herein.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to is also envisaged. Preferably, the amino

acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein. The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW maybe as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalty = 6.66, and Matrix = Identity. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENDGAP = -1, and GAPDIST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino

acid/ polynucleotide/ polypeptide sequences is then calculated from such an alignment as (N/T)*ioo, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating relative percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula: - Sequence Identity = (N/T)*ioo.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to a nucleic acid sequence described herein, or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ o.i% SDS at approximately 20-65°C. Alternatively, a substantially similar polypeptide may differ by at least l, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown herein.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids. Table 1

SEQ ID Description: Represented in:

NO:

1 The SteD sequence of S. Ty phi murium Fig- 2C, 6, and 7A.

2 The SteD sequence of S.Heidelberg Fig- 6

3 The SteD sequence of S.Enteritidis Fig- 6

4 The SteD sequence of S.lnfantis Fig- 6

5 The SteD sequence of S.Dublin Fig- 6

6 The SteD sequence of S. Typhi Fig- 6

7 The SteD sequence of S.Paratyphi A Fig- 6

8 The SteD sequence of S.Paratyphi B Fig- 6

9 The SteD sequence of S.Paratyphi C Fig- 6

10 SteD sequence including all natural variations. Fig- 6

11 SteD sequence specifying only the regions that are Fig- 6 with Fig- 7 important for function.

12 SteD sequence not specifying non-essential region 1. Fig- 6 with Fig- 7

13 SteD sequence not specifying non-essential region 2. Fig- 6 with Fig- 7

14 SteD sequence not specifying non-essential region 3. Fig- 6 with Fig- 7

15 SteD sequence not specifying non-essential region 4. Fig- 6 with Fig- 7

16 SteD sequence not specifying non-essential region 5. Fig- 6 with Fig- 7

17 SteD sequence not specifying non-essential region 6. Fig- 6 with Fig- 7

18 SteD sequence not specifying non-essential region 7. Fig- 6 with Fig- 7

19 SteD sequence not specifying non-essential region 8. Fig- 6 with Fig- 7

20 SteD sequence not specifying non-essential region 10. Fig- 6 with Fig- 7

21 SteD sequence not specifying non-essential region 11. Fig- 6 with Fig- 7

22 SteD sequence not specifying non-essential region 12. Fig- 6 with Fig- 7

23 SteD sequence not specifying non-essential region 14. Fig- 6 with Fig- 7

24 SteD sequence not specifying non-essential region 15. Fig- 6 with Fig- 7

25 SteD sequence not specifying non-essential region 16. Fig- 6 with Fig- 7

26 SteD sequence not specifying non-essential region 17. Fig- 6 with Fig- 7

27 SteD sequence not specifying non-essential region 18. Fig- 6 with Fig- 7

28 SteD sequence not specifying non-essential region 19. Fig- 6 with Fig- 7

29 SteD sequence not specifying non-essential region 20. Fig- 6 with Fig- 7

The invention also encompasses polypeptides having a lower degree of identity but having sufficient similarity to polypeptides of the invention so as to retain some functional activity. Functional activity is the ability to reduce the cell surface levels of pMHCII. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute a given amino acid in a polypeptide by another amino acid of like characteristics. Conserved substitutions are likely to be phenotypically silent. Functional variants of SteD can include one or more conserved amino acid substitutions.

The invention also encompasses polypeptides of the invention with one or more non- conserved amino acid substitutions. Figs. 7A and 7B indicate regions of SteD where non-conserved substitutions do not affect function. As defined by Fig. 7B non- conserved substitutions are less likely to affect function in regions 1, 2, 3, 4, 8, 17, and 18. Non-conserved substitutions can also be tolerated without complete loss of function in regions 5, 6, 7, 10, 11, 12, 14, 15, 16, 19, 20. Polypeptides of the invention also include sequences where any one of these regions is replaced with any amino acid sequence. Polypeptides of the invention also include polypeptides where all of the nonessential regions have been replaced by any amino acid sequence. The inventors have identified natural variations of the SteD sequence in different Salmonella serovars (Fig. 6). Polypeptides of the invention can also contain any combination of these natural variations.

Polypeptides of the invention include functional fragments of polypeptides of the invention that retain some functional activity. Functional activity in this context can be the ability to reduce the surface pMHCII levels of a cell.

Polypeptides of the invention are said to be "isolated" when they are no longer in association with the organism Salmonella. A polypeptide can still be "isolated" and joined to another polypeptide with which it is not normally associated. Polypeptides of the invention can also be isolated and covalently bound, non-covalently bound, or in admixture with other molecules and substances.

The mechanistic data provided in the present application indicate that SteD is applicable to reducing the pMHCII surface levels of both MHCII expressing cells and those which have acquired pMHCII via trogocytosis or similar methods of acquisition.

Alteration of pMHCII levels advantageously allows immune system function to be directly manipulated. As the process of antigen presentation through MHCII is so fundamental to immune activation, alteration of pMHCII levels, either byway of reduction or increase, may have therapeutic effects for many immune mediated diseases. For instance, autoimmune diseases such as type 1 diabetes, multiple sclerosis, autoimmune thyroiditis, systemic lupus erythematosus, Crohn's disease, Celiac disease, lupus, rheumatoid arthritis, Sjogren's syndrome, or psoriasis may benefit from therapeutic reduction in pMHCII levels either systemically or on specific target cells. Other immune mediated disorders which may benefit from the therapeutic reduction in pMHCII levels on all or specific cells include allergies, graft versus host disease, and immune-mediated infertility. Infection can result in immunopathology due to an excessive or prolonged immune response; the present invention may have therapeutic benefits to treatment of such infections. pMHCII is also important for stimulating the suppressive side of the immune system, for instance by the activation of regulatory T cells. The compositions of the invention can therefore be useful for reducing the activity of the suppressive side of the immune system.

There is provided herein a method of treating immune mediated diseases by the reduction of surface pMHCII upon host cells. In an embodiment the reduction of surface pMHCII is achieved by the provision of SteD. SteD may be provided by the transfer of a protein comprising the SteD sequence, or a functional variant thereof, to a recipient in need thereof for the treatment of disease. Alternatively, SteD may be provided via a nucleic acid comprising a sequence encoding SteD, or a functional variant thereof, which is transferred to a recipient for the treatment of disease. The nucleic acid comprising a sequence encoding a molecule of the invention may be transferred as part of a vector. In some embodiments, the SteD can be targeted towards specific cell types. Systemic reduction of pMHCII surface levels can result in immune suppression. The molecules according to the invention can act as alternatives to existing

immunosuppressant drugs, and will be applicable to the same uses.

The present invention is also directly applicable to adoptive cell transfer therapy.

Current issues arising with the use of adoptive transfer therapy include the need to maximise the effector functions of the transferred cells, to maximise the duration of the transferred cells' effector functions, and to minimise the removal of the transferred cells by the host immune system. T cells are one of the major cell populations that are adoptively transferred for therapeutic purposes. Activated human T cells are known to express MHCII and to acquire the molecule from other sources by processes such as trogocytosis. The presence of pMHCII on T cells has been associated with a reduced immune response. In addition, MHCII expressing cells have been shown to be subject to direct killing. The invention therefore provides a method of improving the efficacy of adoptive cell transfer therapy by reducing the pMHCII levels on the transferred cells. In one embodiment this may be achieved by the provision of SteD to the cells to be adoptively transferred. This can be achieved either by the delivery of the SteD polypeptide, or by a vector or expression system incorporating the nucleic acid sequence encoding the SteD polypeptide of the invention.

T cells genetically engineered to express a chimeric antigen receptor (CAR) have been shown to be an effective cell type for use in adoptive cell transfer therapy. Other cell types shown to be effective include: T cells expressing a transgenic TCR, T cells genetically engineered to express effector molecules (for instance IL-12), and tumour infiltrating lymphocytes (TILs). In another embodiment the surface levels of pMHCII are reduced on the cells to be transferred, such as CAR T cells, to improve the efficacy of therapy. One method for achieving the reduction of pMHCII levels may be the provision of a protein comprising the SteD sequence, or a functional variant thereof, to the cells to be transferred. Alternatively, the pMHCII levels may be reduced by the provision of a nucleic acid comprising a sequence encoding SteD or a functional variant under the control of an appropriate (human or other mammalian) promoter sequence, including a moloney murine leukemia retrovirus promoter. The inventive nucleic acid may be provided by transfecting the cells for transfer or alternatively by transduction of the cells for transfer. SteD may be introduced in the same vector as any other potential transgenic constructs, such as the CAR construct. Alternatively, SteD may be introduced in a second vector alongside any potential transgenic constructs or as an additional step to the introduction of the transgenic constructs. For cells to be transferred that are not otherwise genetically engineered, such as TILs, the SteD can be provided during the in vitro expansion and/ or activation of the TILs, or as an additional step. Nucleic acid vaccination is a technique whereby a nucleic acid encoding a target antigen is introduced into a host (alone or delivered by a vector) for the induction of an immune response. The efficacy of nucleic acid vaccination or transgene expression can be reduced by the precocious removal of inoculated host cells. The efficacy of nucleic acid vaccination can also be reduced by the direct presentation of antigen by MHCII expressing non-professional APCs. The present invention provides an improved method of nucleic acid vaccination whereby surface pMHCII is reduced on inoculated host cells, and inventive molecules for use in said method. In an embodiment, surface pMHCII is reduced by the provision of a SteD polypeptide of the invention.

Alternatively, SteD maybe provided by inclusion of a nucleic acid comprising a sequence encoding SteD, or functional variant thereof, under the control of an appropriate (human or other mammalian) promoter sequence, such as a moloney murine leukemia retrovirus promoter, into an expression vector for delivery of the target antigen. The invention is suitable for combination with all types of nucleic acid vaccination and vectors, for instance viral delivery, bacterial vector delivery, nonviral delivery, plasmid DNA vaccines, mRNA vaccines, self-amplifying RNA vaccines, or self- amplifying RNA vaccines based on an alphavirus genome (as disclosed in Geall et al. PNAS Sept. 4,. 2012, Vol 109, No. 36 P 14604-14609; herein incorporated by reference). In a particular embodiment the nucleic vaccination is for the treatment or prevention of HIV. The nucleic acid vaccine may be delivered by any method known in the art. The SteD molecule, or molecule encoding SteD, may be delivered by any method known in the art, including co-delivery with the nucleic acid vaccine. For instance, the SteD molecule may be encoded by the same nucleic acid that encodes the vaccine. Alternatively, the SteD molecule, or molecule encoding SteD, may be separate from the vaccine but co- delivered. The SteD molecule, or molecule encoding SteD, may be associated with, or bound to, the nucleic acid vaccine. Alternatively, the SteD molecule, or molecule encoding SteD, may be associated with, or bound to, the same delivery particle as the nucleic acid vaccine, for instance the delivery particle may be a synthetic lipid nanoparticle or a liposome. Alternatively, delivery of the two components may be entirely separate. The separate delivery may be via the same methods, or each component may be delivered by a different method.

Methods of delivery include injection of naked nucleic acid, delivery via a synthetic lipid nanoparticle, delivery via a liposome, delivery via cationic polymers, delivery via a viral vector, or delivery via a viral replicon particle. Carriers may be any molecule that aids the delivery of the antigen, and may be an ISCOMS, a nanoparticle, an emulsion, a microparticle, a virosome, a micellar delivery system, a denrimer delivery system, a plant vaccine, a melt-in-mouth strip (under the tongue) or an immunostimulatory adjuvant (such as cholera toxin, chitosan). A natural carrier may be a virus-like particle (VLP), a bacterial 'ghosts' bacterium, a bacterial cell or a bacterial spore. In a particular embodiment, the molecules of the invention are used in conjunction with a self-amplifying RNA vaccine delivered via a lipid nanoparticle delivery system.

The vaccine may be used prophylactically or therapeutically. The vaccines of the invention may comprise an adjuvant, which triggers an immune response in a subject to be vaccinated.

The vaccine may comprise an excipient, which may act as an adjuvant. Thus, in another embodiment, the antigenic peptide in the vaccine may be combined with a

microparticulate adjuvant, for example liposomes, or an immune stimulating complex (ISCOMS), virus-like particles or nanoparticles, an emulsion, a microparticle, a virosome, a micellar delivery system, a dendrimer delivery system, a plant vaccine, a melt-in-mouth strip (under the tongue) or an immunostimulatory adjuvant (such as cholera toxin, chitosan).

Gene therapy involves the replacement or removal of an endogenous gene, or the introduction of a new transgene into a host for a therapeutic benefit. The present invention provides molecules and methods for the improvement of gene therapy, specifically for the reduction of an immune response towards the genetically modified cells. Polypeptides of the invention can be provided to the host to improve a gene therapy treatment. Alternatively, a nucleic acid encoding a polypeptide of the invention can be provided to the host to improve the treatment. The nucleic acid encoding a polypeptide of the invention can be incorporated into the gene therapy vector itself, or be provided as part of a separate vector.

Transplantation carries risk of rejection of the transplanted tissue or tissues. The invention is applicable to all grafted tissues, for instance whole organs, tissues, or cellular grafts such as bone marrow. The present invention provides a method of reducing the risk of graft rejection by reduction of pMHCII levels on the cells of a transplant recipient. Alternatively, there is provided a method of reducing the risk of graft rejection by reduction of pMHCII levels on cells of the grafted tissue. In an embodiment the pMHCII levels are reduced by provision of an SteD polypeptide of the present invention. SteD may be provided as a protein comprising the SteD sequence or functional variant thereof. The SteD may also be provided as a nucleic acid comprising a sequence encoding SteD, or functional variant thereof, with either of these under the control of an appropriate (human or other mammalian) promoter sequence, including a moloney murine leukemia retrovirus promoter and delivered to the tissues in question by techniques known to those skilled in the art.

The polypeptides, nucleic acid molecules, vectors, cells, or compositions of the invention may be administered to the subject to be treated on their own, as a monotherapy, or as an adjunct to, or in combination with, other active ingredients and known therapies for treating, ameliorating, or preventing disease. Alternatively, the polypeptides, nucleic acid molecules, vectors, cells, or compositions of the invention may be administered to other cells prior to their use in adoptive cell transfer therapy. They may be administered in a pharmaceutically acceptable vehicle. The molecules according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension, cells to be adoptively transferred, or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given. In another preferred embodiment, therapy may comprise the combination of the polypeptides, nucleic acid molecules, vectors, cells, or compositions of the invention with an extracellular matrix degrading agent, such as enzyme or losartan. Extracellular matrix degrading agents should enhance diffusion in the subject being treated, and especially within tissues such as solid tumour.

Medicaments comprising the polypeptide or nucleic acid molecules of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the molecules may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may be administered by inhalation (e.g.

intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.

The molecules and compositions according to the invention may also be incorporated within a slow- or delayed release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

Molecules, compositions, and medicaments according to the invention maybe administered to a subject by injection into the blood stream or directly into a site requiring treatment. Injections maybe intravenous (bolus or infusion) or

subcutaneous (bolus or infusion), intradermal (bolus or infusion), or enhanced by convection (convection enhanced delivery - relevant to local injections at disease site).

It will be appreciated that the amount of the molecules, agent or medicament that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, vaccine and medicament, and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half- life of the agent within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease, disorder or condition. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10 mg/kg of body weight of agent or medicament according to the invention may be used for treating a disease, disorder or condition, depending upon which agent or medicament is used. More preferably, the daily dose is between 0.01 μg/kg of body weight and 1 mg/kg of body weight, more preferably between 0.1 μg/kg and 100 μg/kg body weight, and most preferably between approximately 0.1 μg/kg and 10 μg/kg body weight.

The molecule, agent or medicament may be administered before, during or after onset of the disease, disorder or condition. Daily doses may be given as a single

administration (e.g. a single daily injection). Alternatively, the molecule, agent or medicament may require administration twice or more times during a day. As an example, agents and medicaments may be administered as two (or more depending upon the severity of the disease, disorder or condition being treated) daily doses of between 0.07 μg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of agents, vaccines and

medicaments according to the invention to a patient without the need to administer repeated doses. Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents and medicaments according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).

The pharmaceutical compositions of the invention may be made, in a further aspect, using a process comprising contacting a therapeutically effective amount of the polypeptides, nucleic acid molecules, vectors, cells, or compositions of the invention, and a pharmaceutically acceptable vehicle.

A "subject" may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a cow), pets, or may be used in other veterinary applications. Most preferably, the subject is a human being.

A "therapeutically effective amount" of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the disease, disorder or condition, or produce the desired effect.

A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions. In an embodiment of the invention, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrating agents. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like. However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active agent according to the invention maybe dissolved or suspended in a

pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers,

preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g.

glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of

administration using sterile water, saline, or other appropriate sterile injectable medium. The molecules, agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

"Pharmaceutically acceptable excipient" means, for example, an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. A person of ordinary skill in the art would be able to determine the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention. Excipients include carriers, diluents, preservatives, colouring agents, and fillers.

It will be appreciated that administration, into a subject to be treated, of a molecule, agent or medicament according to the invention will result in the reduction of cell surface levels of pMHCII, and that this reduction will aid in treating or preventing a disorder, or improving the efficacy of another treatment.

"Immuno-therapy" means the prevention or treatment of a disorder using substances that affect or involve the immune system.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying diagrammatic drawings, in which:

Figure 1 is an illustration showing that Salmonella SPI-2 effector SteD reduces surface levels of peptide-loaded MHCII (pMHCII) molecules. (A) Mel JuSo cells were infected with wild-type or mutant Salmonella strains for 16 h and surface levels of pMHCII were measured by flow cytometry using mAb L243 (that specifically recognises mature peptide-loaded HLA-DR). Error bars represent Standard Deviation (SD) of the geometric mean fluorescence of two independent experiments done in duplicate. (B) Representative FACS plots showing surface levels of pMHCII in infected cells (R2) compared to uninfected cells (Ri). Histograms show surface levels of pMHCII in infected (R2, blue) and uninfected (Ri, dark grey) cells. Cells labelled with isotype control antibody are shown in light grey. (C) Mel JuSo cells were infected with AsteD strain carrying PWSK29, expressing SteD-2HA regulated by its endogenous promoter (psteD). Infected and uninfected cells were discriminated using anti-Salmonella CSA-i antibody after fixation. Error bars represent SD of the geometric mean fluorescence of three experiments done in duplicate. (D) Mel JuSo cells were transfected with vectors encoding GFP-tagged effectors and pMHCII was analysed by flow cytometry. Error bars represent SD of the geometric mean fluorescence of three experiments done in duplicate. **P< 0.01, *P< 0.05 (Student's t test). Figure 2 illustrates that SteD is an integral membrane protein that localizes to the Golgi network after translocation. (A) Immunofluorescence of Mel JuSo cells at 20 h post- invasion with AsteD psteD or post-transfection with M4P plasmid expressing GFP- SteD. Effector is shown in green (anti-HA or GFP) and GM130 Golgi marker is in red. (B) Infected cells at 20 h post-invasion were incubated with Brefeldin A (BFA) for 90 min. Then, BFA was washed out and cells were incubated for another 90 min. Cells were fixed at different times and analysed by immunofluorescence microscopy with anti-HA, anti-TGN46 and anti-GMi30 antibodies. SteD localizes to the intact Golgi network before treatment with BFA and after wash out. Quantification of the percentage of infected cells with a compact, perinuclear accumulation of SteD-2HA and TGN46 in different BFA treatments is shown on the right. (C) Amino acid sequence of SteD showing transmembrane domains (TMD) predicted by TMHMM 2.0 software shaded in grey. (D) Membrane fractionation of Mel JuSo cells infected for 20 h with AsteD psteD-2HA. Soluble proteins were separated from total membrane proteins, which were later treated with 2.5 M urea to discriminate between integral membrane and membrane-associated proteins by ultracentrifugation. Calreticulin and Golgin 97 are membrane-associated proteins and TGN46 is an integral Golgi membrane protein. (E) Mel JuSo cells were infected with AsteD psteD-2HA or transfected with vector encoding SteD fused at its N-terminus to Flag epitope (Flag-SteD). Cells were semi- or completely permeabilized with Digitonin or Triton X-100 to discriminate between cytoplasmic and Golgi luminal antigens, respectively. Antibodies recognizing the luminal portion of TGN46 or cytoplasmic GM130 were used as controls. (F) Schematic representation of SteD topology in the membrane. Scale bars 5 μπι.

Figure 3 is an illustration indicating that SteD increases the internalization and ubiquitination of pMHCII. (A) Flow cytometry analysis showing the internalization rate of pMHCII in Mel JuSo cells infected with WT-GFP or AsteD-GFP strains compared to uninfected cells. Cells were infected for 16 h then labelled with mAb L243 on ice to block internalization. Cells were then exposed to 37 °C to enable resumption of internalization. Surface levels of pMHCII at various time points were normalized to o min, which represents 100%. (B) Cells at 16 h post-invasion were labelled with mAb L243 on ice and incubated for another 5 h in complete medium at 37 °C. Cells were fixed and processed for immunofluorescence microscopy with anti-Salmonella CSA-i (green) and anti-mouse secondary antibody against L243 (red). Images are maximum intensity Z projections showing difference in pMHCII surface levels between cells infected with wild-type or AsteD psteD and its intracellular accumulation (arrows). Scale bar 5 μπι. (C) Mel JuSo cells were infected at an MOI of 300:1 and at 16 h post- invasion, lysed for immunoprecipitation with mAb L243 followed by western blot analysis with anti-ubiquitin (P4D1-HRP), anti-DRa and anti-β tubulin. (D) Stable Mel JuSo cell lines expressing GFP or GFP-SteD were lysed and mAb L243 or IgG2a isotype control were used for immunoprecipitation. Immunoprecipitates were analysed by western blot with the same antibodies as in (C). (E) HA-tagged DR constructs (either wild-type [HA-DR ] or with an arginine substitution of lysine 225 on DR cytoplasmic tail [HA-DR K225R]) were used to transduce Mel JuSo cells or stable cells expressing GFP-SteD. Cells were lysed and immunoprecipitated with anti-HA antibody coupled to agarose beads followed by western blot analysis with anti-HA, anti-ubiquitin (P4D1- HRP) and anti-β tubulin antibodies. (F) Surface levels of pMHCII on stable cells described in (E), measured by mAb L243 labelling and flow cytometry. (G)

Representative histograms showing pMHCII surface levels of cells described in (E) and (F). **P< 0.01 (Student's t test).

Figure 4 shows interactions between SteD, MARCH8 and pMHCII. (A) Quantitative real-time PCR analysis showing mRNA levels of MARCH 8 and MARCH9 in Mel JuSo cells after exposure to a scramble siRNA oligo, two oligos specific to MARCH8, or one oligo specific to MARCH9. (B) Flow cytometry analysis of Mel JuSo cells that were treated with siRNA prior to infection with WT-GFP Salmonella or AsteD-GFP

Salmonella. (C) HEK293T cells transfected with vectors expressing MARCH8-FLAG and GFP-SteD or GFP alone were used for immunoprecipitation with GFP-Trap beads. Immunoprecipitates were analysed by western blot using anti-GFP and anti-FLAG antibodies. (D) Stable Mel JuSo cell lines expressing both MARCH8-FLAG and GFP- SteD or MARCH8-FLAG and an unrelated GFP-tagged bacterial effector were used for immunoprecipitation with mAb L243 (for pMHCII) or isotype control.

Immunoprecipitates were analysed by western blot using anti-GFP, anti-FLAG and anti-DRa antibodies. (E) The stable Mel JuSo cells expressing GFP-SteD and

MARCH8-FLAG were analysed by immunofluorescence with anti-FLAG (red) and mAb L243 against pMHCII (grey). (F) Pearson's correlation coefficient was calculated between pMHCII and SteD or GFP, and between MARCH8 and SteD or GFP. **P< 0.001, *P< 0.02 (Student's t test). Scale bar 5 μπι.

Figure 5 illustrates that reduction of pMHCII surface levels by SteD suppresses T cell proliferation. (A) Mouse bone marrow-derived dendritic cells (BMDCs) were infected with the indicated GFP-expressing bacterial strains and total MHCII surface levels (I- A/I-E haplotypes) were quantified by flow cytometry at 20 h post-uptake. (B) Infected BMDCs were incubated with OVA peptide and co-cultured with T cells labelled with CFSE for three days. T cell proliferation was analysed by flow cytometry after labelling cells with anti-CD4 antibody and using calibration particles for normalization.

Uninfected BMDCs incubated or not with OVA-peptide were used as controls. (C)

Representative FACS histograms showing T cell proliferation measured by CFSE levels in different conditions. (D) Dendritic cells from mesenteric lymph nodes of mice infected with WT-GFP or AsteD-GFP Salmonella by oral gavage were isolated at 48 h post-inoculation and total MHCII surface levels were measured by flow cytometry. (E) Activated T cells (CD25+CD44+ and CD62L-CD44+) as a percentage of total CD4+ T cells isolated from the spleens of mice infected by Salmonella WT or AsteD strains at day 17 post-inoculation. **P< 0.002, *P<0.05 (Student's t test) for (A) and (D) and ***P< 0.0001 (One-way AN OVA) for (B). Data were analysed by two-tailed pair-wise t test using pairs of spleens carrying similar bacterial burdens for (E). ****p< 0.0001, **P<0.01.

Figure 6 is a comparative genomic analysis, showing SteD homologues in several Salmonella serovars. The sequences indicated within this figure represent polypeptides of the invention. Figure 7 identifies regions within SteD important for functional activity. (A) Amino acid sequence of SteD showing transmembrane domains (TMD) predicted by TMHMM 2.0 software shaded in grey, and the locations of the regions (1) to (20). (B) A series of twenty small alanine substitutions (SteD^SteD ²⁰ ) in blocks of five/six amino acids as indicated in (A) were generated. Cells were infected with wild-type or Salmonella mutant strains and surface levels of pMHCII were measured by flow cytometry. (C) and (D) Two constructs were unstable and did not localise to the Golgi network. Apart from SteD^ and SteD^, the sequences represented by this figure are functional SteD polypeptides of the invention.

Examples

The materials and methods employed in the studies described in the Examples were as follows, unless otherwise indicated: Bacterial strains, plasmids and antibodies

All bacterial strains, plasmids and antibodies used in this study are described in Table 2. Strains used were S. enterica serovar Typhimurium NCTC 12023 and its isogenic mutants, which were constructed using a one-step λ Red recombinase chromosomal inactivation system (Datsenko and Wanner, PNAS (2000) 97: 6640-6645). Wild-type and mutant strains were transformed with PFPV25.1 and pFCcGi plasmids for GFP and mCherry expression, respectively, whenever required.

Table 2

S. Typhimurium 12023 strains

Name Description Reference

12023 S. Typhimurium wild- wild-type NTCC

type

AssaV AssaV::km Beuzon et al., 1999

AsseL AsseL::km Mesquita et al., 2012

AslrP AslrP::km Andreas Baumler

AsspHl AsspHl::km Figueira et al., 2013

AsspH2 AsspH2::km Figueira et al., 2013

AgogB AgogB::km Figueira et al., 2013

AsifA AsifA::km Beuzon et al., 2002

AsifA/AsopD2 AsifA::km/AsopD2 Stephane Meresse

AsifB AsifB::km Figueira et al., 2013

AsseJ AsseJ::cm Lossi et al., 2008

AsseF AsseF::km Brumell et al., 2003

AsseG AsseG::km Brumell et al., 2003

AsopD2 AsopD2::cm Figueira et al., 2013

ΔρίρΒ ApipB::km Figueira et al., 2013

ΔρίρΒ2 ApipB2::km Figueira et al., 2013

AsopD AsopD::km Figueira et al., 2013

AsrfJ AsrfJ::km Figueira et al., 2013

AsrfH AsrfH::km Figueira et al., 2013

AsptP AsptP::km Figueira et al., 2013

AsteA AsteA::km Figueira et al., 2013

AsteB AsteB::km Figueira et al., 2013

AsteC AsteC::km Mesquita et al., 2012

AsteD AsteD::km This study

AsteE AsteE::km This study

AspvB AspvB::km Figueira et al., 2013

AspvC AspvC Figueira et al., 2013

AspvD AspvD::km Figueira et al., 2013

AsseKl AsseKl::km Figueira et al., 2013

AsseK2 AsseK2::km Figueira et al., 2013

AsseK3 AsseK3::km Figueira et al., 2013

AsseKl/AsseK2/AsseK3 AsseKl/AsseK2/AsseK3::km Figueira et al., 2013

AcigR AcigR::km Figueira et al., 2013

AgtgA AgtgA::km Figueira et al., 2013

AgtgE AgtgE::km Figueira et al., 2013

AavrA AavrA::km Figueira et al., 2013

Plasmids

Name Description Reference PCR template plasmid with Km

pKD4 Datsenko et al., 2000 resistance cassette (KmR)

Plasmid encoding arabinose-inducible

pKD46 Datsenko et al., 2000 λ-Red recombinase system (CarbR)

Plasmid encoding arabinose-inducible

pCP20 Datsenko et al., 2000

FLP recombinase (CarbR)

rpsM::mCherry and PBAD::gfpmut3a

pFCcGi Figueira et al., 2013 promoter fusions in pFPV25.1 (CarbR) rpsM::gfpmut3a promoter fusion in

pFPV25.1 Valdivia et al., 1996 pFPV25

Retroviral helper plasmid encoding Randow and Sale, pMD.GAGPOL

gag/pol proteins 2006

Retroviral helper plasmid encoding Randow and Sale, pMD.VSVG

vesicular stomatitis virus glycoproteins 2006

pWSK29 containing C-terminus 2HA- psteD tagged steD promoter and open This study

reading frame (CarbR)

M5P retroviral vector containing GFP

GFP Teresa Thurston open reading frame (CarbR/PuroR)

M5P retroviral vector containing N-

GFP-SteD terminus GFP-tagged steD open reading This study

frame (CarbR)

M5P retroviral vector containing N-

McGourty et al., GFP-SifA terminus GFP-tagged sifA open reading

2013

frame (CarbR)

M5P retroviral vector containing N- GFP-SseF terminus GFP-tagged sseF open reading Teresa Thurston frame (CarbR)

M5P retroviral vector containing N- GFP-SseG terminus GFP-tagged sseG open reading Teresa Thurston frame (CarbR)

M5P retroviral vector containing N-

HA-D terminus HA-tagged HLA-DR beta chain This study

(CarbR/PuroR)

M5P retroviral vector containing N-

HA-DR -K225R terminus HA-tagged HLA-DR beta chain This study

with K225 mutated (CarbR/PuroR)

M5P retroviral vector containing C-

MARCH8-FLAG terminus FLAG-tagged MARCH8 This study

(CarbR/PuroR)

Antibodies

Specificity Clones Use Source

HLA-DR L243 FACS/IP Sigma-Aldrich HLA-D a TAL.1B5 WB Dako

HLA-DR DA2 WB Abeam

HA 3F10 IF Roche

FLAG M2 IF Sigma-Aldrich

TGN46 Rabbit Polyclonal IF/WB Lifespan Biosciences

GM 130 35 IF BD Biosciences

Golgin 97 CDF4 WB Invitrogen

Calreticulin Rabbit Polyclonal WB Thermo Scientific

CSA-1 Goat polyclonal IF KPL

Ubiquitin-HRP P4D1 WB Santa Cruz

Tubulin β Rabbit monoclonal WB Abeam

HA Rabbit Polyclonal WB Sigma-Aldrich

FLAG Rabbit Polyclonal WB Sigma-Aldrich

GFP Rabbit monoclonal WB Thermo Scientific

CDllc N418 FACS MiltenyiBiotec l-A/l-E M5/114.15.2 FACS Thermo Scientific

WB: western blot; FACS: flow cytometer; IP: immunoprecipitation; IF: immunofluorescence.

Cell culture and infection

Mel JuSo and HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS) at 37 °C in 5% CO2. L243-producing hybridoma cells were maintained in DMEM containing 10% FCS and reduced to 5% FCS when cell density was high to harvest supernatant containing antibodies. Primary bone marrow-derived dendritic cells (BMDCs) were extracted from C57BL/6 mice (Charles River). Cells recovered from tibias and femurs were grown at 37 °C in 5% CO2 in RPMI-1640 supplemented with 10% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 0.05 M β-mercaptoethanol and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) (Peprotech). After 3 days of culture, fresh complete medium was added to the growing cells. On day 6, medium was replaced by fresh complete medium and cells were harvested on day 8 and seeded 6 h prior to infection. Bacteria were grown in Luria Bertani (LB) medium with shaking at 37 °C, supplemented with carbenicillin or kanamycin as required (50 g/ mL). Mel JuSo cells were infected for 30 min at MOI of 100:1 with late log-phase Salmonella. BMDCs were infected for 30 min at MOI 10:1 with stationary-phase Salmonella opsonized in 20% mouse serum. Cells were washed with PBS twice and incubated in fresh medium containing gentamicin (100 g/ mL) for 1 h to kill extracellular bacteria. After 1-2 h the antibiotic concentration was reduced to 20 g/ mL and the cells were processed 16-20 h post infection. siRNA and DNA transfection and retroviral transduction

Mel JuSo cells were seeded in 6-well plates and transfected on the following day with siRNA oligonucleotides using RNAiMAXTM (Life technologies) according to the manufacturer's instructions with a final concentration of 50 nM. Cells were reseeded 24 h after the first transfection and 24 h later a second round of transfection was done. Cells were infected 24 h after the second transfection and analysed 16-20 h post- invasion. siRNA oligonucleotides for MARCH8 (5'-TCCAGCGGGATTGACACTCAA SEQ ID NO: 30, CTGCTAGAGTCTACAGAAGTA-3' SEQ ID NO: 31) and MARCH9 (5'- CAGGTTGGATGCCGTTGCAGA SEQ ID NO: 32, CAGCACTCCGAGGTATCTAAA-3' SEQ ID NO: 33) were from Qiagen. A scrambled sequence (5'-

AAACTTGTCGACGAGAAGCAA-3' SEQ ID NO: 34) was included in all experiments as a negative control. Knockdown of MARCH8 and MARCH9 mRNA was assessed by Q- PCR using SYBER green and conditions described previously (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). DNA transfection procedures were carried out using Lipofectamine™ 2000 according to the manufacturer's protocol (Life technologies). Samples were prepared for analysis 20 h after transfection. For retrovirus production, HEK293T cells were transfected with proviral plasmid M5P together with helper plasmids using Lipofectamine™ 2000 as described previously (Randow and Sale, Subcellular Biochemistry Vol 40, 383-386, 2006). Mel JuSo cells were transduced with virus in culture supernatant containing 8 μg/ml polybrene (Sigma-Aldrich) by centrifugation at 650 g for 1 h at 37 °C. Transduced cells were selected in 2 μg/ml puromycin or GFP-positive cells were sorted by FACS if required.

Flow cytometry

To measure surface levels of pMHCII, Mel JuSo cells that had been either transfected or infected with Salmonella, were collected using Cell Dissociation Buffer (Sigma- Aldrich) and incubated first with mAb L243 and then anti-mouse secondary antibody diluted in FACS buffer (5% FCS, 2 mM EDTA in PBS) at 4 °C for 30 min. After washings with cold PBS, cells were fixed in 3% paraformaldehyde and analysed using a FACS Calibur™ or Fortessa™ flow cytometers (BD Biosciences) and FlowJo™ vio software. MHCII surface levels were calculated as geometric mean fluorescence of infected cells (GFP-positive)/geometric mean fluorescence of uninfected cells (GFP- negative) ^χ ιοο. For complementation analysis of bacterial mutants, Mel JuSo cells were infected with non-fluorescent bacterial strains and, after labelling of surface pMHCII, cells were fixed and incubated with anti-Salmonella CSA-i antibody diluted in 10% FCS and 0.1% saponin in PBS for 1 h at room temperature, followed by anti-goat secondary antibody labelling under the same conditions. For the internalization assay, Mel JuSo cells were infected with wild-type-GFP or AsteD-GFP Salmonella strains and harvested 16 h post-invasion. The surface pMHCII molecules were labelled with mAb L243 for 30 min at 4 °C. Cells were washed in cold medium, resuspended and split into 1.5 mL tubes containing pre-warmed medium in a water bath at 37 °C. Aliquots were removed at various time points, diluted in ice-cold FACS buffer and kept on ice. At the last time point, cells were centrifuged at 4 °C and resuspended in FACS buffer containing Alexa 647-conjugated goat anti-mouse. After 30 min at 4 °C, cells were washed, fixed in 3% paraformaldehyde, washed and analysed by FACS to quantify PMHCII/L243 complexes remaining at the cell surface. GFP-positive cells were considered infected and GFP-negative uninfected. MHCII surface levels were normalized to those detected at the beginning of the experiment (100%).

Immunofluorescence microscopy

Cells were seeded onto coverslips and infected as described above. For general procedures, cells were collected at 20 h post-invasion, washed with PBS and fixed in 3% paraformaldehyde in PBS for 10 min at room temperature. Cells were incubated with 50 mM NH4CI for 10 min, washed and labelled with appropriate antibodies diluted in 10% FCS and 0.1% saponin in PBS. Antibodies used for IF can be found in Table 2. Co- localization analysis was performed with Zeiss Zen 710 software, with thresholds set using individual controls lacking each analysed primary antibody. For Brefeldin A (BFA) treatment, at 20 h post-invasion new medium containing 5 g/ ml of BFA (Sigma-Aldrich) was added to infected cells and incubated for 90 min. The washout was done by washing cells 5 times in PBS and incubating them in DMEM 10% for another 90 min prior to final wash and fixation for labelling. For selective permealization, digitonin treatment was done using live cells. At 20 h post-invasion, cells were placed on ice, washed with KHM buffer (110 mM KOAc, 20 mM HEPES, 2 mM MgCl ₂ , pH 7.3) and incubated for 5 min with 40 g/ml digitonin diluted in KHM. Cells were washed and incubated with primary antibodies diluted in 10% FCS in PBS for 30 min on ice. After washes, cells were fixed and incubated with secondary antibodies at room temperature under standard procedures. After fixation, cells were incubated with 0.1% Triton X-100 for 5 min at room temperature prior to labelling for 1 h with primary and then secondary antibodies diluted in 10% FCS and 0.1% saponin in PBS. For visualization of internalized pMHCII complexes, cells were placed on ice and incubated with mAb L243 diluted in DMEM containing 10% FCS for 30 min. Cells were washed, incubated with warm medium and returned to the incubator at 37 °C for another 4-5 h. After incubation, cells were washed, fixed and labelled with secondary antibody diluted in 10% FCS and 0.1% saponin in PBS. Samples were mounted and analysed using a confocal laser-scanning microscope LSM 710 (Zeiss). Membrane fractionation

Approximately 5 x 10 ⁷ Mel JuSo cells were infected as described above. At 20 h post- invasion, cells were collected and lysed by mechanical disruption using a Dounce homogeneizer in 600 μΐ of homogenisation buffer containing 250 mM sucrose and 3 mM imidazole (pH 7.4) and 1 mM PMSF. Cell extracts was centrifuged at 1800 g for 15 min and the post-nuclear supernatant was divided in two 1.5 mL ultracentrifuge tubes (300 ΐ, each). Supernatant was centrifuged at 100,000 g for 1 h at 4 °C, giving rise to a pellet containing total membrane proteins and supernatant containing soluble proteins. The pellet comprising membrane proteins was resuspended in 300 μΐ of 2.5 M urea and incubated for 15 min on ice. The suspension was centrifuged again at 100,000 g for 1 h at 4 °C, resulting in a pellet containing integral membrane proteins and a supernatant carrying membrane associated proteins. The volume of all fractions was made to 300 μΐ with homogenization buffer and proteins were analysed by western blot. Imrminoprecipitation and western blot

Analysis of MHCII ubiquitination was done as described previously with minor modifications (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). Briefly, approximately 1 x 10 ⁷ cells were harvested using Cell Dissociation Buffer (Sigma) and lysed in buffer containing 1% Nonidet P-40, 50 mM Tris-Cl (pH 7.4), 5 mM EDTA, 150 mM NaCl, protease inhibitors (Roche) and 10 mM iodoacetamide (LAA) for 30 min at 4 °C. Lysate was centrifuged at 16,000 g for 15 min and post-nuclear supernatant was incubated with 30 μΐ of slurry solution containing CNBr sepharose- coupled mAb L243 or isotype control for 2 h at 4 °C. Immunoprecipitates were washed with lysis buffer and eluted with 100 mM glycine (pH 3.0). Eluates were precipitated with four volumes of cold acetone, resuspended in SDS-PAGE loading buffer and analysed by western blot. For infected cells, the MOI was increased to 300:1 and infection time to 45 min. For co-immunoprecipitations, cells were lysed with 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5% glycerol, 10 mM LAA and 0.5% Triton X-100 containing protease inhibitors for 15 min at 4 °C. Lysate was centrifuged at 16,000 g for 15 min and post-nuclear supernatant was incubated with GFP-Trap

(ChromoTek) or CNBr sepharose-coupled mAb L243 or isotype control for 2 h at 4 °C. Immunoprecipitates were washed with lysis buffer and eluted with glycine as described above. Protein samples were separated by SDS-PAGE and transferred to Immobilon- P™ membrane (Millipore). Membranes were blocked in 5% skimmed milk and 0.1% Tween-20 in PBS and incubated with primary and secondary antibodies in the same solution, which were detected using ECL Plus™ Western Blotting Detection Reagents (Thermo Fisher).

Live-imaging

For live-cell imaging, Mel JuSo cells stably expressing GFP-SteD were seeded at a density of approximately 5 x icH cells per dish onto 35 mm glass-bottom culture dishes (MatTek). Cells were transfected for 20 h with a vector expressing mCherry-MARCH8 using Lipofectamine 2000™ (Life technologies) following manufacturer's instructions. Before imaging, cells were washed and incubated in imaging medium (Opti-MEM containing 10% FCS, 25 mM HEPES pH 7.2). Culture dishes were sealed with parafilm and analysed using a LSM 710 microscope (Zeiss) at 37 °C. Samples were imaged with a 63 x oil objective.

T cell proliferation assay

BMDCs were prepared from C57BL/6 mice as described above. The CDiic-positive cell population was enriched using MACsorting (Miltenyi Biotec) to a purity of 95%. Cells were infected in 15 ml tubes at an MOI 10:1 for 30 min in RPMI 10% FCS, washed and treated with gentamicin as described above. After 16 h of infection, cells were harvested with cold PBS and incubated with ovalbumin (OVA) peptide

(ISQAVHAAHAEINEAGR) at 5 μΜ in RPMI containing 10% FCS for 1 h. Cells were washed, counted and incubated with T cells in 96-well plates. T cells expressing OVA- specifrc T cell receptor (TCR) were isolated from cell suspensions of spleens and lymph nodes of OT-II mice by magnetic sorting of CD4 cells (Miltenyi Biotec) and labelled with carboxyfluorescein diacetate succinimidyl ester (CFSE) as described previously (Quah and Parish, J Vis Exp (2010)). CD4 T cells were incubated with DCs at a ratio of 3:1 in a final volume of 200 μΐ, of medium containing 20 g/ml gentamicin. Three days later, cells were centrifuged and resuspended in 150 μΐ, of FACS buffer containing anti- CD4 antibody and incubated for 30 min on ice. Cells were washed and resuspended in 150 μΐ, of FACS buffer containing 6 μπι blank calibration particles (BD Biosciences) as an internal control for normalization of T cell numbers. Mouse infection and isolation of DCs

Female C57BL/6 mice, 6 to 8 weeks old, were infected by oral gavage with 1 x 10 ¹⁰ colony forming units of late log-phase GFF-Salmonella in 200 ΐ, of PBS containing 3% NaHC03. Mesenteric lymph nodes were isolated 48 h after inoculation and smashed in a 70 μπι cell strainer. Dendritic cells were purified from single cell suspensions using anti-CD 11c antibody-coupled magnetic beads (Miltenyi Biotec) according to the manufacturer's instructions. Purified DCs were labelled on ice with anti-I-A/I-E antibody (recognizing mouse MHC class II molecules), diluted in FACS buffer for 20 min on ice. Purity was assessed by anti-CDiic antibody labelling. Discrimination between infected and uninfected cells was based on GFP fluorescence and MHCII geometric mean fluorescence calculated as described above.

Mouse infection and analysis of T cell activation

For analysis of T cell activation, mice were inoculated intraperitoneally with 5 x 10 ⁵ CFU of virulence-attenuated S. Typhimurium strain SL3261 or SL3261 AsteD that had been grown in LB broth to late exponential phase. Spleens were harvested 17 days later and homogenized in Hanks balanced salt solution (HBSS) supplemented with 10 mM HEPES and 2% FCS. A portion of spleen homogenate was plated to enumerate bacterial CFU and spleens with similar bacterial loads (Table 3) were analysed further.

Erythrocytes were lysed using ACK buffer (150 mM NH4CI, 10 mM KHCO3, 0.1 mM EDTA). After blocking surface Fc receptors using FcR Blocking Reagent (Miltenyi Biotec), the remaining splenocytes were labelled using anti-CD38, anti-CD4, anti-CD25, anti-CD44, and anti-CD62L antibodies. Activation of gated CD4+CD38+ cells was determined on the basis of surface-localised CD25, CD62L, and CD44.

Table 3. Bacterial load (cf.u) per spleen of infected mice.

Group compared WT AsteD

112000 124000

² 9S000 96250

8? 500 94750

4 86000 84500

5 6S750 70000

6 67300 64550

57000 60450

8 54275 54900

53150 54566

10 44700 45475

44S25 47800

1 39000 36733 Example 1 - Salmonella SPI-2 effector SteD reduces surface levels of pMHCII

In an attempt to identify the Salmonella SPI-2 type III secretion system (T3SS) effector(s) involved in the removal of pMHCII molecules from the surface of infected cells, the inventors used a collection of mutant strains lacking individual SPI-2 T3SS effectors, which carried the pFCcGi plasmid for mCherry expression, to infect Mel JuSo cells to measure their pMHCII surface levels by flow cytometry. From the panel of 32 single mutants, a double and triple mutant tested, all strains reduced pMHCII surface levels to approximately the same level as that caused by the wild-type (WT) strain, with the exception of AssaV, AsifA and AsteD (Fig lA). SsaV is an essential protein component of the SPI-2 secretion apparatus and its absence prevents the bacteria from translocating any SPI-2 T3SS effectors. Vacuoles harbouring AsifA bacteria are unstable and loss of the vacuole integrity is also likely to interfere non-specifically with SPI-2 T3SS effector delivery into host cells. Fig. lB shows representative plots of pMHCII levels in Mel JuSo cells infected with WT-GFP, AssaV-GFV and AsteD-GFF Salmonella. A reduction of pMHCII occurred in WT-infected (R2, GFP positive) compared to uninfected cells (Ri, GFP negative), but no difference was detected in AssaV or AsteD infected cells (R2) compared to uninfected cells in the same sample (Ri). To establish if the lack of effect of AsteD on pMHCII was due to the absence of this specific gene and not to an adventitious mutation or polar effect, the mutant strain was transformed with a low copy number plasmid (PWSK29) encoding SteD-2HA under the control of its endogenous promoter (Fig. iC). This strain (AsteD psteD) removed pMHCII from the cell surface to the level observed for the wild-type strain. Transformation of the wild-type strain with the same plasmid increased the ability of Salmonella to reduce pMHCII surface levels, suggesting that the effector works in a dose-dependent manner (Fig. iC). Furthermore, ectopic expression of GFP-tagged SteD (GFP-SteD) in Mel JuSo cells revealed that the protein is sufficient to reduce pMHCII surface levels and its function does not require the expression of other SPI-2 effectors (Fig. lD). The small increase in pMHCII surface levels observed following infection with the AsifA mutant was not detected when a double mutant AsifA/ AsopD2 was tested (Fig. lA). Together with the lack of effect of GFP-SifA on pMHCII (Fig. lD), this confirms that the effect of the AsifA mutant is most likely indirect, probably resulting from instability of the SaZmoneZZa-containing vacuole (Beuzon et al., EMBO J, 2000, 19, 3235-3249)· Example 2 - SteD is an integral membrane protein that localizes in the Golgi complex and has both N- and C-terminal regions exposed to the host cell cytosol

SteD is a small protein comprising 111 amino acids (~12 kDa) and was previously identified as a putative SPI-2 T3SS effector in a study analyzing the secreted proteome of a AssaL mutant, which constitutively secretes SPI-2 T3SS effectors at pH 5.0. SteD translocation into host cells was confirmed using CyaA reporter fusions (Niemann et al., Infect Immun (2011) 79: 33-43). In comparative genomic analyses, the inventors found the steD gene in the genome of several Salmonella serovars, including S.

Typhimurium, S. Enteritidis, S. Gallinarum, S. Paratyphi and S. Typhi (Fig. 6).

However, no similarity of predicted amino acid sequence to proteins of any other bacterial genera was found by BLAST analysis. To determine the subcellular localization of SteD after bacterial translocation into eukaryotic cells and in cells ectopically expressing this protein, the inventors used AsteDpsteD to infect Mel JuSo cells or transfected the cells with a plasmid encoding GFP-SteD. Immunofluorescence microscopy revealed that the majority of SteD localized in the region of the Golgi network, as shown by colocalization with the cis-Golgi marker GM130 (Fig. 2A). To test if SteD localized to Golgi membranes, cells were exposed to Brefeldin A (BFA) to disassemble the Golgi complex, and the localization of SteD was examined by immunofluorescence together with cis- and irans-Golgi markers GM130 and TGN46, respectively (Fig. 2B). Following the same pattern observed for TGN46 and GM130, SteD dispersed after BFA treatment. Furthermore, SteD relocalized to a reformed Golgi network 90 min after BFA removal (Fig. 2B). In addition to its Golgi localization, SteD was also detected in tubules and vesicular structures in both infected and transfected cells (Fig. 2A). Live-cell imaging analysis of Mel JuSo cells expressing GFP-SteD revealed that these vesicles were highly dynamic, moving in anterograde and retrograde directions between the irans-Golgi network and other intracellular regions and the cell surface. According to in silico analysis by TMHMM v2.o software, SteD is predicted to be a membrane protein having two transmembrane domains (Fig. 2C). To test these predictions experimentally, Mel JuSo cells were infected with AsteDpsteD Salmonella and subjected to membrane fractionation. Total membrane proteins were first separated from soluble proteins by ultracentrifugation and then total membrane proteins were treated with 2.5 M urea to extract membrane-associated proteins from integral membrane proteins (Fig. 2D). Using calreticulin (CALR) and Golgin 97 as examples of membrane-associated proteins and TGN46 as an integral membrane protein, western blot analysis revealed that SteD was highly enriched in the fraction comprising integral membrane proteins (Fig. 2D). To establish the membrane topology of SteD, the inventors used C-terminal HA-tagged effector translocated by the bacteria AsteD psteD) and N-terminal FLAG-tagged effector (FLAG-SteD) ectopically expressed in Mel JuSo to carry out selective permeabilization experiments using the mild and harsh detergents Digitonin and Triton X-100, respectively. In the conditions used, Digitonin treatment only permeabilized the plasma membrane and therefore enabled detection of cytosolic epitopes (e.g. GM130), while Triton X-100 treatment permeabilized both plasma and Golgi membranes, allowing the detection of epitopes in the Golgi lumen (e.g. the luminal domain of TGN46). Both HA and FLAG epitope tags of SteD were detected by immunofluorescence microscopy using Digitonin (Fig. 2E), thereby showing that both the N- and C-terminal regions of SteD are exposed to the host cell cytosol (Fig. 2F).

Example 3 - SteD reduces surface pMHCII by increasing ubiquitination of residue K225 on DRj3

It was previously shown that reduction of pMHCII levels on the surface of infected cells was the result of ubiquitination of the cytosolic tail of ϋΡ β (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). To analyse if the pMHCII internalization rate of AsteD-infected cells was different from WT-infected cells, the inventors harvested Mel JuSo cells at 16 h post-invasion and incubated them with mAb L243 on ice. These cells were then incubated at 37 °C for different time points to analyse the amount of pMHCII remaining on the surface by flow cytometry. In agreement with previous results, wild-type SaZmoneZZa-infected cells displayed a higher internalization rate compared to uninfected ones; however, this difference was not observed when cells were infected with the AsteD strain (Fig. 3A). The differential internalization was also detected by immunofluorescence microscopy when infected cells at 16 h post-invasion were labelled with mAb L243 on ice, washed and incubated for another 4-5 h in DMEM 10% at 37 °C (Fig. 3B). After the incubation period, pMHCII molecules were detected in intracellular compartments in cells infected by wild-type Salmonella and AsteD psteD strains, but not in cells infected by the AsteD mutant (Fig. 3B).

Next, the inventors examined the ubiquitination of pMHCII in infected cells and in cells stably expressing fusion proteins by immunoprecipitations of pMHC II using mAb L243 and western blots using an antibody against ubiquitin (P4D1). Ubiquitinated proteins (presumably pMHC II) were present in cells infected by wild-type or AsteD psteD strains, whereas these ubiquitinated proteins were reduced or absent in cells infected by AssaVand AsteD strains (Fig. 3C). Expression of GFP-SteD in stable Mel JuSo cells also caused an increase in ubiquitination of proteins immunoprecipitated by mAb L243 when compared to cells expressing GFP alone (Fig. 3D). To establish if these ubiquitinated products were modified forms of β chain, the inventors used HA-tagged ϋΡ β (either the wild-type version [ΗΑ-ϋΡνβ] or one containing a mutation on the cytosolic K225 residue [ΗΑ-ϋΡνβ K225R]) to carry out immunoprecipitations using anti-HA antibody coupled beads in Mel JuSo cells stably expressing these constructs (Fig. 3E). There was a strong increase in ubiquitination of immunoprecipitated proteins in double stable cells expressing GFP-SteD and ΗΑ-ϋΡνβ (SteD/DR^) compared to single stable cells (ϋΡνβ), but not in cells expressing GFP-SteD and ΗΑ-ϋΡνβ K225R indicating that K225 is the major target of SteD-induced ubiquitination (Fig. 3E). The inventors then analysed the surface levels of pMHCII on these stable cells using mAb L243 (Fig. 3F, G). cells displayed a 4- fold increase in pMHCII on the surface compared to βΐεϋ/ϋΡβ (Fig. 3F, G). This shows that the ability of SteD to reduce surface levels of pMHCII is dependent on K225. The modest increase in pMHCII surface levels in compared to βΐεϋ/ϋΡβ could be explained by the fact that the mAb L243 that was used for labelling would also detect peptide loaded on complexes involving endogenous ϋΡβ as well as ΗΑ-ϋΡνβ- K225R.

Example 4 - SteD complexes with MARCH8 and peptide-loaded MHCII

Bioinformatic analysis of the amino acid sequence of SteD did not reveal any conserved domain or sequence similarity that could indicate possible enzymatic activity or function that would explain ubiquitination of pMHCII. Therefore, the inventors hypothesized that the enzyme driving pMHCII ubiquitination was likely to be derived from the host cell. It was reported that two members of the MARCH ubiquitin ligase family, MARCH 1 and MARCH8, target pMHCII (Bartee et al., J Virol, 2004, 78, 1109- 1120). MARCHi is expressed in inactive dendritic cells (DCs) and its expression is down-regulated upon DC activation, leading to stabilization of pMHCII on the cell surface (De Gassart et al., PNAS (2008) 105: 3491-3496). Mel JuSo cells express MARCH 8 but not MARCH 1 (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). Therefore the inventors used siRNA to knock down MARCH8 and analysed the ability of Salmonella to reduce surface levels of pMHCII in these cells (Fig. 4A). Depletion of MARCH 8 by two different MARCH8-specific siRNA oligos impaired the capacity of Salmonella to reduce surface levels of pMHCII (Fig. 4B).

It was shown previously that Salmonella targets specifically pMHCII and does not affect other surface molecules or MARCH8 substrates such as CDi family proteins, transferrin receptor, B7.1 and B7.2 (Mitchell et al., Eur J Immunol (2004) 34: 2559- 2567; Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). In addition, a general up-regulation of MARCH8 in Mel JuSo cells during infection by Salmonella was ruled out as MARCH8 mRNA levels were similar between infected and uninfected cells (Lapaque et al, PNAS, Vol 106 (33), P14052-14057. 18 August 2009). The inventors hypothesized that SteD might stimulate binding and activity of MARCH8 on pMHCII. To investigate possible interactions between MARCH8 and SteD, HEK293T cells (which do not express pMHCII) were transfected with plasmids encoding

MARCH8-FLAG and GFP-SteD, and subjected to immunoprecipitation with GFP-Trap beads. MARCH8-FLAG co-immunoprecipitated with GFP-SteD but not with GFP alone (Fig. 4C). To investigate possible interactions between SteD, MARCH8 and pMHCII; pMHCII was immunoprecipitated with mAb L243 in stable Mel JuSo cells expressing both GFP-SteD and MARCH8-FLAG. pMHCII formed complexes with MARCH8-FLAG and GFP-SteD (Fig. 4D), but not with a control effector protein. Immunofluorescence microscopy of Mel JuSo cells expressing GFP-SteD and MARCH8-FLAG showed colocalization between these three proteins (Fig. 4E). Pearson's coefficient analysis revealed a greater colocalization between GFP-SteD and MARCH8-FLAG, and between GFP-SteD and pMHCII (mAb L243) compared to GFP only (Fig. 4F). Live cell-imaging analysis of Mel JuSo cells stably expressing GFP-SteD that were transfected with mCherry-MARCH8, showed that both proteins were detected in the same vesicle displaying a dynamic behaviour inside the cell. Vesicles positive for both SteD and MARCH8 left the Golgi network and moved towards the cell surface, and the same vesicles were observed returning to the Golgi region. In an attempt to identify regions of SteD that are important for reducing pMHCII surface levels, the inventors made a series of twenty small alanine substitutions (SteD ¹ - SteD ²⁰ ) in blocks of five/six amino acids, to cover the entire amino acid sequence of SteD (Fig. 7A). Two constructs (SteD? and SteD^) were unstable and did not localize in the Golgi (Fig. 7C, D). FACS analyses of pMHCII surface levels with the remaining eighteen GFP-SteD mutated constructs, which were expressed normally and appeared to localize to the Golgi network, revealed several regions that are important to reduce surface levels of pMHCII (Fig. 7B).

Example 5 - SteD-mediated reduction of surface levels of pMHCII affects T cell proliferation.

Previous reports showed that Salmonella is able to interfere with the ability of bone marrow-derived DCs (BMDCs) to stimulate T cell proliferation in a SPI-2 T3SS- dependent manner (Cheminay et al., J Immunol (2005) 174: 2892-2899; Halici et al., Infect Immun (2008) 76: 4924-4933). To analyse if reduction of pMHCII surface levels by SteD could affect T cell proliferation, T cell proliferation assays were done. First, the inventors confirmed that WT Salmonella reduced overall MHCII (I-A/I-E) surface levels in BMDCs and showed that the reduction did not occur in BMDCs infected with AssaVand AsteD mutants (Fig. 5A). At 16 h post-uptake, infected BMDCs were incubated with OVA-peptide and co-cultured for three days with T cells expressing a T cell receptor specific for OVA. SteD strongly inhibited T cell proliferation (Fig. 5B). Representative histograms of CFSE labelled T cells that were incubated with BMDCs subjected to different conditions are shown in Fig. 5C. To determine if this effect could be detected during infection of a mammalian host, DCs were isolated at 48 h post- inoculation by oral gavage from mesenteric lymph nodes (MLNs). Quantification of total MHCII (I-A/I-E) surface levels by flow cytometry revealed that DCs infected by WT-GFP strain displayed reduced levels of MHCII compared to AsteD-GFP strain (Fig. 5D).

To assess the effect of SteD on T cell activation in vivo, mice were infected for 17 days with WT or steD mutant Salmonella, then spleen cells were recovered and T cells analysed for activation markers by flow cytometry. Analysis of CD4+ T cells from mice with similar bacterial loads revealed that there were significantly more activated T cells in spleens carrying the steD mutant compared to spleens from mice infected with WT bacteria (Fig. 5E). Therefore, the in vitro effects of SteD on mMHCII and T cells are likely to be physiologically important.

Sequence listing

SEQ ID NO: 1

MNVTSGVNAQ TPLLPPSERG NDEKPVAE IV DFNAYGNKPR CLMCLGTTAL FTSVFSGVCS

60

GAVASVSSGA AYTTALTVLG ASFGMGGIGM MGICAGLYLS ANGIRTRPAW P

111 SEQ ID NO: 2

MNVTSGVNAQ TPLLPPSERG NDEKPVAE IV EFNAYGNKPR CLICVGTTAL FTSVFSGVCS

60

GAVASVSSGA AYTTALTVLG ASFGMGGIGM MGICAGLYLS ANGVRTRPAW P

111

SEQ ID NO: 3

MNVTSGVNAQ TPLLPPSERG NDEKPVAE IV DFNAYGNKPR CLMCLGTTAL FAGVFSGVCS

60

GAVASVSSGT AYTTALTVLG ASFGMGGIGM MGICAGLYLS ANGIRTRPAW P

111

SEQ ID NO: 4

MNVTSGVNAQ TPLLPPSERG NDEKPVAE IV DFNAYGNKPR CLMCLGTTAL FTGVFSGVCS

60

GAVASVSSGT AYTTALTVLG ASFGMGGIGM MGICAGLYLS ANGIRTRPAW P

111

SEQ ID NO: 5

MNVTSGVNAQ TPLLPPSERG NDEKPVAE IV DFNAYGNKPR CLMCLGTTAL FTGVFSGVCS

60

GAVASVSSGT AYTTALTVLG ASFGIGGIGM MGICAGLYLS ANGIRTRPAW P

111

SEQ ID NO: 6

MNVTSGVNAQ TPLLPPSEWG DDEKPVAE IV EFNAYGNKPR CLMCLGTTAL FTGAFSGVCS

60

GAVASVSSGA AYTTALTILG ASFGMGGIGM MGICAGLYLS ANGVRTRPAW P

111

SEQ ID NO: 7

MNVTSGVNAQ TPLLPPSERG DDEKPVAE IV EFNAYGNKPR CLMCLGTTAL FTGVFSGVCS

60

GAVASVSSGA AYTTALTVLG ASFGLGGIGM MGICAGLYLS ANGVRTRPAW P

111

SEQ ID NO: 8

MHEEVYMNVT SGVNAQTPLL PPSERGNDEK PVAE IVEFNA YGNKPRCLIC VGTTALFTSV

60

FSGVCSGAVA SVSSGAAYTT ALTVLGASFG MGGIGMMGIC AGLYLSANGV RTRPAWP

117

SEQ ID NO: 9

MHEEVYMNVT SGVNAQTPLL APSERCNDEK PVAEIVDFNA YGNKPRCLMC LGTTALFTGV

60

FSGVCSGAVA SVSSGTAYTT ALTVLGASFG MGGIGMMGIC AGLYLSANGI RTRPAWP

117

SEQ ID NO: 10

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

SEQ ID NO: 11

MXXXXXXXXX XXXXXXXXXX XDEKPVAEIV ( D/E ) FNAYGXXXX XL ( M/ I ) C ( L/V) GTTAL F (T/A) (S/G) (V/A) FSGVCS GAVASVSSG (A/T ) AYTTALT (V/ I ) LG ASFG (M/ I /L ) GGIGM XXXXXXXXXX XNG ( I /V) RTRPAW P

Wherein X can be any amino acid. SEQ ID NO: 12

MXXXXXVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 13

MNVTSGXXXX XPLL (P/A) PSE (R/W) (G/C) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 14

MNVTSGVNAQ TXXXXXSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR

CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 15

MNVTSGVNAQ TPLL (P/A) PXXXX XDEKPVAEIV ( D/E ) FNAYGNKPR CL (M/ I ) C ( L/V) GTTAL F (T/A) (S/G) (V/A) FSGVCS GAVASVSSG (A/T ) AYTTALT (V/L ) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P 111

Wherein X can be any amino acid.

SEQ ID NO: 16

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) XXXXXAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 17

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVXXXX XFNAYGNKPR

CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 18

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) XXXXXNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 19

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGXXXX XL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid. SEQ ID NO: 20

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I ) C (L/V) GXXXX XX ( S/G) (V/A) FSGVCS GAVASVSSG (A/T ) AYTTALT (V/L ) LG ASFG (M/I/L) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P 111 Wherein X can be any amino acid.

SEQ ID NO: 21

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) XXXXXXCS GAVASVSSG (A/T ) AYTTALT (V/L ) LG ASFG (M/I/L) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P 111 Wherein X can be any amino acid.

SEQ ID NO: 22

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVXX XXXXSVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 23

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T ) AXXXXXXXLG ASFG (M/I/L) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P 111 Wherein X can be any amino acid.

SEQ ID NO: 24

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) XX XXXX (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 25

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFGXXXXXX MGICAGLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 26

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM XXXXXXLYLS ANG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 27

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGXXXX XNG ( I /V) RTRPAW P

111

Wherein X can be any amino acid.

SEQ ID NO: 28

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) ( G/C ) (N/D ) DEKPVAE IV ( D/E ) FNAYGNKPR CL (M/I ) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T )

AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS AXXXXXRPAW P

111

Wherein X can be any amino acid. SEQ ID NO: 29

MNVTSGVNAQ TPLL ( P/A) PSE ( R/W) (G/C) (N/D ) DEKPVAE IV ( D /E ) FNAYGNKPR CL (M/I) C (L/V) GTTAL F ( T/A) ( S /G ) (V/A) FSGVCS GAVASVSSG (A/T ) AYTTALT (V/L) LG ASFG (M/ I /L ) GGIGM MGICAGLYLS ANG ( I /V) RTXXXX X

111

Wherein X can be any amino acid.