WO2010061226A1 - Composition and method for enhanced secretion of peptides and proteins from bacteria - Google Patents

Abstract

This invention provides compositions and methods for achieving enhanced delivery of desired protein or peptide products from an expression system delivered by means of an appropriate bacterial host where a novel leader peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) is operatively linked to the peptide or protein sequence to achieve enhanced expression and secretion of the product from the bacterium. Included in the system according to this invention is a bacterium which auto-induces expression of a genetic construct encoding the leader peptide in operative association with a peptide or protein product the expression and secretion of which is desired from the bacterium.

Description

COMPOSITION AND METHOD FOR ENHANCED SECRETION OF PEPTIDES AND PROTEINS FROM BACTERIA

FIELD OF THE INVENTION

An isolated peptide comprising or consisting of the amino acid sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1), or a variant thereof, providing enhanced secretion of proteins in bacteria.

BACKGROUND OF THE INVENTION

The delivery of gene products to animals or animal cells is desirable for a variety of applications. Such applications include treatment of infectious diseases, therapy of acquired or inherited diseases or conditions, induction of an immune response to a protein antigen and the study of various cellular functions. A range of bacteria have therefore been developed and used for the delivery of therapeutic and non-therapeutic molecules.

One of these bacteria is Lactococcus lactis, a food grade, Gram- positive bacterium which is an attractive host for heterologous protein production due to its well developed genetics and long record of safe use. Many heterologous proteins and peptides have been successfully expressed in L. lactis for different biotechnological applications. Despite its lack of invasiveness, L. lactis is able to deliver heterologous antigens and cytokines to the systemic and mucosal immune system (1). Cytokines have been extensively targeted for heterologous production in L. lactis, in order to increase host immunization (2) and/or for in vivo immuno-modulation (3). In this context, Steidler et al. (4) provided the first example for the use of a cytokine-secreting L. lactis vaccine vector to enhance immune responses to a co-expressed heterologous antigen. More recently, the in vivo therapeutic efficacy of this mode of cytokine delivery has been shown using a L. lactis both as a treatment of murine colitis (3) and as a preventative for food induced IgE sensitisation (5).

This mode of delivery has been used in the digestive and respiratory tract of humans and animals, facilitating the interaction between secreted cytokines and the mucosal immune system. Cytokine mucosal delivery is therefore dependent upon their release into the mucosa when L. lactis cells are in contact with the mucosa-associated lymphoid tissue. Secretion of recombinant cytokines has generally involved fusion of the relevant gene to a signal peptide recognized by the general secretory (Sec) pathway. In L. lactis the leader sequence of the abundant Usp45 protein (6) is often the secretion signal of choice for efficient protein secretion (3, 4, 5). Although, this signal peptide has been used in the production of a variety of heterologous proteins, including cytokines such as IL-2 (4), IL-12 (7) and IL-10 (3), in some of these cases inefficient secretion was observed (6). Cytokines are non-structural, intercellular regulatory proteins that mediate a multiplicity of immunologic as well as non-immunologic biological functions. The cytokine mlL-12 (p70) is a heterodimeric glycoprotein composed of two disulphide-linked subunits p40 and p35. It is associated with a number of biological activities including: the differentiation of ThO cells into Th1 cells, use as an adjuvant in vaccine therapies, antitumor activity and for conferring a protective effect against specific bacterial, viral and parasitic infections (8). It is recognized that the final level of the immune response is highly dependant upon the amount of cytokine presented. With this in mind, improvement of secretion efficiency is a desirable aim for researchers.

Research is still ongoing in order to develop more efficient secretory systems for the delivery of proteins. S-layers are crystalline monomolecular assemblies of proteins, covering the cell surface of bacteria at all stages of cell growth and division. Due to the short generation times of bacteria it can be concluded that their expression and secretion via the general secretory pathway has to be highly efficient. S-layer proteins have been identified in a number of firmicutes including bacilli, Clostridia and lactobacilli, but so far have not been described in lactococci. For expression of proteins and peptides the nisin inducing system has been widely described (9, 10, 11 , 12). For the activation of such an inducible expression system in this host, the exogenous addition of nisin is required. The majority of these studies are based upon the intranasal administration of recombinant L lactis (2, 3, 12, 14). To date, no data exists where an autoinducible version of this expression system has been utilised for oral immunization.

In terms of future development of functional foods and neutraceuticals, expression of bioactive molecules is important as is the development of 'smart' probiotics that are designed to provide specific health benefits. Therefore, and in view of the shortcomings of the systems presently used, there is a long-felt need for improved systems for delivery of biologically active peptides, proteins, antigens, and the like, including, delivery of cytokines or antigens. The present invention is aimed at providing an improved and efficient secretion system for the delivery of therapeutic and non-therapeutic agents.

SUMMARY OF THE INVENTION

The invention relates to an isolated peptide comprising the signal sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1), or a variant thereof and uses of said peptide in increasing secretion of a fusion protein comprising said peptide from a bacterium. The invention is also directed to related methods and uses.

In a first aspect, the invention relates to an isolated peptide comprising the signal sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof wherein said signal peptide mediates secretion of a peptide or protein in a bacterium.

In another aspect, the invention relates to a nucleic acid comprising or consisting of a sequence encoding a peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof.

In a third aspect, the invention relates to a recombinant nucleic acid molecule comprising a nucleic acid encoding a peptide comprising the sequence

MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) and operably linked thereto a second nucleic acid wherein the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide.

In a forth aspect, the invention relates to a vector comprising a nucleic acid encoding a peptide comprising the sequence

MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof.

The invention also relates to a host cell comprising said vector. A host cell according to the invention may express a fusion protein wherein said fusion protein comprises a peptide of SEQ ID: 1 or a variant thereof.

In another aspect, the invention relates to a method of delivering a medicament comprising administration of said host cell.

The invention also relates to a pharmaceutical composition comprising a host cell as defined herein. Furthermore, the invention relates to the use of a peptide or nucleic acid encoding a peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof for increasing secretion of a peptide or protein and/or in the delivery of vaccines, antiviral, antimicrobial or antiallergenic agents, enzymes or other non-therapeutic agents.

In another aspect, the invention relates to a method for enhancing secretion of a fusion peptide or fusion protein from a bacterial host wherein said peptide comprises a signal peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof.

In a further aspect, the invention relates to a method for mucosal, in particular oral, delivery of a peptide or protein which comprises encoding said peptide or protein in vector in operative association with a leader peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof, and optionally regulatory sequences in operative association therewith, introducing said vector into a bacterial host and administering said bacterial host to a mammal.

The invention also relates to a food stuff comprising a host cell as defined herein.

Finally, the invention relates to an isolated bacterial strain designated FI10611 and deposited under depositors number NCIMB 41596 at NCIMB Ltd on 17 November 2008.

The secretion of recombinant cytokines by Lactococcus lactis has predominantly involved the use of the signal peptide of the protein Usp45. However, low secretion efficiency of large proteins, such as the murine interleukin-12 (mlL-12), has been observed. We designed the novel signal peptide, designated "SLPmod", on the basis of a consensus sequence of lactobacillal S-layer proteins. The L. lactis strains, FI5876 (nisin producer) was used as final hosts for mlL-12 secretion. Using SLPmod with FI5876 surprisingly, a more than four-fold increase in secretion was observed when compared to using their respective Usp45 signal peptide counterparts. In addition, mice were orally administered parental strain FI5876 carrying the control vector, their mlL-12 secreting derivatives, or a buffer solution. In a preferred embodiment, a new composition, expression system and method according to this invention comprises use of the autoinducible host FI5876, and SLPmod-driven protein secretion, for which we show results in significantly increased levels of mlL-12 in plasma. Our data show that this novel signal peptide is able to enhance the mlL-12 secretion in L. lactis and we expect it to be useful in enhancing the level of expression for other proteins where the secretion efficiency is low. In order to avoid the side effects associated with the systemic administration of IL-12 in the treatment against some tumors, the autoinducible strain secreting IL-12 developed here is anticipated to be useful for the oral delivery of this cytokine. This new system will also have application in prophylactic use in the vaccination against some tumors, allergies and autoimmune diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Alignment of sequences the analysis of which led to the design of the leader peptide MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) according to this invention.

Fig. 2. Cloning strategy used for the introduction of the fragment PnisA_SignalPeptide_p40_Linker_p35 into the vector pTG262 (not to scale).

Fig. 3. Western blot detection of mlL-12 in the intracellular content (A) and culture supernatants (B). Lane 1 , mlL-12; lane 2, L. lactis FM 0615; lane 3, L lactis FH 0611 ; lane 4, L. lactis FM 0632; lane L, MagicMark Western protein standard.

Fig. 4A. Secretion of β-galactosidase by L lactis.

Fig. 4B. Detection of β-galactosidase in L. lactis culture supernatant.

Fig. 5. Lysis of L. monocytogenes by ΦLM4 endolysin engineered L. lactis strains.

Fig. 6. Murein hydrolase activity of culture supernatant samples from L. lactis.

Fig. 7. Detection of ΦLM4 endolysin in intracellular content and culture supernatants of L. lactis. Fig. 8. Lysis of L. monocytogenes by ΦLM4 endolysin engineered Lb. johnsonii strains

DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS OF THE INVENTION

We describe the use of a novel signal peptide for enhanced secretion of biologically active protein from bacteria, for example lactic acid bacteria. To date, the signal peptide predominantly used for heterologous secretion in L lactis has been that of the major extracellular lactococcal protein Usp45 (6). Although several cytokines have been successfully secreted by L. lactis using the Usp45 signal peptide, poor secretion efficiency with the large cytokine protein mlL-12 was observed (7). Moreover, in our laboratory we have found that the secretion efficiency of large proteins is reduced when the Usp45 signal peptide is used.

In a first aspect, the invention relates to an isolated peptide comprising the signal sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof wherein said peptide mediates secretion of a peptide or protein in a bacterium. In one embodiment, the isolated signal peptide consists of MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof.

The term variant is used to describe a signal peptide which retains the biological function of the peptide defined in SEQ ID NO:1 , that is its ability to mediate secretion of a peptide, protein or agent as described herein in a bacterium. As shown herein, using said peptide in a fusion protein increases the secretion of said protein from a bacterial cell. A skilled person would know that the sequence of SEQ ID NO:1 can be altered without the loss of biological activity. In particular, single like for like changes with respect to the physio-chemical properties of the respective amino acid should not disturb the functionality. Furthermore, the deletion of the glycine residue in position 2 will not alter the efficiency.

As shown herein, said signal peptide is capable of mediating secretion of a protein from a bacterial cell. As shown, the signal peptide enhances secretion of the protein when compared to conventionally used signal peptides. Secretion may be enhanced two-, three- or fourfold or more.

In one embodiment, the bacterium is a Gram positive bacterium. Preferred embodiments of bacteria according to the invention are set out in detail below.

In another aspect, the invention relates to a nucleic acid comprising or consisting of a sequence encoding the peptide as described above.

The invention also relates to a recombinant nucleic acid molecule comprising a nucleic acid encoding comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof and operably linked thereto a second nucleic acid wherein the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide.

The invention also relates to a fusion protein encoded by a nucleic acid as described herein.

Said second nucleic acid may encode an antiviral, antimicrobial or antiallergenic agent, a protein used as a food additive or enzyme. For example, said agent may be selected from a cytokine or antigen. In one embodiment, the cytokine is an interleukin.

The inventors have shown that the signal peptide disclosed herein can enhance secretion of a number of proteins from various bacteria as set out in the examples. These proteins include mlL-12, β-galactosidase and ΦLM4 endolysin. The cytokine may be selected from IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11 , IL-12, IL-13, GM-CSF, M-CSF, SCR, IFN-γ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN.alphaV.beta.

In one embodiment, the cytokine is an interleukin. The interleukin may be selected from IL-2, IL-6, IL-10, IL-12 or IL12-family members (IL-12, IL-23 and IL-27). A preferred interleukin is IL-12. Thus, a fusion protein according to the invention may comprise the signal peptide described herein and IL-12.

In one embodiment according to the present invention, we demonstrate the ability of a new signal peptide (SLPmod), designed on the basis of an S- layer protein consensus sequence from Lb. acidophilus, Lb. crispatus and Lb. helveticus, to secrete the cytokine mlL-12 in L. lactis. A significant and surprising result was that in comparison to the secretion achieved when the signal peptide of Usp45 was used, the presence of the alternative SLPmod signal peptide lead to more than a four-fold increase in mlL-12 secretion which in turn resulted in an eight-fold increase of IFN-γ production in the biological activity assays using mouse splenocytes (see examples below). This important finding demonstrates that improvements in the secretion of large proteins are achievable when SLPmod is employed as the secretion signal in L. lactis.

Although mlL-12 secretion had been achieved previously using the Usp45 signal peptide, a low efficiency had been observed (7). The potential prophylactic and therapeutic uses of cytokine mlL-12 secreting lactococci have already been reported by Bermudez et. al (16). They were able to show that promising therapeutic results against a human papilloma-virus induced tumor were obtained with L. lactis expressing an E7 antigen and mlL-12 (17). However, no such relevant outcomes were seen when L lactis secreting mlL- 12 was used in a mouse model of allergy (18, 19). For instance, Cortes-Perez et al (18) carried out an intranasal co-administration of mlL-12-secreting L. lactis and a major cow's milk allergen. Their study showed that mlL-12 was inefficient in breaking tolerance induction despite the observation that mlL-12 had exhibited a Th1 adjuvant effect in other studies. The authors speculated that the mlL-12 dose administered could have been too low to develop a Th1 adjuvant effect. In addition to this, we hypothesize that that the nature of the strains used and the route of administration applied may also have played a role in the less than optimal results achieved. We provide herein solutions to both of these problems by combining a signal peptide which results in enhanced secretion of biologically active mlL-12 and an optimized strain for expression and delivery of the cytokine.

Systemic effects after oral immunization have already been shown by Fossard et al., (5) who reported that increased plasma levels of murine interleukin-10 (mlL-10) were obtained when mice were intragastrically gavaged with L. lactis secreting mlL-10 under the control of a constitutive promoter. We disclose herein the engineering a strain of L. lactis, derived from FI5876 (20) to achieve secretion of mlL-12 based on the nisin controlled gene expression system. This inducible system, under the control of the strong nisin promoter, requires the presence of nisin in the media in order to be activated (9). We analysed compared the ability of the mlL-12-secreting derivative strain of FI5876 (autoinducible) to influence the plasma levels of mlL-12 after their oral administration in mice. Comparable mlL-12 levels of secretion were detected in the culture supernatants of FI5876 (185 or 40 pg ml^"1; SLPmod or Usp45 signal peptide respectively) derivative strains in ELISA assays. Nevertheless, elevated mlL-12 plasma levels in mice were only obtained when the autoinducible strain FM 0611 was used. This strain is a derivative of FI5876 (nisin producer) and therefore, nisin is likely to be present when the metabolically active strain is in contact with the intestinal mucosa. This supply of nisin is able to autoinduce the expression system, leading to the subsequent secretion of mlL-12 and its delivery to the mucosal tissue. In addition, nisin is known to be partially degraded or inactivated in the gastrointestinal tract (21). We conclude that a nisin producing strain is the ideal host for oral delivery. Thus, in one exemplary embodiment according to this invention, we have engineered the self-inducing nisin producing L lactis strain FI5876 to express mlL-12 constitutively. We demonstrate the ability of mlL-12-secreting FI5876 to orally deliver mlL-12. Other uses and applications of this invention will be evident to those skilled in the art. The new SLPmod signal peptide will be a very useful instrument for the heterologous production of proteins not only in L lactis but we anticipate successful utilization in other Gram-positive bacteria, where an enhancement of protein secretion is required.

Although mlL-12 has been shown to be highly effective against different tumors (22), more attractive procedures for treatment need to be developed due to the fact that systemic administration of this cytokine can cause a number of side effects. Intragastrically, administering a recombinant non-colonizing L. lactis that secretes biologically active mlL-12 effectively is anticipated to be of great benefit in reducing toxic side effects. In addition, mucosal delivery of mlL-12 has been shown to possess great properties as an adjuvant in vaccine delivery which might be enhanced by the application of the FI10611 strain developed here. For example, the autoinducible mlL-12- secreting strain may be used in ameliorating food allergies.

A variety of antigens may also be used according to the invention, for example antigens as diverse as antigen from Plasmodium falciparum malaria and tetanus toxin fragment C from Clostridium tetani, as well as urease subunit B from H. pylori. The skilled person will appreciate that the methods of the present invention could be used to deliver a range of biologically active polypeptides. Thus, in the recombinant nucleic acid molecule, any protein that elicits an immune response may be used. In one embodiment, the molecular weight of the polypeptide encoded by the second nucleic acid is below 116kDa (the molecular weight of β-galactosidase).

In another embodiment, the second nucleic acid may encode enzymes, such as proteases, glycoside hydrolases and lysins. For example, the enzyme may be an endolysin. Endolysins are phage-encoded enzymes that break down bacterial peptidoglycan at the terminal stage of the phage reproduction cycle. Endolysins have been employed in food science, in microbial diagnostics and for treatment of infections by bacteria and prevention thereof. The recombinant nucleic acid molecule of the invention may also further comprise regulatory sequences, for example promoter sequences. In one embodiment, said promoter is an inducible promoter. An example of an inducible promoter is the nisin promoter that is widely used for regulating gene expression in L. lactis. This inducible system, under the control of the strong nisin promoter, requires the presence of nisin in the media in order to be activated (9).

In another aspect, the invention relates to a vector comprising a nucleic acid as described herein. Suitable vectors comprising a nucleic acid for introduction into bacteria can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. ^'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.

In a further aspect, the invention relates to a host cell comprising a nucleic acid or vector as described herein. The invention also relates to a host cell expressing one or more fusion proteins wherein said fusion protein comprises the signal as defined herein and encodes for therapeutically beneficial protein or non therapeutic agent as described herein. The inherent signal peptidase I of the host strain will remove the signal peptide during the transport process through the general secretory system. Moreover, a skilled person would know methods for removal of the signal peptide to generate a desired mature protein unencumbered by the leader - e.g. via cyanogen bromide cleavage; endopeptidase cleavage; factor X activity, etc; as well as His(n)-Nickel affinity or other affinity column (antibody to the leader) methods for purification of the fusion peptides and/or removal of the cleaved leader from the mature protein of interest. A host cell according to the invention may express one or more fusion proteins or "heterologous" polypeptides wherein said fusion protein or "heterologous" polypeptide comprises the signal peptide as defined herein. A "heterologous" polypeptide is one not native to the bacterium.

The bacterium according to the various aspects of the invention is preferably a Gram positive bacterium. In one embodiment, the Gram positive bacterium is one which does not invade the digestive tract of a vertebrate host into which it is introduced. Thus, the bacterium according to the invention is preferably non-invasive or non-pathogenic bacterium. However, the bacterium may also be a non-pathogenic attenuated strain of a Gram-positive pathogenic bacterium. In one embodiment, the bacterium is selected from lactic acid bacteria. As used herein the term "lactic acid bacterium" designates a Gram-positive, microaerophilic or anaerobic bacterium which ferments sugars with the production of acids including lactic acid as the predominantly produced acid. The industrially most useful lactic acid bacteria are found among Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium and Propionibacterium. Additionally, lactic acid producing bacteria belonging to the group of the strictly anaerobic bacteria, bifidobacteria, i.e. Bifidobacterium, which are frequently used as food probiotic cultures alone or in combination with other lactic acid bacteria, are generally included in the group of lactic acid bacteria. Thus, according to the invention, the bacterium may be a lactic acid bacterium selected from Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium and Propionibacterium or Enterococcus. In one embodiment, the bacterium is Lactococcus or Lactobacillus. For example, Lactobacillus may be Lactobacillus johnsonii or Lactobacillus lactis and Lactococcus may be Lactococcus lactis.

In one embodiment, the bacterial host is FI10611. This strain has been deposited as NCIMB 41596 at NCIMB Ltd. In another embodiment, the strain is FI5876. Another aspect of the invention relates to a method of delivering a medicament comprising administration of a host cell as defined herein.

Moreover, the invention relates to a pharmaceutical composition comprising a host cell as defined herein. The pharmaceutical composition may be a vaccine. Administration of the pharmaceutical composition may be orally. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions). A pharmaceutically acceptable carrier may be included. The skilled person will appreciate that methods of administration depend on the heterologous protein included in the composition.

The invention also relates to the use of a peptide or nucleic acid as defined herein for increasing secretion of a heterologous peptide or protein as defined herein. For example, secretion of an antiviral, antimicrobial or antiallergenic agent, a food additive or an enzyme may be increased. Such agents and enzymes are defined elsewhere in here. In addition, the secretion of unmodified bacteriocins may be increased.

The invention also relates to the use of a peptide or nucleic acid as defined herein in the delivery of vaccines, antiviral, antimicrobial or antiallergenic agents. Such agents are defined elsewhere in here. For example, the agent may be a cytokine, in particular an interleukin such as IL- 2, IL-6, IL-10, 11-12 or IL12-family members (IL-12, IL-23 and IL-27). The agent may be an antimicrobial peptide. Antimicrobial peptides (AMPs) are small molecular weight proteins with broad spectrum antimicrobial activity against bacteria, viruses, and fungi. Examples of antimicrobal peptides include cathelicidins, magainins, alamethicin, pexiganan or Template:MSI-78, and other MSI peptides like Template:MSI-843 and Template:MSI-594, Template:Polyphemusin, Template: LL-37, defensins or protegrins. According to the invention, it is also possible to deliver an antibiotic.

Delivery is preferably mucosally. The mucosa refers to the epithelial tissue that lines the internal cavities of the body, such as the gastrointestinal tract, the respiratory tract, the lungs, and the genitalia. Thus, according to the invention, the mucosal surface includes nasal epithelium and the luminal surface of a gastrointestinal organ selected stomach, small intestine, large intestine, and rectum. In one embodiment, delivery is preferably orally.

The invention also relates a method for enhancing secretion of a fusion peptide or fusion protein or heterologous polypeptide from a bacterial host wherein said peptide comprises a signal peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1). Such agents are defined elsewhere in here. For example, the agent may be a cytokine selected from the group consisting of IL-2, IL-6, IL-10, 11-12 or IL12-family members (IL-12, IL-23 and IL-27).

In another aspect, the invention relates to a method for mucosal delivery of a peptide or protein which comprises encoding said peptide or protein in a vector in operative association with a leader peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1), and optionally regulatory sequences in operative association therewith, introducing said vector into a bacterial host and administering said bacterial host to a mammal.

As described elsewhere, the bacterial host in these methods may be a lactic acid bacterium, for example Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium, Propionibacterium or Enterococcus. In one embodiment, the host is FI10611. In another embodiment, the host is FI5876.

The invention further relates to food stuff comprising a host cell as defined herein. Such food stuff may be solid or liquid and includes yoghurt and drinks.

The invention also relates to an isolated bacterial strain designated FI10611 , deposited under deposit number NCIMB 41596 at NCIMB Ltd.

Based on this disclosure, including the specifics of the examples provided below, and the general teachings provided herein above, those skilled in the art will appreciate that a wide range of applications and modifications of the essential features of the invention may be employed, without departing from the hear of this invention. Thus, for example, those skilled in the art will appreciate based on this disclosure that a wide range of peptides, antigens, proteins and the like may be expressed to advantage by including the signal peptide, or a modification or variant thereof. Those skilled in the art will appreciate that different host strains may be used for delivery of appropriate constructs by following the guidelines provided herein, for example, to achieve regulated expression of proteins or peptides which might otherwise be toxic. In addition, where this is desirable, those skilled in the art are by now well familiar with methods for achieving cleavage of signal peptides from mature proteins, in the event that this is considered necessary. Accordingly, in light of the guidance provided herein, those skilled in the art are fully enabled to practice the entire scope of the invention as reflected in the claims appended to this disclosure.

Materials and methods are as disclosed in the non limiting examples below. Wilcoxon rank tests were used for statistical analysis of the animal study data. A result was considered significant when p < 0.05.

EXAMPLES

Having generally disclosed how to make and use the invention, including the best mode and equivalents thereof, those skilled in the art are provided with the following written description in the form of non-limiting examples to assist in the fully comprehending and practicing the invention. The scope of the invention should not, however, be construed as being limited to the specifics of these examples. Rather, reference should be had to the appended claims and their equivalents for that purpose.

EXAMPLE 1

Bacterial strains and culture conditions

Lactococcal strains and plasmids employed are listed in Table 1. L. lactis FI5876 (nisin producer) was used as the final host for mlL-12 secretion. These bacterial strains were grown in M17 medium (Oxoid, Basingstoke, UK) supplemented with 0.5% (wt/vol) glucose (GM17 medium) at 3O⁰C without agitation. E. coli was grown in L broth at 37⁰C on an orbital shaker. Antibiotics were added as selective agents when appropriate at the following concentrations: chloramphenicol (Cm), 5 μg ml^"'' for Lactococcus, 7.5 μg ml^"^ for Lactobacillus and 15 μg ml^"'' for E. coli, ampicillin (Ap), 200 μg ml^"'' .

Strains were grown to an OD₆oo ~1 -0 before supernatants and cell pellets were collected.

Table 1 mlL-12 concn Reference or

Strain Host ³ Plasmid signal peptide (pg/mi) ^b source

FI5876 - 0 20

FM 0632 FI5876 pTG262 - 0 This study

FI10611 FI5876 pFI2596 SLPmod 185 This study

FH 0608 FI5876 pFI2602 Usp45 40 This study

FI10610 FI5876 pFI2595 None - This study

^a Entire nisin gene cluster b The concentration of mlL-12 (pg ml^"1) in culture supernatants of lactococcal strains, as determined by sandwich ELISA. The values represent the mean from quadruplicate samples (standard deviation <8%).

EXAMPLE 2 Molecular techniques

Recombinant plasmids were recovered by transformation of E. coli as described previously by Dodd et al. (15) or by electroporation of L lactis according to the method of HoIo and Nes (23) with the modifications of Dodd et al. (15). Primers were purchased from Sigma-Genosys Ltd, Haverhill, UK. Fragments generated for the construction of vectors were amplified using Phusion DNA polymerase (Genetic Research Instrumentation Ltd; Braintree, UK), and cloned into pCR2.1 (Invitrogen Ltd, Paisley, UK). Confirmation of nucleotide sequences was carried out on purified plasmid DNA using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit and an Applied Biosystems DNA sequencer. EXAMPLE 3

Design of the SLPmod signal peptide

This novel signal peptide was developed on the basis of an alignment of lactobacillal S-layer proteins. The respective S-layer protein sequences were retrieved from the UniProt database (http://www.uniprot.org/). Incomplete sequences were removed, and the first 70 amino acids of the remaining proteins were aligned using clustalW (24). Upon inspection of the alignment, we organized the sequences into 3 groups or clusters (see Fig. 1 , SEQ ID. 2-33), the largest of which contained sequences from Lactobacillus acidophilus, Lactobacillus crispatus and Lactobacillus helveticus. Because of a clear majority of available sequences clustering into one group and the fact that we noted strong sequence similarity, especially around the signal peptidase cleavage site of the S-layer signal peptides from this group with that of a secreted lactococcal protein (llmg_0851) unrelated to this work, we chose the respective consensus sequence as basis for the design of SLPmod, MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1).

EXAMPLE 4

Construction of mlL-12 expression vectors

The regions encoding the mlL-12 p40 and p35 subunits were amplified by PCR employing mouse spleen cDNA as template (AMS Biotechnology, Abingdon, UK). Primer pairs δ'-ATGTGGGAGCTGGAGAAAGAC-S', SEQ ID. 34, and S'-CCTGGATCCGACCCTGCAG-S', SEQ ID. 35, for the p40 fragment, and 5'-AGGTGGAGGAGGATCTAGGGTCATTCCAGTCTC-S', SEQ ID. 36, and δ'-CTGGATCCTTTCAGGCGGAGC-S', SEQ ID. 37, for the p35 fragment were used to create fragments of 938 bp and 578 bp respectively. Each primer was designed to carry restriction enzymes sites strategically located for cloning purposes. In order to allow the protein subunits to interact and acquire the appropriate biologically active conformation, an artificial linker coding for (GIy₄ Ser)₃, SEQ ID. 38, previously described by Huston et al., (25) was used. In our construction, the sequence coding for this artificial linker was designed taking into account the L. lactis codon usage. The final fragments PnisA_SignalPeptide_p40_Linker_p35 were achieved through a two step procedure (Fig. 2). Initially, spliced overlap extension PCR was used to create two fragments; the first representing SignalPeptide_p40 under the control of the nisin A promoter and the second representing Linker_p35. Finally, the two sub-fragments were cut using restriction enzymes and then ligated. The signal peptides included were either SLPmod or that of Usp45. The resulting fragments (PnisA_SignalPeptide_p40_Linker_p35) were cloned into the vector pTG262 (26) using EcoRI and Bam H I restriction enzymes. Subsequently, L. lactis strain FI5876 was transformed with the pTG262 derivatives, pFI2596 or pFI2602 encoding for SLPmod or Usp45 signal peptide respectively, in order to generate the FI5876 derivatives Fl 10611 and FI10608.

EXAMPLE 5

Immunological detection of mlL-12 expression

Tris-SDS-PAGE and Tris-Glycine protein electrophoresis was carried out in NuPAGE 10% Bis-Tris pre-cast or Novex 4-20% Tris-Glycine gels (Invitrogen Ltd) using a Xcell SureLock™ unit (Invitrogen Ltd). Culture supernatants were concentrated using an Amicon filter device (Millipore, UK). In order to obtain intracellular content a standard bead beating procedure was followed using 10 mM Tris pH 7.5 as buffer. MagicMark Western Protein Standard (Invitrogen Ltd) and mlL-12 (R&D Systems, Abingdon, UK) were used as molecular weight markers.

Gels were transferred to a nitrocellulose membrane (Hybond ECL₁ GE Healthcare Bio-Sciences Ltd) employing a Western Transfer Apparatus (Invitrogen Ltd) and subsequently submitted to Western blotting analysis using the ECL Advance Western Blotting Detection kit (GE Healthcare Bio- Sciences Ltd). Polyclonal anti-mlL-12 antibodies (Cambridge Biosciences, Cambridge, UK) were used as primary antibodies (Fig. 3). Finally, the mlL-12 concentration in the culture supernatants of recombinant strains was quantified by sandwich ELISA using a mlL-12 kit (BioSource Europe). Duplicate sandwich ELISA assays were performed with four independent culture supernatants for each of the strains tested. In this study the nisin producing FI5876 L. lactis strain secreting the heterodimeric mlL-12 was tested using either the signal peptide of the Usp45 protein or the novel SLPmod.

Initially, Western blot analysis was carried out on both the intra- and extracellular contents (Fig. 3). Culture supernatants from FI5876 derivatives carrying the plasmids pTG262, pFI2596 or pFI2602 revealed that when an anti-mlL-12 polyclonal antibody was used, a clear band of approximately ~ 70 kDa was only detected in the strains where the plasmids pFI2596 or pFI2602 were present (Fig. 3B; data shown for pFI2596). L. lactis FI10611 utilising the SLPmod signal peptide for secretion produced the highest level of secreted mlL-12 (185 pg mr¹) (Table 1).

A significant finding is that the new SLPmod signal peptide in conjunction with the host strain FI5876 produced more than a four-fold increase in secretion when compared to the routinely used Usp45 signal peptide counterparts (Table 1).

In addition, we observed that the buffering of media with 2% NaHCO₂ lead to a further 3-fold increase of mlL-12 production. Furthermore, an additional plasmid (pFI2595) encoding mature mlL-12 (no signal peptide) was also included. The resulting Western blot of an intracellular content sample of Fl 10610 revealed a strong single band (Fig. 3A) indicating the presence of an intact cytokine, unlike the result published by Bermudez et al., (7) which showed a smeared range of different molecular weight bands suggesting that some degradation had occurred.

EXAMPLE 6

Biological activity of mlL-12

Twenty miililitres of L. lactis culture supernatant were concentrated 100-fold using an Amicon filter device. Splenocytes from several mice were isolated using Histopaque (Sigma-Aldrich, UK). Spleen cells (2 x 10⁶ cells ml^" 1) in AIM-V medium (Gibco) were added to the wells of a microtiter plate.

Aliquots (10μl) of concentrated L. lactis supernatants or 100 pg mH of commercial recombinant mlL-12 (Biosource Europe) were incorporated into wells in triplicate. Following 48h incubation at 37 ⁰C under 5% CO₂, the supernatants were collected and the IFN-γ level was measured by ELISA (BioSource Europe).

Culture supernatants of the L. lactis strain secreting mlL-12 as well as from the respective parental strain were assessed for mlL-12 bioactivity by measuring their capacity to induce splenocyte IFN- γ secretion. Spleen cell suspensions of three independent experiments were inoculated with either culture supernatants or a known concentration of pure mlL-12. After incubation, IFN- γ was measured. Addition of culture supernatants of the L. lactis FI5876 derivative strain secreting mlL-12 to mouse splenocytes resulted in induction of IFN-γ production. These induction levels were comparable to those obtained using standard mlL-12 indicating that the majority of the mlL- 12 cytokine secreted by the L. lactis FI5876 derivative remained bioactive.

EXAMPLE 7 Animal study

Two groups of C3H/HeJ mice were intragastrically gavaged with 50 μl of freshly prepared 10⁹ cfu ml^"'' L. lactis cultures of the vector-only control strain FM 0632 (FI5876/pTG262) or the FI5876 mlL-12-secreting derivative strain FI10611. A third (control) group received only 2% NaHCO₂ buffer. Blood samples were collected 6 hours after intragastric administration of the LAB strains or the buffer control. Blood was recovered and mixed with potassium- EDTA to a final concentration of 0.75%. Samples were centrifuged at 20Og for 10 min at 4°C; the subsequent plasma samples were stored at -70°C. An ELISA kit (Biosource Europe) was used to measure mlL-12 levels.

a. Plasma levels of mlL-12

Three groups of animals were intragastrically gavaged with either the L lactis parental strains FI5876 carrying the control vector, the mlL-12 engineered derivative or a control buffer solution. Plasma samples were collected 6 hours after gavaging and mlL-12 levels were measured. Statistical data analysis was carried out and a significant increase in mlL-12 levels was obtained when the mlL-12 secreting FI5876 strain was used (FI10611 ; an auto inducer strain). Independent studies were carried out and a mean value of 40 ± 11 pg ml^"1 was obtained when animals had been gavaged with Fl 10611. In contrast, a mean value of 30 ± 10 pg ml^"1 was exhibited when mice had been gavaged with the controls.

EXAMPLE 8

Secretion of β-galactosidase by Lactococcus lactis

Initially primer pair δ'-ATTCATGAAAGGGGCCGTCG-S' and 5'- TTATTTTTGACACCAGACCAACTG-3' were used to PCR amplify a 3058bp lacZ region of pORI280 (27) encoding for β-galactosidase. The lacZ fragment was BspHI digested and cloned into Λ/col and Sma\ sites of pFI2590, a derivative of the P_nJ5A expression vector pUK200 (10), which allows the addition of 6 histidine residues at the n-terminus of the expressed protein. The resulting plasmid was designated pFI2610. Using spliced-overlap extension PCR a 96 bp SLPmod (5'-ATCCATGGGTAAAAAAAATTTAAGAATTG-S' and 5'- TGATGGTGATGACTCATAGCAGCATTAACTGGC-S') or 81 bp Usp45 (5'- TATTCATGAAAAAAAAGATTATCTCAGC-S' and 5-

TGATGGTGATGACTCATAGCGTAGACCCCTGA-3') signal peptide coding region was fused to the 3087 bp 6x his-tagged lacZ (5'- ATTCATGAAAGGGGCCGTCG-3' and 5'-

TTATTTTTGACACCAGACCAACTG-3') fragment of pFI2610. The SignalPeptide_6xhis_/acZ PCR fragment was digested with Λ/col or BspHI respectively and cloned into the /Vcol and Sma\ sites of pUK200, to create pFI2611 (SLPmod) and pFI2612 (Usp45) respectively. Subsequently, L lactis strain UKLd 0 (10) was transformed with pFI2611 or pFI2612 in order to generate the UKLdO derivatives Fl 10653 and Fl 10654 respectively.

L. lactis cultures were grown to an ODβoo of 0.5 prior to the addition of inducing nisin at a final concentration of 2.0 ng ml^"1. Following a further 18- hour period of incubation, culture supernatant proteins were concentrated 1000-fold using an Amicon filter device, β-galactosidase activity measurements were performed on duplicate 10μl samples. A more than three-fold increase was seen in β-galactosidase activity when the SLPmod signal peptide was employed as compared to the activity observed with the respective Usp45 derivative (Fig. 4A). Tris-SDS-PAGE protein electrophoresis was carried out a NuPAGE 4-12% Bis-Tris precast gel. BenchMark Protein Ladder (Invitrogen Ltd) was used as the molecular weight marker. The resulting gel shows a band equivalent in size to the 6xHis_β-gal protein (117 kDa) only in the track relating to the SLPmod sample Fl 10653 (Fig. 4B).

EXAMPLE 9

Secretion of the Listeria monocytogenes bacteriophage ΦLM4 endolvsin by

Lactococcus lactis

Primer pair 5'- ATCCATGGCATTAACAGAGGCATGG-3' and 5'- TTATTTTAAGAAGTAGTTCGCTG-3' was used to PCR amplify a fragment coding for the ΦLM4 endolysin using pF!322 (28) as template. This 868bp fragment was digested with Λ/col and cloned into the Λ/col and Sma\ sites of pFI2590 in order to create pFI2591. Using spliced-overlap extension PCR the 96 bp SLPmod-encoding (5'-ATCCATGGGTAAAAAAAATTTAAGAATTG-S' and 5'-GCCTCTGTTAATGCCATAGCAGCATTAACTGGC-S') signal peptide was fused to the ΦLM4 endolysin gene (5'- GCCAGTTAATGCTGCTATGGCATTAACAGAGGC-3' and 5'-

TTATTTTAAGAAGTAGTTCGCTG-S'). The SLPmod_ΦLM4 PCR fragment was digested with Ncol and cloned into Λ/col and Sma\ sites of pFI2590 to create pFI2608. L lactis strain UKLd 0 was transformed with pFI2591 or pFI2608 in order to generate the UKLdO derivatives FI10606 and FI10648 respectively.

For the detection of murein hydrolase activity L. lactis strains Fl 10606 and Fl 10648 together with a vector-only control strain Fl 10544 were spread on to the surface of 20ml 0.2M potassium phosphate, pH7 buffered GM17 (plus chloramphenicol) agar plates containing 100μl of 200-fold concentrated autoclaved stationary phase L. monocytogenes NCTC 12453 cells. Duplicate plates were prepared one of which contained 1 ng ml^"1 of inducing nisin. Plates were incubated at 3O⁰C for 2 days. Zones of lysis were detected only where the gene for ΦLM4 endolysin was present (Fig. 5). A considerably larger zone radius was visible in the case of the SLPmod signal peptide related strain FH 0648. The difference in murein hydrolase activity between the two ΦLM4 endolysin expressing L lactis strains was clearly visible with or without nisin induction.

EXAMPLE 10

Detection of murein hydrolase activity in culture supernatants of Lactococcus lactis

L. lactis cultures were grown in 0.2M potassium phosphate buffered GM17 (pH7) plus chloramphenicol, to an OD₆oo of 0.5 prior to the addition of inducing nisin at a final concentration of 1 ng ml^"1. After an additional 2-hour period of incubation, culture supernatant proteins were concentrated 500-fold by precipitation using an equal volume of chilled ethanol. Precipitated proteins were recovered by centrifugation at 14,000g and resuspended in 0.1 M pH7.5 Tris buffer.

Samples (5μl) of the concentrated supernatant proteins were added to wells prepared in 0.1 M pH7.5 Tris buffered agar (1.5%) containing autoclaved cells of stationary phase L. monocytogenes NCTC 12453. Plates were incubated for 2 days at 3O⁰C. Lysis zones were detected only for ΦLM4 endolysin expressing strains (Fig. 6). The size increase of the zone diameter produced by the SLPmod derivative strain Fl 10648 in comparison to the no signal peptide strain FM 0606 was considerable.

Concentrated culture supernatant proteins from FH 0648 were dialysed against 0.1 M pH7 Tris buffer, using 10 kDa MWCO dialysis tubing. Using a cell disruption technique cell-free extracts were prepared from cultures of Fl 10606 and Fl 10648 which had been induced for 2-hours. Tris-SDS-PAGE protein electrophoresis was carried out in a NuPAGE 10% Bis-Tris precast gel. SeeBlue Plus2 pre-stained protein standard (Invitrogen Ltd) was used as the molecular weight marker. In the cell-free extract samples, protein bands relating to either the 34 kDa 6x his-tagged_ΦLM4 endolysin (Fl 10606) or the 37 kDa unprocessed SLPmod_ΦLM4 endolysin (Fl 10648) were visible (see Fig. 7). Bands of similar sizes were not present in the negative control (data not shown). A single band equivalent in size (33 kDa) to processed ΦLM4 endolysin (no additional histidine residues) is clearly present in the dialysed culture supernatant protein sample. The correct processing of SLPmod_ΦLM4 endolysin through signal peptidase I was verified by MALDI- TOF analysis.

EXAMPLE 11

Secretion of ΦLM4 endolysin by Lactobacillus johnsonii

Use of the P_ni_SA based expression system requires the presence of a two-component regulatory system encoded by nisR and nisK (nisRK). A position in the genome of the Lb. johnsonii strain FI9785 was chosen for the integration of nisRK. Initially a 1033 bp integration region was PCR amplified (5'- AGTAGTAAATGAACTTATTCAACC-S' and 5'-

ATTAATGTCTCATATACCAATGAC-3') and cloned into the blunt-ended Spel site of pGhostθ (29) (pFI2657). A 2.4 kb fragment encoding for nisRK was PCR amplified (5'- CCCGGGAGAATCTTAAAGAGTCTAGGG-3' and 5'- AAAAAGTAATCCTTAGAGATTAC-S¹) from the genome of FI5876 and cloned into the blunt-ended SsfEII site of the pGhostθ derivative pFI2657 to generate pFI2652. Lb. johnsonii FI9785 was transformed (30) with pFI2652 and a gene replacement protocol (31) was used to integrate nisRK into the Lactobacillus genome creating Fl 10744.

L. monocytogenes ΦLM4 endolysin expression plasmids pFI2591 (no signal peptide) or pFI2608 (SLPmod) were introduced into the NisRK expressing Lb. johnsonii strain Fl 10744, generating FM 0753 and FM 0754 respectively.

For the detection of murein hydrolase activity Lb. johnsonii strains Fl 10753 and Fl 10754 together with a vector-only control strain Fl 10752 were spread on to the surface of 0.1 M pH7.5 Tris buffered agar (1.5%) containing autoclaved cells of stationary phase L. monocytogenes NCTC 12453. Duplicate plates were prepared one of which contained 10 ng ml^"1 of inducing nisin. Plates were incubated at 37⁰C for 2 days. Zones of lysis were detected only where the gene for ΦLM4 endolysin was present (Fig. 8) and not in the vector control strain Fl 10752 (data not shown). A measurable increase in the zone radius of the SLPmod derivative strain Fl 10754 is evident. Equivalent results from either the no induction or nisin induced samples suggest that the NisRK expressing Lb. johnsonii strain Fl 10744 conveys constitutive expression from P_nIsA- Example 12

Engineering of strains FI5876 and FI10611

L. lactis FI5876 was constructed by the conjugal transfer of the nisin determinants from NCFB 894 (31) to the plasmid-free strain MG1614 (32). Selection was made for transconjugants able to metabolize sucrose, as described by Gasson (31).

Fl 10611 was created by transforming FI5876 with the mlL-12 expression plasmid pFI2596.

A skilled person would know how to create "FI5876" by using Fl 10611 in a simple curing method. This could be achieved by repeatedly sub-culturing FM 0611 in culture medium without the addition of the plasmid selective antibiotic. After a number of subcultures, the resulting culture would be plated out diluting to single colonies. Individual colonies could then be screened for the retention of the plasmid by patching onto plates with/without selective antibiotic (Cm @ 5mg/ml). Cm sensitive colonies would represent FI5876.

Sequence listing

SEQ ID: 1

MGKKN LRIVSAAAAALLAVAPVAATAM PVNAA

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Claims

WHAT IS CLAIMED IS:

1. An isolated peptide comprising the signal sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof wherein said peptide mediates secretion of a peptide or protein in a bacterium.

2. An isolated signal peptide according to claim 1 consisting of MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1) or a variant thereof.

3. A nucleic acid comprising a sequence encoding the peptide of claim 1 or claim 2.

4. A recombinant nucleic acid molecule comprising a nucleic acid encoding the peptide of claim 1 or claim 2 and operably linked thereto a second nucleic acid wherein the recombinant nucleic acid molecule encodes a fusion protein comprising the signal peptide.

5. A recombinant nucleic acid molecule according to claim 4 wherein said second nucleic acid encodes an antiviral, antimicrobial or antiallergenic agent, food additive or enzyme.

6. A recombinant nucleic acid molecule according to claim 5 wherein said agent is selected from a cytokine or antigen.

7. A recombinant nucleic acid molecule according to claim 6 wherein said cytokine is an interleukin.

8. A recombinant nucleic acid molecule according to claim 7 wherein said interleukin is selected from IL-2, IL-6, IL-10, or IL12.

9. A recombinant nucleic acid molecule according to any of claims 4 to 8 further comprising regulatory sequences.

10. A recombinant nucleic acid molecule according to claim 9 wherein said regulatory sequences are promoter sequences.

11. A recombinant nucleic acid molecule according to claim 10 wherein said promoter is an inducible promoter.

12. A recombinant nucleic acid molecule according to claim 11 wherein said promoter is a nisin promoter.

13. A fusion protein encoded by a recombinant molecule according to any of claims 4 to 12.

14. A vector comprising a nucleic acid according to any of claims 3 to 12.

15. A host cell comprising a nucleic acid or vector according to any of claims 3 to 12 or 14.

16. A host cell expressing a fusion protein wherein said fusion protein comprises a peptide as defined in claims 1 or 2.

17. A host cell of claims 15 or 16 wherein said host cell is a bacterium.

18. A host cell of claim 17 wherein said bacterium is a Gram positive bacterium.

19. A host cell of claim 17 or 18 wherein said Gram positive bacterium is one which does not invade the digestive tract of a vertebrate host into which it is introduced.

20. A host cell of any of claims 15 to 19 wherein said bacterium is selected from lactic acid bacteria.

21. A host cell of claim 20 wherein said bacterium is selected from Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium, Propionibacterium or Enterococcus.

22. A host cell of claim 21 wherein said bacterial host is selected from FI5876 or FI10611.

23. A method of delivering a medicament comprising administration of a host cell as defined in any of claims 15 to 22.

24. A pharmaceutical composition comprising a host cell as defined in any of claims 15 to 22.

25. A pharmaceutical composition according to claim 24 wherein said composition is a vaccine.

26. Use of a peptide or nucleic acid as defined in any of claims 1 to 3 for increasing secretion of a peptide or protein from a bacterium.

27. A use according to claim 26 wherein secretion of an antiviral, antimicrobial or antiallergenic agent, vaccine, food additive or an enzyme is increased.

28. A use of a peptide or nucleic acid as defined in any of claims 1 to 3 in the delivery of vaccines, antiviral, antimicrobial or antiallergenic agents.

29. A use according to claim 28 wherein said agent is a cytokine.

30. A use according to claim 28 or 29 wherein said agent is delivered mucosally.

31.A use according to any of claims 27 to 30 wherein said bacterial host is a lactic acid bacterium.

32. A method for enhancing secretion of a fusion peptide or fusion protein from a bacterial host wherein said peptide comprises a signal peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1).

33. A method according to claim 32 wherein said peptide or protein is a cytokine selected from IL-2, IL-6, IL-10, or IL12.

34. A method for mucosal delivery of a peptide or protein which comprises encoding said peptide or protein in a vector in operative association with a leader peptide comprising the sequence MGKKNLRIVSAAAAALLAVAPVAATAMPVNAA (SEQ ID: 1), and optionally regulatory sequences in operative association therewith, introducing said vector into a bacterial host and administering said bacterial host to a mammal.

35. A method according to any of claims 32 to 34 wherein said bacterial host is a Gram positive bacterium.

36. A method according to claim 35 wherein said bacterial host is a lactic acid bacterium.

37. A method according to claim 36 wherein said bacterial host is selected from Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, Pediococcus, Brevibacterium, Propionibacterium or Enterococcus.

38. A method according to claim 37 wherein said bacterial host is selected from FI5876 or FI10611.

39. A food stuff comprising a host cell as defined in any of claims 15 to 22.

40. An isolated bacterial strain designated FI10611 , deposited under deposit number NCIMB 41596 at NCIMB Ltd.