WO2009040529A1

WO2009040529A1 - Bacterial vaccine

Info

Publication number: WO2009040529A1
Application number: PCT/GB2008/003245
Authority: WO
Inventors: School Of Tropical Medicine Liverpool; Anthony Hart; Alistair Craig
Original assignee: Ulive Enterprises Limited
Priority date: 2007-09-28
Filing date: 2008-09-25
Publication date: 2009-04-02
Also published as: GB0718966D0

Abstract

The present invention relates to peptide mimeotopes of bacterial capsule carbohydrates, and nucleic acids encoding such peptides, that may be used as vaccines for treating bacterial infections.

Description

BACTERIAL VACCINE

The present invention relates to medicaments suitable for use in the prevention and/or treatment of bacterial infection and particularly bacteria with a polysaccharide capsule or long chain polysaccharide attached to lipids of the bacterial membrane (lipopolysaccharides).

A number of important bacterial pathogens possess a thick hydrophilic capsule, which gives them a selective advantage in avoiding the human immune defences. The capsule envelopes the outer surface of the bacterium of Gram Positive (e.g. Streptococcus pneumoniae, Steptococcus agalactiae) and Gram Negative (e.g. Neisseria meningitidis, E. coli Kl) bacteria. The capsule expresses different epitopes that differ between different bacterial genera, species and serogroups. Other important gram negative bacterial pathogens (e.g. Salmonella typhimurium) do not possess a capsule and express lipopolysaccharide on their surface. This lipopolysaccharide enables them to escape killing by complement and neutrophils. It is known that antibodies to each of these carabohydrate structures (capsule or lipopoylsaccharide) provides protection against such bacteria.

Carbohydrates are important distinguishing antigens of many infectious agents and antibodies to carbohydrate epitopes are known to confer protection against several pathogens. Therefore they are potential targets for preventive or therapeutic vaccination. However, carbohydrates alone are generally weak immunogens and pose problems for vaccine development because of their propensity to stimulate immune responses that are T-cell independent. Consequently they generally induce low affinity antibodies (primarily IgM) at low titre, and a humoral response that neither undergoes Ig class switching nor boosting following re-exposure to antigen. Certain populations, such as children <2 years of age and the elderly >65 years of age often do not respond well to these antigens. This greatly limited the introduction of capsular vaccines since their associated disease impact was greatest in young children.

A number of approaches have been used to overcome these problems based on converting antigens from T-cell independent to T-cell dependent. For example, conjugating the capsular polysaccharide to a carrier protein elicits a good protective antibody response with the generation of B-memory cells. However, conjugate vaccines are relatively expensive to make since they require purification of the capsular polysaccharide and then chemical coupling to the carrier proteins. In addition, there are problems with the nature of the carrier, for example:

(1) concerns about over-immunization of the carrier proteins used;

(2) variation in the immune response to conjugate polysaccharide vaccine; and

(3) some capsular polysaccharides (notably Group B Meningococci and E. coli Kl) overlap considerably with human tissue antigens and thus are either poorly immunogenic or have the potential to induce auto-antibodies.

Neisseria meningitidis (i.e. a meningococcus) is an example of a capsulate bacterium that is the most common cause of bacterial meningitis and septicaemia. It is also associated with other severe infections including meningococcal arthritis and rarely pneumonia. Risk groups include infants and young children, refugees, household contacts of patients, military recruits, college students and microbiologists who work with live isolates. Case fatality rates are around 15% but survivors may have serious long-term effects such as brain damage, hearing loss, learning disability and limb amputation. Twelve serogroups of N. meningitidis have been identified and four (A, B, C and W-135) are the major pathogens. Groups B and C are the commonest types causing meningitis in Europe and the Americas. Group Y is associated with pneumonia and incidence is currently increasing in the US.

Known meningococcal vaccines are primarily paediatric vaccines that target group C only. Other vaccines are being developed which offer protection against groups A, C, Y and W- 135. However, these multi-valent vaccines contain 4 different antigens which need to be purified from each of the 4 separate strains. This multivalent approach is both time-consuming and expensive. At present there is no vaccine available against group B meningococcus, one of the main causative strains of meningitis in the US and Europe.

Group B streptococci are also capsulate bacteria that cause infection primarily among newborns, pregnant women or women after childbirth. It is the most common cause of blood septicaemia and meningitis among newborns. 10% of newborns with group B streptococcal disease die, and up to 50% suffer long-term damage.

There is also at present no vaccine against the group B streptococcus. Treatment is currently by antibiotics administered to the mother during childbirth. 30 percent of American women harbour group B streptococci, resulting in more than 1 million women treated with antibiotics every year. However, not all disease is prevented and additionally there is a risk of harmful antibiotic side effects and development of resistance.

It will be appreciated from the above that there is a need to develop new and improved vaccines against capsulate bacteria.

According to a first aspect of the invention there is provided a peptide with the sequence EIFTN (SEQ. ID. NO. I) or derivative thereof.

As explained in more detail below, the present invention is based on work carried out by the inventors in connection with a peptide: EQEIFTNITDRV (SEQ. ID. No. 2). They established that the peptide of SEQ ID No. 2 is a mimeotope of a bacterial capsule carbohydrate. The peptide may be administered to a subject and will immunise the subject against subsequent exposure to a bacteria bearing the capsule carbohydrates mimicked by the peptide.

The inventors have identified such mimeotopes (Peptide mimics) by exploiting a monoclonal antibody directed to a polysaccharide epitope to probe peptides of random amino acid sequence in a phage display library. It will be appreciated that mimeotopes may also be identified by utilising solid phase synthesis in micro-scale format. Alternatively, peptide mimeotopes may be deduced using anti-idiotype technology. BLAST searches show that this peptide is not found in a natural protein and in particular is not found encoded by human or bacterial genomes. Accordingly the inventors have found a unique peptide. The peptide EQEIFTNITDRV (SEQ. ID. No. 2) is considered to be an important feature of the invention and is highly suitable for use as a bacterial vaccine (as discussed below). However the inventors carried out further development work (see Examples 3 and 4) which identified that the core antigenic amino acids were those as defined according to the first aspect of the invention. They therefore believe that peptides consisting of, or comprising, EIFTN (SEQ. ID. No. 1) represent a particularly useful peptide for use as an antibacterial vaccine as discussed below.

Using a monoclonal antibody protective against Streptococcus agalactiae, (Group B streptococcus) Pincus et al ( 1998, J. Immunol. 160: 293-298) managed to derive a peptide mimic of the group B streptococcal type III capsular polysaccharide by means of a phage display approach. However to date no clinically useful mimeotopes have been identified. The inventors' experiments identified a number of peptides that also had limited immunogenic activity. However they were surprised to find that peptides based on EQEIFTNITDRV (SEQ. ID. No. 2) were capable of producing a strong immunogenic response that made them suitable for use as vaccines against capsular bacteria. Example 3 illustrates some of the tiling experiments conducted by the inventors to identify the important amino acids in SEQ ID No.2 that need to be conserved in order that immunogenicity may be maintained. The inventors established that it was most important to conserve at least amino acids 3 and 4 (from the N-terminal end) of SEQ ID No. 2. They also found that other positions appeared to have some significance and therefore established that peptides of the generic sequence EXEIFTNXXDXX (SEQ ID NO. 3) were useful for inducing immune responses. They then proceeded to conduct further tiling experiments (see Example 4) which established that EIFTN (SEQ. ID. No. 1) was a core antigenic component of SEQ ID No.s 2 or 3 .

By the term "derivative thereof we mean any variant of the peptide that retains the immunogenicity thereof. Such variants may comprise SEQ ED No. 1 with amino acid deletions, additions or substitutions. Preferred derivatives of SEQ ID No.l include peptides containing conservative amino acid substitutions that retain the immunogenic activity of SEQ ID No.l (as characterised by their ability to induce an immune reaction in a host suitable to prevent or reduce bacterial infection in the host).

Alternatively or additionally the variant may be a peptoid, retropeptoid or may comprise D-amino acids as discussed below.

The peptide according to the first aspect of the invention may consist of the amino acid sequence EIFTN (SEQ. ID. No. 1). However it will also be appreciated that the present invention includes peptides that comprise EIFTN (SEQ. ID. No. 1). Therefore according to a second aspect of the invention there is provided a peptide that comprises EIFTN (SEQ. ID. No. 1).

Peptides according to the second aspect of the invention may be up to 30 amino acids long, up to 20 amino acids long and are preferably up to about 12 amino acids long. The peptide may be 11, 10, 9, 8, 7 or 6 amino acids long provided that it comprises EIFTN (SEQ. ID. No.l). It is most preferred that the peptide is less that 8 amino acids long (i.e. the peptide comprises

EIFTN (SEQ. ID. No.l) and an additional 1, 2 or 3 other amino acids).

Preferred peptides according to the second aspect of the invention are: X_nEIFTNX_n (SEQ. ID. No. 4), X_nQEIFTNX_n (SEQ. ID. No. 5), X_nQEIFTNK_n (SEQ. ID. No. 6) or X_nEIFTNITX_n (SEQ. ID. No. 7) wherein "X" is any amino acid and "n" may be 0 or any number of amino acid up to 30. It is preferred that "n" is 0, 1, 2 or 3.

Suitable variant forms of the peptide of first or second aspects of the invention may be ones in which certain of the amino acids, or X in the case of the peptide of the second aspect of the invention, are replaced with amino acids having 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. X may be any of these amino acids in the peptide according to the second aspect of the invention. It is preferred that X is the amino acid found in the corresponding position of SEQ ID No. 2 or 3 or that X is an alanine residue.

Other modifications in protein sequences (such as those which occur during or after translation, e.g. by acetylation, amidation, carboxylation, sulphation, phosphorylation, proteolytic cleavage or linkage to a ligand) may provide further variant forms of the peptides of the invention.

Derivatives of the peptides may include derivatives that increase or decrease the peptides' half-life in vivo. Examples of derivatives capable of increasing the half-life include peptoid derivatives, D-amino acid derivatives and peptide-peptoid hybrids.

Preferred derivatives are peptoid derivatives that have greater resistance to degradation than do the unmodified peptides. Such derivatives may be readily designed from knowledge of the peptides' structure. Commercially available software may be used to develop suitable peptoid derivatives according to well-established protocols.

Other preferred derivatives are retropeptoids based on the peptides of the first or second aspect of the invention, (but in which all amino acids are replaced by peptoid residues in reversed order). A retropeptoid may be expected to bind a reactive antibody in the opposite direction, as compared to a peptide or peptoid-peptide hybrid containing one peptoid residue. As a result, the side chains of the peptoid residues are able to point in the same direction as the side chains in the original peptide.

A further preferred derivative comprises D-amino acid forms of the peptides of the first or second aspect of the invention. In the case of D-amino acid forms, the order of the amino acid residues comprising the derivative is reversed as compared to those in the original peptide. The preparation of derivatives using D-amino acids rather than L-amino acids greatly decreases any unwanted breakdown of such a peptide derivative by normal metabolic processes. This decreases the amount that needs to be administered and also the frequency of its administration.

It will be appreciated that peptides according to the first or second aspects of the invention (as well as many of the possible variants and derivatives thereof) may be subject to degradation by a number of means (such as protease activity in subjects treated with the peptide). This degradation may limit the bioavailability of peptides and hence the ability of the peptides to achieve a clinical effect. There are many well-established techniques by which peptide derivatives that have enhanced stability in biological contexts can be designed and produced. Such peptide derivatives may have improved immunogenicity as a result of increased resistance to protease-mediated degradation. Preferably, a peptide derivative or analogue suitable for use according to the invention is more protease-resistant than the peptide from which it is derived.

Protease-resistance of a derivative of peptides according to the first or second aspects may be compared with protease-resistance of the artificial proteins themselves by means of well- known protein degradation assays. Suitable assays for the investigation of protease-resistance are described in the prior art. According to a third aspect of the invention there is provided a vaccine comprising a peptide according to the first or second aspects of the invention.

It is preferred that the vaccine comprises: EIFTN (SEQ. ID. No. l),X_nEIFTNX_n (SEQ. ID. No. 4), X_nQEIFTNX_n (SEQ. ID. No. 5), X_nEIFTNIX_n (SEQ. ID. No. 6) or X_nEIFTNITX_n (SEQ. ID. No. 7) wherein "X" is any amino acid and "n" may be 0 or any number of amino acids up to 30. It is preferred that "n" is 0, 1, 2 or 3.

The inventors have established that peptides according to the first or second aspects are particularly effective mimeotopes that mimic the capsular carbohydrates or lipopolysaccharides found on many strains of bacteria. Furthermore, immunisation of mice with this mimeotope generated good antibody responses that showed reactivity against all strains of capsulate or gram negative bacteria tested to date (these include group B streptococcus and meningococcal groups A, B, C, X, Y, Z and W-135).

The peptides have therefore been demonstrated to be surprisingly active as broad-acting vaccines and can provide protection against many different types of disease-causing bacteria. There are no existing vaccines that target multiple pathogenic bacterial agents and as such vaccines based on the peptide according to the first aspect of the invention represent a valuable contribution to the art.

Vaccines according to the third aspect of the invention are particularly effective for immunising subjects against meningococcal and streptococcal bacteria. Accordingly the vaccines are useful for preventing a number of infections including meningitis, septicemia and pneumonia. Furthermore the vaccine has efficacy against a host of other disease-causing capsulate strains including Haemophilus influenzae (another causative agent of meningitis), Streptococcus pneumoniae (pneumonia, bacteraemia, otitis media, meningitis), and E. coli Kl (neonatal meningitis and septicaemia).

The vaccine is also effective against gram negative non-capsulate bacteria that expresss lipopolysaccharide such as Salmonella enterica, Shigella spp., Yersinia spp., Burkholderia spp., Pseudomonas spp., The vaccines are preferably used to vaccinate against the pathogenic bacteria identified in

Table 2 below and most preferably to vaccinate against N. meningitidis (Types A, B & C) and pathogenic E.coli (e.g. K12).

Advantages of peptide mimeotope vaccines according to the invention include:

(a) In some embodiments a single inoculation (effective against a broad spectrum of bacteria) can replace multiple vaccines (against individual bacterium). There are currently no vaccine products on the market with activity against multiple bacterial pathogens and the vaccine according to the invention is effective against a wide range of different strains.

(b) Vaccines according to the invention represent the first effective vaccine against group B meningococcus (one of the main causative strains of meningitis in the US and Europe)

(c) Vaccines according to the invention represent the first effective vaccine against group B streptococcus, for vaccination of mothers during pregnancy

(d) Vaccines according to the invention constitutes a defined single-component vaccine, rather than an attenuated whole organism, with a reduced risk of adverse reaction in patients

(e) Vaccines according to the invention are less expensive to manufacture than current products. Current marketed meningococcal and pneumococcal vaccines are either polysaccharide or polysaccharide-protein conjugates. Polysaccharide vaccines are inherently weakly immunogenic plus carbohydrate chemistry is expensive, hi conjugate vaccines the capsular polysaccharide antigens are attached to carrier proteins to elicit greater immune response. Conjugate vaccines are expensive to make and problems can arise due to the nature of the carrier.

(f) Vaccines according to the invention comprises just one peptide and thus will be cheaper and simpler to produce. Multi-strain meningococcal vaccines being developed by Aventis- Pasteur and Chiron are multi-valent vaccines, that is, contain different polysaccharide antigens which need to be purified from each of the different strains, which is both time consuming and expensive.

(g) It is desirable to replace multiple vaccinations with a single vaccine. For instance, the paediatric vaccination schedule is overcrowded, requiring children to have over a dozen vaccinations in the first year of life. The solution of combining multiple vaccines into one inoculation has solved some of these problems but has raised concerns among the public regarding safety of these strategies. An inoculation with Vaccines according to the invention the single component mimeotope will have none of these dangers. According to a preferred embodiment of the invention, the vaccines are used as a paediatric prophylactic vaccine. The vaccine may be administered to babies and infants to protect them through life from meningitis, pneumonia and other serious infections.

In another preferred embodiment, pregnant women may be vaccinated to protect both mother and child from group B streptococcus disease.

Other applications of the vaccine according to the invention include the prevention of a range of diseases in adults and the aged. The vaccines are particularly useful as a travel vaccination to prevent bacterial infection when individuals travel abroad.

The broad-spectrum nature of vaccines according to the invention also offer significant advantages in biodefence applications where the specific nature of the threat pathogen is undefined.

In a preferred embodiment of the invention the peptides are provided in the form of a multiple antigenic peptide molecule (MAP peptide). MAP peptides can be prepared from the peptides of the first or second aspects of the invention. MAP peptides may comprise an 8 to 18 kDa molecule consisting of a central core of lysine residues with four identical peptide chains extending outward from the core. Individual peptide subunits are attached to the central core via the C- terminal carboxyl groups. The MAP peptides are often more immunogenic than using short peptides alone and can be used as an immunogen without the need to couple it to a carrier molecule, due to its large molecular weight. The MAP peptide molecule consists of approximately 90-95% of its mass as the desired peptide sequence, with only 5-10% of the mass representing the lysine core.

One method by which the suitability of peptide vaccines in accordance with the invention may be usefully investigated is by assessment of the ability of such peptides to bind to antigen presenting cells (APCs). For example, the peptide of SEQ ID No. 1 may be fluorescently labelled and incubated with suitable APCs. Examples of suitable APCs will include APCs derived from the species to which the vaccines are to be administered, and particularly APCs from tissues in which it is desired to induce immunity. Binding of peptides to the APCs may then be assessed. Preferred vaccines of the invention may be those exhibiting increased uptake by, or binding to, the APCs. It will be appreciated that these protocols may also be used to determine whether or not a variant, fragment or derivative of a peptide of SEQ DD No. 1 may be suitably used as a vaccine according to the invention exhibits Fragments or derivatives exhibiting increased APC binding may be selected as preferred vaccines. Furthermore, investigation of the binding characteristics of such derivatives or fragments may allow the design and production of derivatives or fragments having preferred APC binding characteristics. For example, such a procedure may involve the selection of fragments or derivatives having preferred APC binding characteristics; production of further fragments or derivatives ("second generation" fragments or derivatives) based on the selected derivatives; and selection of those second generation fragments or derivatives having improved binding characteristics. Such a procedure may be further repeated through third and subsequent generations of fragments or derivatives.

According to a fourth aspect of the invention there is provides a nucleic acid molecule encoding a peptide according to the first or second aspects of the invention.

The nucleic acid molecule may be a DNA molecule or an RNA molecule.

Preferred nucleic acid molecules encode the peptide of SEQ ID. No.l or a derivative thereof. It will be appreciated that due to degeneracy in the genetic code that several different nucleic acid molecules may encode the peptide of SEQ ID. No.l.

According to a fifth aspect of the present invention, there is provided an expression cassette comprising a nucleic acid molecule according the fourth aspect of the invention.

The expression cassette according to the invention preferably also comprises regulatory elements that facilitate expression of the immunogen. By the term "regulatory element", we mean a nucleic acid sequence that regulates the transcription of a gene with which it is associated, i.e. the DNA sequence encoding the immunogen. The regulatory element may comprise a promoter and other elements that facilitate translation of the protein(s) encoded by the cassette. The regulatory element may be "operatively linked" to the nucleic acid of the fourth aspect of the invention, by which we mean that the regulatory element is able to induce the expression of immunogen. Preferably, the regulatory element induces RNA polymerase to bind to the cassette and start transcribing the DNA encoding the peptide. The regulatory element may comprise promoters, leader sequences, transcription termination signals and the like. A skilled person will appreciate that such elements will be selected based upon the vector, and then subject, into which the cassette is to be inserted. Most preferred regulatory elements are discussed in Example 1.

The regulatory element may comprise an inducible promoter that allows a clinician to modulate the production of the peptide. This has the advantage that the production of the immunogenic proteins may be controlled by separate addition of a modulator of such a regulatory element.

The 5' and 3' ends of the expression cassettes may also be designed to include restriction sites that allow easy splicing of the cassettes into vectors.

According to a sixth aspect of the present invention, there is provided a vector comprising a nucleic acid molecule according to the fourth aspect of the invention or an expression cassette according to the fifth aspect of the invention.

It will be appreciated that vaccines may take the form of a "peptide" vaccine, as discussed in relation to the third aspect of the invention, or a "DNA" vaccine. DNA vaccines represent an important further embodiment of the invention. Therefore, according to a seventh aspect of the present invention, there is provided a nucleic acid according to the fourth aspect of the invention, an expression cassette according to the fifth aspect of the invention or a vector according to the sixth aspect of the invention for use as a DNA vaccine.

DNA immunization was first reported in 1992 and research into the potential applications of DNA immunization has progressed rapidly across the infectious disease spectrum (from virus to helminths). Phase I and II clinical trials of a number of DNA vaccines have already taken place. High stability and low production costs are particularly appealing features of DNA as a vaccination vehicle. The early reports suggested that DNA immunization primarily stimulates cell-mediated immunity, but it is now clear that antibody production is also induced. A few investigations have demonstrated the feasibility of peptide mimicry in DNA vaccine development, moreover, they have shown that DNA vaccination can re-direct the immune responses to carbohydrates into a ThI response profile, which is thought to be a desirable response for targeting many pathogens and tumour cells.

The inventors therefore developed DNA vaccines in accordance with the invention and have found that they are particularly effective in preventing and/or treating bacterial infection of a subject in need of vaccination. The vaccine may be administered to the subject such that a vector according to the invention transfects cells of the subject. The peptide of the first or second aspect of the invention is translated and processed inside the cell. The peptide is then transported to the plasma membrane of the transfected cell and may subsequently be released from the cell into the extracellular environment. The presence of the peptide in the extracellular environment results in an immune response that causes the subject to become immunised to subsequent exposure to a capsular, or lipopolysaccharide bearing, bacterium.

It is preferred that the expression cassette according the invention is ultimately inserted into vector that may be used as a DNA vaccine according to the invention.

It is preferable that the vaccine vector is optimised (e.g. codon optimisation) for transformation of mammalian cells; preferably primate cells; and most preferably human cells.

It will be appreciated that the vector according to the invention may comprise the regulatory element discussed above. When this is the case the expression cassette need not comprise duplicate elements. Furthermore it will be appreciated that the expression cassette may be formed by inserting nucleic acids according to the fourth aspect of the invention into an expression vector that already comprises the requisite regulatory elements.

The vector may further comprise at least one selectable marker to assist in isolation of the vectors during the manufacturing process. For example, if the vector is to be replicated in bacterial hosts, it is preferred that the selectable marker confers resistance to an antibiotic.

Vectors can also include an origin of replication (e.g., a prokaryotic ori) and a transcription cassette that, in addition to containing one or more restriction endonuclease sites, into which a DNA vaccine insert can be cloned, optionally includes a promoter sequence. Promoters known as strong promoters can be used and may be preferred. One such promoter is the cytomegalovirus (CMV) intermediate early promoter, although other (including weaker) promoters may be used

Vectors and expression cassettes according to the invention may comprise a leader sequence. A leader sequence should be matched to the recipient by species (e.g for vaccination in mice the Igkappa leader sequence is frequently used). Leader sequences are preferred for antigen presentation by MHC-class 2 for a CD4 response that tends to favour antibody production. In some cases the leader sequence is omitted to favour a MHC-class 1/ CD8 response that tends to produce CTL. Vectors for human use can include a leader sequence that is a synthetic homolog of the tissue plasminogen activator gene leader sequence (tPA) and/or an intron sequence such as a cytomegalovirus intron A.

Vectors and expression cassettes according to the invention may comprise expressable adjuvants such as CpG sequences and CTLA4.

Vectors, eg nucleic acid vectors (e.g., a plasmid) may contain a terminator sequence (i.e., a nucleotide sequence that substantially inhibits transcription, the process by which RNA molecules are formed upon DNA templates by complementary base pairing). A useful terminator sequence is the lambda T.sub.O terminator sequence. The terminator sequence is positioned within the vector in (a) the same orientation as, and in-frame with, a selectable marker gene (i.e., the terminator sequence and the selectable marker gene are operably linked) and in (b) the opposite orientation from a sequence encoding an antigen when that sequence is inserted into the vector's cloning (or multi-cloning) site. By preventing read through from the selectable marker into the vaccine insert as the plasmid replicates in prokaryotic cells, the terminator stabilizes the insert as the bacteria grow and the plasmid replicates.

Vectors may be designed such that the vector will autonomously replicate in a cell or can be used to integrate into the genome of a host cell which is a different species to the subject to be treated. This has the advantage that vectors according to the second aspect of the invention can be propagated in such a host (e.g. a prokaryote, yeast or even a different eukaryotic host) to produce bulk amounts of the vector. The vector can then be purified and formulated for use as a vaccine in a subject to be immunised. When used in such a subject it is preferred that the vector is constructed such that translation of the peptide(s) encoded by the cassette is possible but replication of the vector will not occur in the subject. Alternatively, after the expression cassette has been replicated in such a host, it may be spliced into a different vector that will then be used as a vaccine according to the invention.

When replication in a host cell is desired, elements that induce DNA replication may be required in the recombinant vector (e.g. a bacterial or yeast origin of replication). Suitable elements are well known in the art, and for example, may be derived from well known existing plasmids such as pBR322.

The vector according the invention may be a recombinant vector optimised for expression of the peptide encoded on the vector. The vector may be a plasmid, cosmid, phage or viral vector.

Preferred DNA-based vaccines may comprise vectors according to the sixth aspect of the invention that are bacterial plasmids that express protein immunogens in vaccinated hosts. Recombinant DNA technology may be used to clone cDNAs encoding immunogens of the first or second aspects of the invention into eukaryotic expression plasmids. Vaccine plasmids may then be amplified in bacteria, purified, and directly inoculated into the hosts being vaccinated. DNA typically is inoculated by a needle injection of DNA in saline, or by a gene gun device that delivers under high pressure DNA-coated gold beads into skin (see example below). The plasmid DNA is taken up by host cells, the vaccine protein is expressed, processed and presented in the context of self-major histocompatibility (MHC) class I and class II molecules, and an immune response against the DNA-encoded immunogen is generated.

Attenuated bacteria vectors may be used according to the invention (e.g. ARO-A mutants).

The vectors most often used as vaccines are viral vectors that are optimised for infecting cells of a specific subject species being treated; comprise a regulatory element that is operative in that species; and allows expression of the peptide from target cells in the subject. It will therefore be appreciated that it is preferred that vectors according to the invention are viral vectors. Such vectors may comprise sufficient nucleic acid to encode proteins that, when processed within the cell of a subject, will assemble to form a Virus Like Particle (VLP). Such vectors may be derived from Adenoviruses, Alphaviruses, Herpes simplex virus (HSV) or Adeno-associated virus

(AAV).

It is preferred that the vector is a DNA virus or derivative thereof and more preferred that the vector is a pox virus or derivative thereof. Pox viruses are large DNA viruses that have no apparent restriction in the quantity of additional recombinant DNA they can accommodate.

Preferred pox viruses include Vaccina viruses (e.g. Modified Vaccinia Ankara - MVA) or attenuated fowlpox viruses (FPV - e.g. FP9). FPV may be safely administered to humans. Other pox viruses suitable for use in accordance with the invention include Canarypox, and the highly attenuated pox virus vectors NYVAC and ALVAC. Such viruses can be advantageously grown in chick embryo fibroblasts to enable production of commercial quantities of vaccine but do not replicate in human cells. This allows doses of the vaccine to be controlled when administered to a human subject.

It is preferred that a DNA vaccine according to the invention comprises a FPV vector containing an expression cassette encoding the peptide of SEQ DD No. 1.

Examples of other suitable vectors for use as vaccines according to the invention include DNA encoding the peptide of SEQ ID No. 1 operatively inserted in to pSecTagB (Invitrogen, Carlsbad, CA. USA). An oligonucleotide encoding the peptide of SEQ ID No. 1 may be incorporated into HindIII and BamHI sites (to aid directional cloning) in the vector. Methods of producing such preferred vectors are described in Example 1.

Other preferred vectors include vectors such as pcDNAl and series, and pVax and series (plus pVaxSec) may be used a DNA vaccines. These vectors use pCMV as a promoter. Other suitable vectors are discussed in Daudel et al. (Expert Rev. Vaccines 6(1) 97-110 (2007)) Joseph et al. (Expert Rev. Vaccines 5(6) 827-838 (2006)) or Yang et al.(J. Immunol. 1059-1067 (2006)).

Vaccines of the invention may take a number of different forms depending, in particular on the manner in which they are to be administered. Thus, for example, they may be in the form of a liquid, ointment, cream, gel, hydrogel, powder or aerosol. The vaccines may be formulated following known procedures. For instance, known procedures conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc), may be used to establish specific formulations of compositions comprising peptides or vectors of the invention and precise therapeutic regimes for administration of such compositions (such as effective immunising doses of the active agent and suitable frequency of administration).

In one embodiment a pharmaceutical vehicle used in a vaccine of the invention may be a liquid and a suitable pharmaceutical composition would be in the form of a solution. Liquid vehicles may be used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The peptide or expression vector of the invention can be 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 solubilizers, emulsifϊers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water, 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 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, intradermal or subcutaneous injection. Sterile solutions can also be administered intravenously. The vaccine of the invention may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. Vehicles are intended to include necessary and inert binders, suspending agents, lubricants and preservatives. In an alternative embodiment of the invention the vaccine may be formulated for mucosal delivery. This approach has the benefit that needleless immunisation will be possible. Suitable formulations for use in mucosal delivery include nasal sprays or inhaled nebulised suspensions.

hi another embodiment, the pharmaceutically acceptable vehicle is a solid and a suitable composition is in the form of a powder or tablet. A solid vehicle can include one or more substances that may also act as flavouring agents (for oral consumption), lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the vehicle is a finely divided solid which is in admixture with the finely divided peptide or expression vector of the invention, hi tablets, the peptide or expression vector is 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 agent of the invention. 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 the situation in which it is desired to administer an agent of the invention by means of oral ingestion, it will be appreciated that the chosen vaccine will preferably be comprise a peptide and derivatives thereof or an expression vector that has, or is formulated to have, an elevated degree of resistance to degradation. For example, the vaccine may be protected such that its rate of degradation in the digestive tract is reduced.

Vaccines according to the invention may be administered to the eye, in which case a vaccine of the invention may be formulated as an eye drop.

It may be preferred that peptide vaccines in accordance with the present invention further comprise adjuvants capable of increasing and prolonging the specific immune response to antigens in the vaccine. It is particularly preferred that vaccines in accordance with the present invention may further comprise known adjuvants that are capable of intensifying T-cell proliferation and the cellular immune response. Examples of suitable adjuvants that could be used include inorganic adjuvants in gel form (aluminium hydroxide/aluminium phosphate, calcium phosphate); bacteria-derived adjuvants such as monophosphoryl lipid A and muramyl peptides; particulate adjuvants including immunestimulatory complexes, liposomes and biodegradable microspheres; adjuvants based on oil emulsions and emulsifiers, such as Freund's adjuvant or Incomplete Freund's adjuvant (IFA); saponins such as QS-21; squalene; synthetic adjuvants such as non-ionic block copolymers, muramyl peptide analogues, synthetic lipid A, synthetic polynucleotides and polycationic adjuvants such as polyarginine or polylysine.

Vaccines in accordance with the present invention may also comprise buffering agents in order to produce vaccines that are isotonic with respect to the subject to whom the vaccines are to be administered. Suitable agents that may be used in ensuring that vaccines in accordance with the present invention are isotonic may include sugars, sugar alcohols, oligosaccharides, polysaccharides, polyhydric alcohols, amino acids or lipids.

Vaccines in accordance with the present invention may be buffered to an appropriate pH level. Suitable buffers known to those skilled in the art include phosphate-buffered saline (PBS) and HEPES-buffered saline (HBS).

It will be appreciated that the amount of a vaccine of the invention that must be administered to achieve an effective immunising dose depends on a number of factors including the biological activity and bioavailability of the peptide or expression vector present in the vaccine, which in turn depends, among other factors, on the specific formulation and the mode of administration of the vaccine. Other factors in determining an effective immunising amount of a vaccine of the invention may include:

A) The half- life of the agent in the subject being treated.

B) The specific condition to be treated (e.g. immunisation to prevent bacterial colonisation or infection).

C) The age of the subject.

Frequency of administration will depend upon on, among other considerations, the biological half-life of the agent used. Typically a vaccine of the invention should be administered such that the concentration of the agent at a tissue at risk of bacterial colonisation or infection is maintained at a level sufficient to achieve an effective immunising dose.

Vaccines of the invention, may be administered by any suitable route capable of achieving the desired effect of preventing or reducing bacterial infection, but it is preferred that the vaccines be administered orally, or by injection or inhalation.

The inventors have found that the prevention or reduction of bacterial infection may be effected by the administration of a vaccine of the invention by injection. For instance, vaccines of the invention may be administered by means of intradermal intravenous or subcutaneous injection. Thus a preferred vaccine in accordance with the invention comprises an injectable solution of a peptide of the invention. Suitable formulations for use in this embodiment of the invention are considered below.

Alternatively vaccines according to the invention may be administered to the skin following standard scarification techniques.

The optimal concentration of peptide or vector to be used will be determined by a range of factors, including the nature of the medicament, the route of administration, and the tissue in which immunisation is to be achieved. The ways in which preferred concentrations may be calculated based on such factors are conventional, and will be well known to those skilled in the art.

It will be appreciated that vaccines in accordance with the invention should be administered so as to provide an effective immunising dose. For the purposes of the present invention an effective immunising dose should be considered to be a dose of a vaccine in accordance with the present invention sufficient to bring about the production of neutralising antibody and/or protection from bacterial infection, colonisation, or disease.

It is recognised that administration of an effective immunising dose may be achieved by way of a single administration (i.e. administration of a single dose of a vaccine, said dose constituting an effective immunising dose), or by way of multiple administration (i.e. administration of two or more doses of a vaccine, said two or more doses combining to constitute an effective immunising dose). The use of multiple administrations of vaccines (for example a primary dose followed by one or more booster doses) is well known, particularly in the context of live vaccines.

Vaccines in accordance with the present invention may preferably be provided in the form of dosage units. For example, in the case where a vaccine is to be administered such that a single administration is sufficient to provide an effective immunising dose, such a single administration may constitute a suitable dosage unit. Alternatively, when multiple administrations of a vaccine are to be required in order to provide an effective immunising dose (for example a first "primer" and second "booster" administration) a suitable dosage unit may comprise sufficient vaccine to provide the necessary administrations.

The amount of a peptide according to the first or second aspects of the invention to be contained in a dose of a vaccine of the invention in order to provide an effective immunising dose may be determined with reference to a number of factors. Suitable factors may include the immunogenicity of the peptide, fragment or derivative, contained in the vaccine; the route of administration by which the vaccine is administered; the relative size of the subject receiving the vaccine; and the extent of bacterial infection or colonisation from which the subject may suffer, either before or after administration of the vaccine.

In the case of peptide vaccines according to the invention, an effective immunising dose may contain between 0.1 micrograms to 10,000 micrograms of the protein, fragment or derivative. Preferably an effective immunising dose may contain in the region of 1 microgram to approximately 1,000 micrograms of the protein, fragment or derivative, and more preferably may contain about 10 micrograms to about 500 micrograms.

It will be appreciated that the amount of a DNA vaccine required for successful vaccination will depend on a number of factors. For instance, the amount required will depend upon: the efficiency of the vector for transfecting cells in the subject being treated; whether or not the expression vector is allowed to replicate in the cells of a subject being treated; the efficiency of the promoter driving expression of the peptide; and the activity of any regulatory elements that modulate expression. The dose of DNA needed to raise a response depends upon the method of delivery, the host, the vector, and the encoded antigen, and may contain from 0.2 micrograms to 20 micrograms of DNA for gene gun deliveries of DNA.

Vaccines according to the present invention may be used to prevent or reduce bacterial infection or colonisation as a monotherapy (e.g. through use of vaccines of the invention alone). Alternatively the vaccines may be used in combination with other compounds or treatments to prevent or reduce bacterial infection. Suitable treatments that may be used as parts of such combination therapies will be well known to those skilled in the art, and may include the use of applicable antibiotic and antimicrobicidal agents.

The vaccines of the invention may preferably be provided in pre-filled vessels containing the composition. Such pre-filled vessels provide advantages in terms of their ability to readily deliver vaccines, and particularly pre-sterilised vaccines, to the location at which the vaccine is to be employed. Suitable vessels may be selected with reference to the chosen formulation and the method or route by which the vaccine is to be administered, and will typically include vessels such as vials or syringes.

Vaccines in accordance with the invention may be provided in readily stored forms, such as in sealed vessels. Such readily stored forms, and/or sealed vessels, may advantageously be sterilised or sterilisable. Suitable sealed vessels may be selected with reference to the manner in which the vaccine contained therein is to be used. By way of example, in the case of vaccines to be administered by injection, suitable sealed vessels may include vials, ampoules, prefilled syringes, or the like.

Vaccines of the invention may be administered in the form of a liquid or suspension. In the event that vaccines in accordance with the invention are stored in a dried form vaccines may then be dissolved or suspended in a suitable diluent, such as distilled water prior to their use.

It is preferred that the immune response caused by vaccines, according to any aspect of the invention, produces a neutralising antibody response in addition to a cytotoxic T lymphocyte (CTL or "killer T cell") response. According to a further aspect of the invention there is provided a method of preventing, treating or reducing a disease associated with bacterial colonisation or infection, the method comprising administering to a subject in need of such prevention, treatment or reduction an effective amount of a vaccine as defined herein.

Vaccines according to the invention may be used to treat any human or non-human subject in need of treatment or reduction of disease. In particular the vaccines may be used to treat subjects such that they become immunised and will not develop an infection when subsequently exposed to a pathogenic bacteria. It is preferred that the subject is a human although it will be appreciated that the invention may be applied to other animals of veterinary importance (e.g farm animals such as cattle, sheep and pigs or pets such as dogs and cats).

The present invention will now be further described by way of illustration with reference to the accompanying Experimental Results section, Figures and Tables in which:

Figure 1 illustrates the reactivity of antibodies generated in mice immunised with a MAP of the peptide of SEQ ID No.l isolated by biopanning as described in Example 1. The data represent pooled sera from MAP immunised mice reacted with 15 bacteria strain listed in order as in Table 2 of Example 1. The data are presented in OD units (405nm). The control represents an irrelevant-MAP control based on a helminth peptide DAQPEDID. The sera were collected 2 weeks after the immunisation. Fig. IA and IB represent two separate experiments on different groups of mice.

Figure 2 illustrates types of antibody generated in mice immunised with a MAP of the peptide of SEQ ID No. 1 as described in Example 1. Fig. 2A shows the different IgG subclasses raised reacting against group Z Meningococus, group A Meningococcus, E.coli K12, E.coli K15, group B S. agalactiae, group C Meningococcus C and group B Meningococcus respectively. Fig. 2B shows IgM, IgA and IgE antibodies raised reacting against the same panel of bacteria.

Figure 3A illustrates the antibody responses as indicated by ELISA OD in mice immunised with pSecTagB plasmids containing DNA constructs of SEQ ID No. 3 (i.e. a DNA vaccine according to the invention) by GeneGun as described in Example 1. Pooled sera were used in each group (n=5) and reacted against 15 bacterial strains as listed in Table 2. Fig 3B shows the control antibody response following DNA vaccination with DNA vector alone (minus DNA constructs of SEQ ID No. 3). X-axis indicates time following start of prime-boost immunisation programmes. Mice were bled one day prior to immunising, with 2 weeks in between immunisations.

Figure 4A illustrates changes of IgG subclasses in mice immunised by GeneGun with pSecTagB plasmids containing DNA constructs of SEQ ID No. 3 as described in Example 1. Pooled sera (n=5) were used. X-axis indicates time following start of prime-boost immunisation programme. Mice were bled one day prior to immunising, with 4 weeks in between immunisations. Fig. 4B shows the IgG profile of the final antibody sample (taken at week 16) reacted against group Z Meningococus, group A Meningococcus, E.coli K12, E.coli K15, group B S. agalactiae, group C Meningococcus C and group B Meningococcus respectively.

Figure 5 illustrates the targeting of antibodies to a range of bacteria using indirect immunofluorescence as discussed in Example 1. Fig. 5 A shows that a representative sample of bacterial strains (from Table 2) bound antibodies from pooled sera from mice immunised by GeneGun with pSecTagB plasmids containing DNA constructs of SEQ ID No. 3 (i.e. a vector according to the iunvention). Fig. 5B shows binding to meningococci bacteria of pre- immunisation sera and sera from mice immunised with the DNA vector alone (minus DNA constructs of SEQ ED No. 3). It shows no antibody binding to the bacteria. Fig. 5C shows the reactivity of antibodies raised to MAP of peptide of SEQ ID No. 1 against a range of pathogenic bacteria. Data are given for antibody titres in pre-bleed and test bleed sera samples following immunisation.

Figure 6 shows the survival of mice in the challenge study described in Example 2 for control (group 1 in the study) and MAP immunised mice (A= group 2 a and B= group 2b from the study) challenged with N. meningitidis group B. N=5 mice in each of the control and two MAP groups.

Figure 7 shows pooled blood sera antibody titres as indicated by ELISA OD from control (group 1, n=5) and MAP immunised mice (groups 2a + 2b pooled, n=10) reacted against N. meningitidis group B as discussed in Example 2. Figure 8 shows the survival of mice in the challenge study described in Example 2 for control (group 3) and MAP immunised mice (groups 4a and 4b) challenged with N. meningitidis group C. N=5 mice in each of the control and two MAP groups.

Figure 9 shows pooled blood sera antibody titres as indicated by ELISA OD from control (group 3, n=5) and MAP immunised mice (groups 4a + 4b pooled, n=10) reacted against N. meningitidis group C as discussed in Example 2.

Figure 10 illustrates the effect of pooled blood sera antibody titres in mice immunised with MAPs for Hl -H 12 on the bacterium described in Table 5 of Example 3.

Figure 11 illustrates the reactivity of antibodies generated in mice immunised with a MAP of each of the small peptides shown in Table 6 in Example 4. Each graph represents the pooled sera from two MAP immunised mice, reacted with the bacterial strains as listed. The data are presented in OD units (405nm) and are shown for week 0 (pre-immunisation) and 7 and 10 weeks (after final boost) during the immunisation protocol described in Example 4.

EXAMPLE 1

The inventors investigated whether or not peptides that bind carbohydrate antigens of capsular bacteria (in this Example Streptococcus and Neisseria meningitidis) are capable of generating an immune response and are therefore useful as vaccines. The inventors further test the usefulness of expression vectors for such peptides as putative DNA vaccines.

This was carried out by selection of mimeotopes by biopanning of a phage display peptide library. A phage peptide display library was screened using a panel of antibodies to the polysaccharides of Streptococcus agalactiae (group B Streptococcu) and Neisseria meningitidis. A limited number of mimeotopes were identified. However of those tested a few peptides showing the highest binding capacity and strongest ELISA reaction were selected for immunization experiments. These mimeotopes were either synthesized as MAP (Multiple Antigenic Peptide) or oligodeoxynucleotides for constructing plasmids for DNA immunization. Mimotope-MAP immunization produced immediate (after the first injection) humoral immune responses to the corresponding antigens and also showed significant levels of surface labelling to S. agalactiae and N. meningitidis. DNA vaccination induced a range of antibody responses. The peptide of SEQ ID No. 2 exhibited a surprisingly good antibody response. This mimeotope was shown by ELISA to be able to elicit significantly greater amount of antibodies against target bacteria than the vector-only control plasmid. This response started from the first injection at week 2, was strongly enhanced after boost injection at week 6 and showed a ThI -associated profile, which was dominated by IgG2a, followed by IgGl. Antibodies from DNA immunisation with the peptide according to the first aspect of the invention reacted with the surface molecules of S. agalactiae, N. meningitidis and E.coli K5 in indirect immunofluorescence staining, indicating a possible localisation to the bacterial capsule. The inventors concluded that carbohydrate mimeotopes based on SEQ ID No.2 represent useful vaccines for the induction of humoral responses against a braod spectrum of encapsulated bacteria.

Material and methods Antibodies:

Antibodies used in screening the phage display library were purchased commercially from Biogenesis (Poole, UK) and are listed with their details in Table 1.

Bacteria:

Various strains of Streptococcus, Neisseria and E.coli strains used for this study are summarised in Table 2. Table 2

Phage-displayed random peptide library screening

Antibodies listed in Table 1 (diluted to 100 μg/ml in 0.1 M NaHCO₃, pH 8.6) were immobilised onto an ELISA plate overnight. Biopanning of plate-coated antibodies with ph.D-12 phage display peptide library kit (New England BioLabs, Beverly, MA, USA) was according to the manufacturer's instructions. Briefly, 4 X 10¹⁰ phage in 100 μl of TST (5OmM Tris- HCl/150mMNaCl/0.01% Tween-20) were added to the plate coated with antibodies. Unbound phage were washed away and bound phage were eluted with 0.2M Glycine-HCl (pH 2.2). The eluted phage were titrated, amplified and the resulting phage were subjected to the process again for a total of four rounds. From the fourth elution, 10 individual clones were isolated and sequenced.

DNA sequencing and peptide selection

The selected phage clones were amplified and purified with the rapid purification method for sequencing template according to manufacturer's instructions. Then, the templates were sequences using the -96 primer supplied with the Ph.D-12 kit. DeltaTaq cycle sequencing was performed in conjunction with an ABI Technology 373 sequencer. Each insert of phage DNA was translated by Gene Jockey II and selected peptides were aligned with each other using the multiple align program of Gene Jockey II.

MAP (Multiple Antigen Peptide) synthesis and immunisation

Four MAPs were synthesised based on the amino acids sequences of selected peptides by Merrifield solid phase chemistry (Alta Bioscience, Birmingham), comprising a lysine core linking 8 repeats of a specific 12 amino acid peptide in tandem with a promiscuous T helper cell epitope from tetanus toxin P2 (36). For immunisation, Groups of Balb/c mice (n=8) were immunised with lOOug of MAP emulsified in Freund's complete adjuvant (for the first inoculation) or Freund's incomplete adjuvant (for subsequent inoculations). MAP was administered by subcutaneous injection on each of three occasions, at intervals of 2 weeks. Mice were bled 7 days after each boosting.

Preparation of DNA constructs

The mammalian expression vector, pSecTagB (Invitrogen, Carlsbad, CA. USA) was used in DNA vaccination. Five oligonucleotides encoding peptide mimics (shown in Table 3) were synthesised by Qiagen Operon DNA synthesis Company (West Sussex UK). The ends of the oligodeoxynucleotides encoding the mimotopes were incorporated in HindIII and BamHI sites to aid directional cloning. 50 pmoles of each pair of oligonucleotides in 50 μl 1 X annealing buffer (10 niM Tris-HCl, pH 7.5/10 mM MgCl₂/! mM DTT) were heated to 9O⁰C for 5 min and then gradually cooled to room temperature over 30 minutes. 2μl of annealed oligonucleotides were used to react with 50ng pre-cut pSeTagB for ligation. The ligation wtls transformed into DH5α, colonies propagated in LB medium and plasmid DNA purified by Qiagen-tip 500. The resulting recombinant DNA constructs were verified by sequencing the flanking regions and the inserts using T7 promoter primer.

Table 3

Production of DNA gold beads for GeneGun immunisation

The protocol used in our study for producing DNA gold beads for GeneGun immunisation is derived from Eisenbraum et al (DNA Cell Biol. 1993 Nov;12(9):791-797) and Haynes et al (AIDS Res Hum Retroviruses. 1994;10 Suppl 2:S43-5). Briefly, DNA of recombinant constructs was precipitated onto 2.6 μm gold beads and used to coat the inner surface of plastic tubing. The tubing was cut into half inch lengths and stored dry at 4⁰C until required. The quantity of gold and DNA comprising each immunising 'shot' was adjusted to produce the 1 μg DNA/0.5 mg gold. The DNA pellets were expelled under a burst of Helium gas at 300psi into the epidermal layer of mice abdomen using the GeneGun.

Reverse Enzyme-linked immunosorbent assays (ELISA) for measuring the binding activities

Binding of selected phage clones was determined by reverse ELISA as instructed in ph.D-12 phage display peptide library kit. Briefly, Maxisorp plates (Nalge Nunc International) were coated overnight with selection antibodies at a concentration of 50μg/ml in 0.1M NaHCO₃ (pH8.6). Wells were blocked by overnight incubation with Blocking buffer (0.5% BSA in 0.1M NaHCO₃). 10¹² phage in 200μl TST were added and incubated for 2 hours at room temperature. The wells were washed in TST, and 200 μl of HRP-coηjugated anti-M13 antibody

(Pharmacia) were added at 1: 3000 dilution in blocking buffer for 1 hour at room temperature. Plates were washed and the assay was developed using 0.02% 2,2'-azino-bis(2- ethylbenzthiazoline-6-sulfonic acid) (ABTS) (Sigma). The absorbance at 405 nm was read on a Dynatech MR 5000 plate reader.

ELISA for mice antibody responses

Maxisorp plates (Nalge Nunc International) were coated overnight with antigens at a concentration of 2μg/ml in 0.05M carbonate buffer (pH9.6) at 4⁰C. Wells were blocked by 0.5% BSA in TST for 1 hour at 37⁰C. Mice sera were diluted 1 : 200 in 0.5%BSA/ TST and applied to plate for 3 hours at room temperature. The wells were washed in TST, and goat anti-mouse IgG (H+L) horse radish peroxidase conjugate (Nordic) was added at 1 : 3000 dilution for 1 hour at room temperature. Plates were washed and the assay was developed as described above.

IgG subclass ELISA

Isotype-specifϊc antibodies were measured by using ELISA employing isotype-specifϊc conjugates. Briefly, plates were coated overnight with bacterial antigen and blocked. Mice sera were diluted 1 : 200 and applied to plate for 3 hours. The plates then were probed with goat anti- mouse antibody conjugated with horseradish peroxidase (Bio-Rad, Hemel Hempstead, UK), IgGl (1:200), IgG2a (1:200), IgG2b (1:200) and IgG3 (1:200) for 2 hours at room temperature. Plates were washed and the assay was developed as described above.

Gel electrophoresis and immunoblotting

Proteins were extracted from parasites by boiling for 5 min in electrophoresis sample buffer (3% [w/v] SDS, 62 mM Tris-HCl pΑ 6.8, 15% [v/v] glycerol) containing 5% 2-mercaptoethanol. Insoluble material was removed by centrifugation for 5 min at 16,00Og. Extracts were fractionated on 12.5% polyacrymide gels using the Tris-glycine-SDS system with molecular mass markers (M_r; 94kDa, phosphorylase b; 67kDa, bovine serum albumin; 43kDa, ovalbumin;

3OkDa, carbonic anhydrase; 2OkDa, soybean trypsin inhibitor; 14kDa, a-lactalbumin). Separated proteins were electrophoretically transferred to nitrocellulose and the membranes were blocked by overnight incubation in 5% foetal calf serum in Tris/saline/Tween (TST: 0.01 M Tris pH 8.5 /0.15 M sodium chloride /0.1% Tween 20(39). Blots were incubated with rabbit anti-chitinase and anti-chitinase fragment antibodies at 1 :3000 dilution in TST. Goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (Nordic, 1:2000) was used to localise antibody-antigen complexes. The blot was developed using 0.05% (w/v) 3,3'-diaminobenzidine tetrahydrochloride solution.

Indirect immunofluorescence

To localize the target of DNA vaccine-derived antibody on bacteria, indirect immunofluorosence staining was used on Streptococcus, Meningicoccus and E.coli . The bacteria were grown on plates and collected by washing with PBS. The thin smear was made from bacterial suspension and left to dry. The slides were then fixed and permeabilized by exposure to absolute methanol at -20°C for 20min. After washing with PBS and blocking with 5% FCS, the slides were incubated with antibodies from DNA vaccination group 3 and group 6 in a dilution of 1 :200 for 1 hour. Fluorescence -conjugated goat anti-mouse IgG diluted in 1 :1000 were used to detect the primary antibodies and scored by immunofluorescence microscopy.

Statistical analysis

Data from the vaccination experiments were analysed for statistical significance using the student's t-test. P<= 0.05 was considered statistically significant.

Results

Five antibodies specific for carbohydrate structures on Streptococcus or Neisseria meningitidis were able to bind to the Ph.D.- 12 phage Display Peptide Library, although with different affinities as shown by the number of plaques on the first panning plates (data not show). A total of 150 plaques (30 for each antibody) were sequenced and the number of consensus sequences for each antibody were 2, 5, 5, 3 and 2 for antibodies 1 to 5 respectively (Table 1). Clone 1-6 shared same sequence with clone 2-8; clone Gl -2 screened from antibody 1 shares same sequences with a few clones screened from antibodies 3, 4 and 5. Clone G3 (EQEIFTNITDRV i.e SEQ DD No. 2) was repeatedly selected, when experiments were replicated, by antibody 3 but only with this antibody. A total of 17 phage clones containing consensus sequences were further characterised by reverse ELISA and of these six clones yielded strong ELISA signals (OD > 1.0) in the highest concentration of 10¹² phage , with good dose responses. The signal was reduced to the background levels in most clones when the dilution reached 10⁸ to 10⁴. Cross reaction for some clones among the antibodies raised against different strains and subgroups was seen in the in vitro binding experiments, but the titre of the cross reaction never reached that of the original reaction (data not show).

Three sequences obtained from the strongest ELISA reading were chosen for MAP and DNA vaccination (Table 3). MAPs of each mimeotope were synthesised as 8-branched multipeptides as well as a control MAP (MAP 4) containing a helminth peptide: DAQPEDID. Immunisation with the MAPs generated good antibodies to the MAP itself and there was some cross-reaction among four antibodies to the four MAPs (data not show). However, when the anti-MAP antibodies were reacted with a number of different bacterial extracts, there was a dramatic difference. The three anti- mimeotope MAP antibodies derived from biopanning (based on clones . 2-8, 3-2 and G3 and designated MAP 1, MAP 2 and MAP 3 respectively)) showed varying levels of reactivity (data not shown).

The inventors were surprised to find that MAP 3 (a MAP of SEQ ID NO. 2) caused an immune response to every capsular bacterium tested (see Fig. 1). Its antibody response against seven bacterial strains was mainly dominated by IgG2a, IgM and IgA and was increased compared with the reaction from preimmune-sera ( Fig. 2). The MAP 3 sequence (i.e. a peptide of SEQ ID No. 2) also exhibited significant responses when delivered by GeneGun to mice (see below) and the inventors therefore decided to characterise this peptide further in order that its suitability as a bacterial vaccine may be assessed.

DNA constructs encoding the selected peptides corresponding to MAPs 1-4 were sequenced and tested for expression by in vitro transfection of COS cells using Fugene-6 (Roche, East Sussex, UK). All constructs were in correct reading frame as checked by sequencing and positive peripheral fluorescence staining were seen when using anti- myc antibody as a probe (data not show). This indicated that all the constructs were competent to express the selected mimeotopes when introduced into animals. DNA constructs encoding mimeotopes were introduced into animals by GeneGun delivery (see Table 3). A regimen of 2μg of DNA for GeneGun delivery was used throughout the study. The antibody responses were measured using pooled sera of each group and verified by the individual responses. The mice showed weak antibody responses to all DNA constructs from the first immunisation. However, from the second immunisation onwards, mice immunised with expression vectors encoding the peptide of SEQ ID No. 2 consistently produced a significant antibody response to bacteria compared with a control (immunised with vector only). These responses were enhanced after boosted immunisation (Fig. 3) and were significant higher than the group immunised with vector alone.

To verify the responses from the pooled sera, the individual sera from the second boost immunisation were used to measure antibody responses to 15 bacteria strains. The responses in individuals vaccinated according to the invention (Group 3) were higher than that in controls (Fig. 3), the mean OD for each mouse in group 3 were: 0.7149, 0.5675, 0.5339, 0.4216, 0.5073 and in group 6 (control) were: 0.1439, 0.2219, 0.2740, 0.2161, 0.2463 respectively. The increased responses in group 3 compared to those in the vector control group 6 were statistically significant (pO.OOl).

The antibody responses of mice with GeneGun immunisation were dominated by IgG2a followed by IgGl. IgG2b and IgG3 levels were negligible. The IgG2b and IgGl titres in mice immunised with expression vectors according to the invention rose subsequent to each immunisation and remained constant following the final immunisation (Fig. 4).

To further characterise the antibodies generated by the GeneGun immunisation, the inventors used indirect immunofluorescence to show the targets of antibodies localized in the bacteria. Fluorescence can be seen in Meningococcus and Streptococcus slides (Fig. 5A), and is consistent with localization to the bacteria surface or capsular structures. Positive staining was also seen on E.coli. The slides stained with sera from mice immunisated with vector control DNA were negative (Fig. 5B).

EXAMPLE 2 Challenge study

The inventors investigated whether or not peptides based on SEQ ED No. 2, which are capable of inducing antibodies which bind carbohydrate antigens of bacteria, are capable of protecting mice from bacterial disease, and are therefore useful as a vaccine. This study involved immunisation with the peptide vaccine using a prime/triple boost vaccination protocol, then challenging with Neisseria meningitidis groups B & C to ascertain any protective effect when compared to control mice. Test bleeds were included in the protocol so that levels of induced antibodies following peptide vaccination could be monitored.

Methods Peptide vaccine

A synthetic multiple antigen peptide (MAP) repeat of SEQ. ID. No.2 was used as the vaccine substance. Freeze-dried preparations of the MAP were reconstituted with an equivalent weight/volume of Phosphate Buffered Saline (PBS) to provide a stock solution containing 5mg/ml. Aliquots of 0.1ml (each containing 500 μg) were prepared and stored frozen at -20⁰C until required. At the time of use, aliquots were thawed and mixed with the appropriate adjuvant (Complete Freund's for prime, Incomplete Freund's for boosts). The antigen preparation (for lOOμg) is lOOμl (1 in 5 dilution) antigen + lOOμl adjuvant. Control substance used was PBS with equal volume of relevant adjuvant.

Challenge substance

Neisseria meningitidis Group B (strain K454) and Group C (strain Faml8) were grown overnight and prepared on the day of challenge to produce challenge doses of 10⁸ colony forming units (CFU) per animal in a volume of 0.5ml. Each challenge dose contained lOmg human transferrin.

Animals

6 to 8 weeks old female mice (NIH strain) were used in the study. Animals' health and welfare were monitored on a daily basis, and they were fed and watered ad-libitum. Study Protocol

Table 4. Challenge study protocol. N=5 mice in each group.

Immunisation:

Aliquots of stock solution of 5mg/ml were thawed on each vaccination occasion and diluted 1 in 5 in PBS to give an antigen concentration of lOOμg per lOOμl. For the priming vaccination this antigen preparation was mixed in an equal volume of Complete Freund's Adjuvant (CFA). For subsequent boost vaccinations the antigen preparation were mixed with an equal volume of Incomplete Freund's Adjuvant (IFA). Volumes given per animal were as follows:

Prime: give 200μl in total (150μl subcut/ 50μl im) in CFA Boost: give lOOμl in total (125μl subcut/ 25μl im) in IFA

Animal Procedures:

Mice were allocated to boxes of 5 mice and allowed to acclimatise for a minimum of 3 days. On the first day of the study, T=O days, all animals were bled from the tail vein to provide a minimum of lOOμl of blood into microtainer tubes. These tubes were stored overnight at +40C and then centrifuged to provide serum. The separated serum was stored frozen at -20⁰C prior to performing antibody titre assays.

At T=O animals in Groups 2a + 2b and 4a + 4b were primed with lOOμg test vaccine in Freund's Complete Adjuvant (FCA) by injection of 150μl by the subcutaneous route and 50μl by the intramuscular route in one limb only. Animals in the negative control groups 1 and 3 were vaccinated with adjuvant only.

At T= 14 days animals were boosted with the appropriate test or control substance in IFA (each dose to contain 50μg of test substance), using a total of lOOμl (125μl by the subcutaneous route and 25 μl by the intramuscular route in one limb only. This was repeated at T=35 days and T=56 days.

At T=63 days a test bleed for serum was taken from all animals. Animals were bled from the tail vein to provide a minimum of lOOμl of blood into microtainer tubes. These tubes were stored overnight at +40C and then centrifuged to provide serum. The separated serum was stored frozen at -2O⁰C prior to performing antibody titre assays.

Challenge

At T=70 days animals in groups 1, 2a and 2b were challenged with 10⁸ CFU N. meningitidis group B by the intraperitoneal route. Animals in groups 3, 4a and 4b were challenged with 10⁸ CFU N. meningitidis group C.

Challenge stocks were prepared such that the dose was contained in a volume of 0.5ml: each dose contained lOmg human transferrin. Human transferrin was given again to all surviving animals at 24 hours post challenge.

Animals were monitored for signs of ill health twice daily and a record made of any clinical observations such as piloerection, depressed or withdrawn behaviour or other signs of clinical distress. At each monitoring point a health score was calculated for each animal based on the observations on the animals. The scoring system starts with each healthy mouse given a score of 5. The following observable symptoms resulted in cumulative deductions in health score: ruffled fur, -1, eyes shut, -1; ruffled fur and eyes shut, -2; immobile, -4. Health was regularly monitored and the mice were humanely killed when immobile.

Results

Figure 6 demonstrates that MAP immunised mice (groups 2a and 2b) challenged with N. meningitidis group B survived longer than control animals. Pooled blood sera antibody titres (reactive against N. meningitidis group B) was also higher in the MAP immunised mice when compared to controls (Fig. 7).

Figures 8 demonstrate that MAP immunised mice (group 3) challenged with N. meningitidis group C also survived longer than control animals. Pooled blood sera antibody titres (reactive against N. meningitidis group C) was also higher in the MAP immunised mice when compared to controls (Fig. 9).

The data indicate a right-hand shift in the survival curves in mice vaccinated with peptide according to the present invention compared to controls, that is, the MAP vaccinated mice demonstrated an increased survival time (delay in time to death) compared to controls. These MAP vaccinated groups also showed enhanced levels of antibodies compared to the controls.

hi conclusion, mice vaccinated with the MAP peptide elicited a greater antibody response which was correlated with increased survival time when challenged with disease-causing bacteria. This provides evidence for a protective effect of this mimeotope peptide vaccine. EXAMPLE 3

Tiling path study

This study further characterised the functional component of the peptide of SEQ ID No. 1 by defining the critical amino acid residues required for antibody reactivity. The inventors designed a study to examine the effect of changing individual residues in the peptide, specifically a range of peptides with a single alanine replacement at each position, and raising antibodies in mice from these modified peptides (see Table 4).

Table 4: Range of peptides used in tiling path study

(* Residues in the peptide mimeotope are labelled Hl to H12 from the amino-terminal)

MAPs based on the peptide sequences with single alanine replacements were used to immunise two mice using the regimen; Day 0 Immunisation lOOug MAP + FCA, IP; Day 14 Boost lOOug MAP + FIA, S/C and IP; Day 35 Boost lOOug MAP + FIA, S/C and IP; Day 56 Boost lOOug MAP + FIA, S/C and IP; Day 70 Terminal bleed. The reactivity of antibodies generated in the resulting sera were then tested by capacity for binding to a range of bacteria by ELISA.

Results

Table 5: Summary of tiling path studies

The table illustrates that several residues were found to be important in a range of bacteria, particularly Hl, H3 and H4

Figure 10 illustrates the reactivity of antibodies generated, following immunization of mice with each of peptides (Hl -H 12), for binding to the range of bacteria described in Table 5.

It can be noted from this data that in reactivity to N. meningitidis the amino acid residues 3, 4 and 6 appear to be particularly important. In view of Example 4 and the first aspect of the invention this would suggest that the critical motif to target N. meningitidis would be EIXTX. Accordingly in another aspect of the invention the peptide EIXTX may be used as a vaccination against N.meningitides. Likewise, in reactivity to e coli, n. mucosa and klebsiella oxytoca, residues 3, 4, 5 and 7 were important and according to a further aspect of the invention the peptide EIXTN may be used as a vaccination against e coli, n. mucosa and klebsiella oxytoca. However as illustrated in Example 4 the peptide according to the first aspect of the invention, IFTN (SEQ ID NO. 1), may preferably be used as an effective vaccine for all of these bacterial species.

DISCUSSION

These data illustrate that peptide and DNA vaccines based on SEQ ID No. 2 have utility as anti- carbohydrate vaccines for immunizing against a broad spectrum of bacterium. Antibody raised to the mimeotope was able to bind to a diverse array of bacteria both gram positive and gram negative, capsulate or non-capsulate and long or short chain saccharides expressed on the bacterial surface.

The inventors were able to identify a variety of peptides (see table 5) that bound to antibodies raised against capsular carboydrates. However they were surprised to find that peptides according to the invention had particular efficacy for initiating an immune response in mice that were subsequently challenged with a variety of capsular bacteria. Hence, a DNA vaccine based on SEQ ID No. 2 may be used to drive immune responses directed to the carbohydrate target.

A peptide based on EQEIFTNITDRV (SEQ ID No. 2) was selected by polyclonal antibody against serogroups A, B and C of Neisseria meningitides and also showed cross reaction with antibodies raised to the carbohydrate of type B Streptococcus. The inhibition ELISA or competition studies in this work indicated that the peptide binds at or near the carbohydrate binding site. Database comparison of the amino acid sequence of SEQ ID No. 2 did not reveal any significant homology to known molecules, indicating a novel peptide or mimic.

EXAMPLE 4

Small Peptide Study

Further tiling experiments were conducted to establish the active (i.e. antigenic) fragment of the peptide of SEQ ID No. 2 and thereby identify the peptide according to the first aspect of the invention.

A number of small MAPs were made based on the G3 peptide sequence (i.e. the peptide of SEQ ID No. 2) and used to immunize mice (as described below).

Table 6: Ran e of e tides used in tilin ath stud

MAPs based on the peptide sequences S1-S6 were used to immunise two mice using the regimen set out in Table 7. The reactivity of antibodies generated in the resulting sera were then tested by assessing their capacity for binding to a range of bacteria by ELISA. The sera were screened against a sonicated extract of 4 bacterial strains (N. meningitidis (Types A, B & C) and a pathogenic E.coli Kl 2).

Table 7: S ecific rotocol 2 mice)

DISCUSSION

Fig. 11 illustrates the reactivity of antibodies generated in mice immunised with a MAP of each of the small peptides shown in Table 6. Peptides Sl, S2 and S3 were surprisingly effective for generating antibodies that will bind to N. meningitidis (Types A, B & C) and a pathogenic E.coli Kl 2. The inventors noted that the common sequence motif for these peptides was the peptide according to the first aspect of the invention (i.e. SEQ ED No. 1) and therefore realised that this peptide was a useful carbohydrate mimeotope that could be used to immunise individuals against a subsequent challenge by a wide range of bacteria .

Claims

1. A peptide with the sequence EIFTN (SEQ ID NO. 1) or a derivative thereof.

2. A peptide comprising the sequence EIFTN (SEQ ID NO. 1) or a derivative or fragment thereof.

3. The peptide according to claim 2 wherein the peptide has the sequence: X_nEIFTNX_n wherein "X" is any amino acid and "n" is 0,1,2 or 3.

4. The peptide according to claim 3 wherein at least one X is an alanine residue.

5. The peptide according to claim 3 wherein the peptide is X_nQEIFTNX_n, X_nQEIFTNDC_n or X_nEIFTNITX_n.

6. The peptide according to claim 5 wherein the peptide is QEIFTN (SEQ. ID. No. 5), QEIFTNI (SEQ. ID. No. 6) or EIFTNIT (SEQ. ID. No. 7).

7. The peptide according to any preceding claim comprising a peptoid derivative.

8. The peptide according to any one of claims 1 - 6 comprising a retropeptoid derivative.

9. The peptide according to any one of claims 1 - 6 comprised of D-amino acids.

10. The peptide according to any preceding claim wherein at least one amino acid, or derivative thereof, is modified by acetylation, amidation, carboxylation, sulphation or phosphorylation.

11. A vaccine comprising a peptide according to any preceding claim.

12. A nucleic acid encoding a peptide according to any one of claims 1 -10.

13. An expression cassette comprising a nucleic acid sequence according to claim 12.

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

15. A DNA vaccine comprising a nucleic acid according to claim 12, an expression cassette according to claim 13 or a vector according to claim 14.

16. The use of a vaccine according to claim 11 or 15 for preventing or treating bacterial infections.

17. The use according to claim 16 wherein the bacteria are encapsulated bacteria.

18. The use according to claim 17 wherein the bacteria is a Group B meningococcus.

19. The use according to claim 17 wherein the bacteria is a strain of Neisseria meningitides.

20. The use according to claim 17 wherein the bacteria is a strain of streptococcus.

21. The use according to claim 16 wherein the bacteria is a smooth gram negative bacterium with lipopolysaccharide on the cell surface.

22. The use according to claim 21 wherein the bacteria is a strain of salmonella.

23. The use according to any one of claims 16 -22 wherein the vaccine is used to prevent or treat meningitis, septicaemia or pneumonia.

24. A method of preventing, treating or reducing a disease associated with bacterial colonisation or infection, the method comprising administering to a subject in need of such prevention, treatment or reduction an effective amount of a vaccine as defined by claims 9 or 15.