"Chlamydia"is the familiar name for organisms belonging to the order Chlamydiales.

Chlamydiae are obligate intracellular bacteria that can infect a wide variety of hosts and cause many diverse forms of disease.

Chlamydia trachomatis belongs to the genus Chlamydia of the family Chlamydiaceae (Everett et al. , 1999). C. trachomatis is a parasite of humans and is not found naturally in other hosts. C. trachomatis is subdivided in three different ways. First, according to their serological reactivities with hyperimmune polyclonal or monoclonal antisera. This subdivision system enables the recognition, currently, of 15"serovars"and several serovar variants, namely A, B, Ba, C, D (and variants Da, D-and D*), E, F, G (and variant Ga), H, I (and variants la and I-), J (and variant Jv), K, LI, L2 (and variant L2a) and L3. New variants continue to be described and this list is not exhaustive. Serological differences are usually detected using a microimmunofluorescent antibody test (Wang et al. , 1973,1985) and are largely if not entirely due to differences in the Outer Membrane Protein 1 (OMP1) of the different serovars. The second system of subdividing C. trachomatis is by recognising"serogroups", of which there are three. Serogroups are clusters of serovars which are closely related serologically. Serogroup B contains serovars B, Ba, D (and its variants), E, LI and L2. Serogroup C contains serovars A, C, H, I (and its variants) and J (and its variant); and an intermediate serogroup, termed F/G, contains serovars F, G, K and L3.

The third system of subdividing C. trachomatis is into"biotypes"according to the disease induced. On this basis, three biotypes can be distinguished. The first contains the serovars A, B, Ba and C and is associated with trachoma, a transmissible condition of the eyes. The second"biotype"contains the serovars D, E, F, G, H, I, J and K; the Ba serovar is also sometimes included in this biotype and will be assumed to constitute a member of this second biotype for the purposes of this application. This biovar is associated with genital tract disease and is usually sexually transmitted, although vertical transmission from mother to infant can occur. The third"biotype"contains the serovars LI, L2 and L3; this biovar is also sexually transmitted, but the disease caused is lymphogranuloma venereum.

The family Chlamydiaceae currently contains a second genus (the Chlamydophila), within which is placed a second human pathogen, Chlamydophila pneumoniae. Of the three biovars of C. pneumoniae one ("TWAR") is an important cause of respiratory and other conditions in humans. The other two biovars of C. pneumoniae infect koalas and horses respectively. Also placed within the genus Chlamydophila are the species Chlamydophila abortus and Chlamydophila psittaci. The former is usually found in ruminants, in which it causes abortion or the production of weakly offspring; the latter is found in birds. Both C. abortus and C. psittaci can occasionally infect humans. Since all members of the family Chlamydiaceae share several antigens/epitopes in common (most notably lipopolysaccharide and heat shock proteins of-lOkDa,-57kDa and-75kDa), it is important that serological tests for C. trachomatis are capable of distinguishing between antibodies against this organism and those against other chlamydiae.

The 9"genital tract"serovars (and variants) of C. trachomatis, namely Ba, D, E, F, G, H, I, J and K, are generally sexually transmitted and cause diseases which, as primary infections, involve the genital tract. An exception is the infection of newborn infants during birth, with consequent development of ophthalmia neonatorum, a conjunctival condition. In the majority of infected people, the initial, genitally-acquired infection is

asymptomatic. In these, and in those patients which show acute signs of disease (cervicitis or urethritis), the infection may resolve without further signs developing. However, in a proportion of untreated cases, infection progresses into chronic form (s) involving other parts of the genital tract or body. In the female, the chronic manifestations of chlamydial infection include Tubal Factor Infertility (TFI), Pelvic Inflammatory Disease (PID) and Ectopic Pregnancy (EP). In the male, urethritis may by followed by prostatitis. In both sexes, arthritis ("Sexually Acquired Reactive Arthritis"or SARA) may occur as a sequel to the primary infection.

C. trachomatis is now the most common cause of genital tract disease in the world. In the UK, some 5% of the population is estimated to be infected. In the USA, the level of infection is much higher and estimated to be 8-14%, with an estimated 4 million cases annually, resulting in costs exceeding $2 billion per annum (Pearlman and McNeely, 1992). Serovars D, E, F, H and K account for nearly 85% of genital infections (Batteiger et al., 1989).

An important issue with any vaccine against Chlamydia infections is the need for any such vaccine to be effective in the absence of complement, i. e. the vaccine must be protective in the absence of the complement based components of the immune system. Such components are not present in the genital tract.

There thus exists a need to provide suitable immunogenic components for the provision of a vaccine against Chlamydia trachomatis which will provide effective protection in the absence of complement and which will also provide protection against the various serovars. We have now identified such components.

In a first aspect, therefore, the present invention provides an epitopic peptide which binds specifically to human antibody against Chlamydia trachomatis, having the sequence of any of SEQ ID NOs : 1-40.

Peptides having the sequences of SEQ ID NOs: 33-35 and 37-40 are cyclised through covalent linkages between the disulphide bridges of the residues at each end of the peptide The above epitopic sequences, or epitopes, induce the formation of antibodies to C. trachomatis in humans to which the said antibodies bind as part of the natural antigen. The term"epitopic sequence"is defined herein as that part of an antigen which reacts with an antibody and which is responsible for eliciting production of that antibody in antibody- producing B cells. The term"peptide"is used herein in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds.

It therefore includes within its scope substances which may sometimes be referred to in the literature as peptides, polypeptides or proteins (whether or not they are covalently bound to other moieties e. g. to form fusion proteins). The epitopic peptides of the invention may also be described as"immunogenic", ie the peptides are capable of eliciting a protective immune response in a subject.

As can be seen from the experiments detailed below, each of the peptides of SEQ ID NOs: 1-40 shows greater than 50% neutralisation against at least one serovar of C. trachomatis.

The sequences are derived from the Outer Membrane Protein 1 (OMP1) of C. trachomatis and the OMP2 protein. OMP1 of C. trachomatis is approximately 40 kDa and comprises some 372 amino acids (the different serovars show minor variations in this aspect). OMP1 constitutes approximately 60% of the outer membrane of Chlamydia trachomatis by weight and acts as a porin in regulating intake/expulsion of metabolites. The molecule

comprises 5 constant and 4 variable regions, the latter known variously as Variable Segments (VS) or Variable Domains (VD) 1 to 4.

It should be noted that the above epitopic sequences are specific to antibodies found in human serum. Surprisingly, epitopes for C. trachomatis show a great deal of inter-species specificity, so that, for example, epitopes identified using rabbit serum may not react with human serum at all.

The skilled person will appreciate that homologues or derivatives of the peptides of the invention will also find use in the context of the present invention, i. e. as immunogenic material. Thus, for instance peptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar"type". For instance replacing one hydrophobic amino acid with another.

The"percent identity"of two amino acid sequences can be or is generally determined by aligning the sequences for optimal comparison purposes (e. g. gaps can be introduced in either sequence for best alignment with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The"best alignment"is an alignment of two sequences that results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i. e. , % identity = # of identical positions/total # of positions x 100).

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified as in Karlin and Altschul (1993) Proc.

Natl. Acad. Sci. USA 90 : 5873-5877. The NBLAST and XBLAST programs of Altschul,

et al. (1990) J. Mol. Biol. 215: 403-410 have incorporated such an algorithm. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.

Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e. g., XBLAST and NBLAST) can be used. See www. ncbi. nlm. nih. gov.

Another example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the GCG sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci. , 10: 3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

In the case of homologues and derivatives, the degree of identity with the peptides described herein is less important than that the homologue or derivative should retain the immunogenicity of the original peptide. However, suitably, homologues or derivatives having at least 60% similarity (as discussed above) with the peptides described herein are provided. Preferably, homologues or derivatives having at least 70% similarity, more preferably at least 80% similarity are provided. Most preferably, homologues or derivatives having at least 90% or even 95,96, 97,98 or 99% similarity are provided.

In particular, the peptide may have the sequence of any of SEQ ID NOs: 3 and 33-40.

In the second aspect, the present invention provides a compound comprising one or more of the epitopic sequences of the first aspect. The compound may be a peptide.

The sequences in the compound may be the same or different. Thus, compounds which contain two or more epitopic sequences, and are therefore multimers (such as dimers, trimers or tetramers) and/or chimeric are also encompassed within the present invention.

In particular, the compound may comprise at least one of SEQ ID NOs : 3 and 33-40. For example it may comprise two, three, four, five or all six of those sequences.

In total, the compound may comprise at least 5, for example at least 10,15, 20,25, 30 or 35 of SEQ ID NOs: 1-40.

In multimers, the repeated epitopic sequences may be linked together with one or more linker molecules. The linker molecule may comprise amino acid residues which are considered not to contribute directly to the epitope reactivity. For example, a single amino acid may be used, such as serine, lysine, glycine, asparagine, tyrosine or arginine. Glycine, asparagine, serine and lysine are preferred. A linker comprising more than one amino acid, such as a serine-glycine motif, may also be used. The linker molecule may also be, for example, biotin, which may act as both a spacer and as an attachment link to, for example, streptavidin.

Separation of epitopic sequences within a compound with a linker has the advantage of ensuring better epitope definition. The linker molecule may also be the amino acids found in the natural sequence. Three or more of such amino acids may represent ancillary epitopes.

It will be recognised by the skilled person that, within the compound defined above, substitutions and/or deletion of one or possibly more of the amino acids constituting the compound can be made without excessive decrease in the reactivity and/or specificity.

Such variants of the compounds described above, which are functionally equivalent to the present compounds but contain certain amino acid residues which may be non-naturally occurring, modified and/or synthetic, are within the scope of the present invention, if they are recognised by antibodies specific to C. trachomatis or to a specific serovar of C. trachomatis. Such derivatives/homologues form another aspect of the invention. The skilled person would be aware that the antibody binding ability of epitope analogues containing, for example, single amino acid substitutions, may be determined using a suitable scanning technique.

The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that peptide. For example, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains).

Other amino acids that can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).

Substitutions of this nature are often referred to as"conservative"or"semi-conservative" amino acid substitutions. Amino acid deletions and insertions relative to a peptide as

defined above can also be made. However, substitutions can also be made which are not explicable by side-chain morphology alone.

Epitopes incorporating amino acid changes (whether substitutions, deletions or insertions) relative to the epitopic sequences as defined above can be provided using any suitable techniques. The epitopic sequences or the compounds of the present invention can be prepared by any suitable conventional processes for synthesising peptides. Such techniques generally utilise solid phase synthesis. Chemical synthesis techniques that allow peptides having particular sequences to be produced have now been automated. Apparatus capable of chemically synthesising polypeptides is available, for example, from Applied Biosystems. If necessary, short peptides can be synthesised initially and can then be ligated to produce longer peptides. The peptides or the compounds may also be produced as larger fusion proteins, comprising more than one of the above-defined peptides, of the same type or different type, which are then split up by appropriate techniques.

In a third aspect, the present invention provides a composition comprising at least one epitopic sequences or compounds as defined in the first or second aspects, and optionally one more excipients, carriers, diluents and/or adjuvants. The epitopic sequences or compounds in the composition may be the predominant component present (i. e. where it is present at a level, when determined on a weight/weight basis, of more than 50% of the total composition; preferably at a level of more than 75%, of more than 90%, or of more than 95% of the total composition).

Alternatively, when incorporated in polymer microspheres comprising e. g. poly-L-lactide for administration by the nasal/oral route, the proportion of compounds is generally of the order 80% polymer: 20% peptides. Thus polymer microspheres incorporating the epitopic sequences or compounds of the present invention may comprise, by weight of the total

composition, at least 15% epitopic sequences or compounds according to the present invention, for example at least 20% or 25%.

Preferred features of the first and second aspects apply mutatis mutandis.

In particular, the compositions of the third aspect of the present invention may comprise at least one of SEQ ID NOs: 3 and 33-40. For example it may comprise two, three, four, five or all six of those sequences.

The composition may comprise at least 5, for example at least 10,15, 20,25, 30 or 35 of SEQ ID NOs : 1-40.

In a fourth aspect, the present invention provides the use of an epitopic sequence or a compound or composition as set out in the first, second or third aspects of the invention in medicine. The term"medicine"used herein includes diagnosis, prognosis and treatment, as applied to humans, i. e. includes any treatment which is designed to cure, alleviate, remove or lessen the symptoms of, or prevent or reduce the possibility of contracting any disorder or malfunction of the human body. The invention finds particular use in the prevention of infection of a subject by C. trachomatis.

Preferred features of the first and second aspects apply mutatis mutandis. As above, the epitopes used in the fourth aspect of the present invention may comprise at least one of SEQ ID NOs: 3 and 33-40. For example it may comprise two, three, four, five or all six of those sequences.

In a fifth aspect, the present invention provides a vaccine for Chlamydia trachomatis, comprising at least one epitopic sequences or compounds of the invention.

The epitopic sequences in the vaccine of the present invention all bind to a human antibody against C. trachomatis.

Thus, at least one epitopic sequences from any or all of the above may be included in the vaccine. In particular, the vaccine may comprise at least one of SEQ ID NOs: 3 and 33-40.

For example it may comprise two, three, four, five or all six of those sequences.

The vaccine may comprise at least 5, for example at least 10,15, 20,25, 30 or 35 of SEQ ID NOs: 1-40.

The above epitopic sequences are B cell epitopes and as such are capable of inducing antibody formation if presented correctly to the designated host. Correct peptide presentation for the induction of antibody production usually requires its conjugation to a carrier protein. A simple, commercially-available system (Perbio) comprises coupling the peptide to a maleimide-activated carrier protein such as keyhole limpet haemocyanin.

A skilled person would be well aware of the relevant procedures.

This protein-conjugated water-soluble compound may then be formulated with an adjuvant such as alum or oil adjuvants e. g. Incomplete Freund's Adjuvant, Seppic formulations, MDP. An immunostimulant such as Quil A may additionally be included. Another adjuvant system uses so-called Immune Stimulating Complexes [ISCOMS], which are formulated from cholesterol, phosphatidyl choline and Quil A. Oil, alum and ISCOM adjuvanted formulations are generally administered by the parenteral route, most commonly subcutaneously.

Preferably, the vaccine is administered by a mucosal route such as the nasal-oral, rectal or vaginal. One formulation system suitable for administration by the mucosal route comprises the use of microspheres, which contain the relevant antigens, enable adhesions

of the particles to the mucosal surface and also often contain an immunostimulant such as cholera toxin p-subunit or Escherichia coli heat labile toxin.

In a further aspect, the present invention provides a method of selecting an epitope for inclusion in a vaccine against a target organism, comprising the steps of : i) attaching a compound, which comprises an isolated epitopic sequence, to a device; ii) exposing the device to (e. g. contacting it with) a biological fluid; iii) removing any antibody which has bound to the peptide; and iv) optionally assaying the antibody to determine its reactivity against the target organism.

The biological fluid may contain relevant antibodies-for example it may be a serum sample from a patient. The compound which is attached to the device may comprise one of SEQ ID NOs : 3 and 33-40.

The peptide specific antibody preparations thus prepared are termed"Mono-Epitope- Specific, Polyclonal Antibody Preparations"or MESPAPs.

The use of isolated, specific epitopic sequences, as opposed to antigen preparations, in the selection of antibodies enables the provision of a preparation of antibodies which is specific to a single epitope, rather than a spectrum of different antibodies, each antibody reactive to a different epitope found in the antigen. This method allows the obtaining of monospecific but polyvalent antibodies which are functionally equivalent to monoclonal antibodies, from the biological fluid, e. g. polyclonal serum.

In step (i), an epitope is formulated as a compound. As defined above, an epitopic sequence, or"an epitope", is that part of an antigen which reacts with an antibody and

which is responsible for eliciting production of that antibody in antibody-producing B cells. Thus, for example, the epitopic sequences shown above react with antibodies against C. trachomatis. These epitopic sequences may be used in this method to determine suitability for inclusion in a vaccine against C. trachomatis.

A compound is formulated from an epitopic sequence. Preferably the compound is a peptide.

The compound is attached to a device. The attachment may be by any suitable means but preferably involves a biotin molecule which is attached at the N-terminal end of the compound. Biotin has a strong affinity for avidin and streptavidin, which enables the attachment of the compounds to a device. Any suitable system which provides good attachment of the compound to the device may be used.

More than one copy of a compound may be attached to a device. Preferably, at least ten copies of a particular compound are attached to a device. More preferably, the compounds are attached to the device at saturation level. As discussed above, only one type of epitopic sequence is found in one compound.

The device may be any device which is suitable for exposing the compounds attached thereto to a biological fluid. Preferably, the device is a magnetic bead (e. g. Dynal RTM).

It may however also be a gel formulation (e. g. Perbio RTM).

Depending on the method used to attach the compounds to the device, the device may be coated with an appropriate reagent or molecule. Thus, for example, if biotin has been attached to the compounds, then the device will need to be coated with streptavidin or avidin to allow binding of the compounds to the device.

In step (ii), the device is exposed to a biological fluid. Preferably, the biological fluid is serum and is more preferably human serum. The serum may be constituted from individual serum samples each containing high titres of polyclonal antibodies against the target organism. The biological fluid may however also be any fluid which contains or is likely to contain an antibody for the target organism.

The exposure of the device to the biological fluid may take any form which is suitable for the device. Thus, for example, if the device is a magnetic bead, it may be added to a serum then withdrawn using a magnet. If the device is avidin or streptavidin-containing gel, then it would be appropriate to add a serum to the gel which is contained within a column.

The exposure of the epitopic sequences to the biological fluid results in the binding of any antibody which is present in the fluid to the device via the compound, if that antibody recognises the epitopic sequence attached to the device. In this way, antibodies which recognise a particular epitopic sequence are"fished out"of the fluid.

In step (iii), the antibody which attached to the device is removed from the device. The device may be washed with buffer and the antibody eluted off under appropriate conditions which would be apparent to the person skilled in the art, for example under acid pH (2.5), which would disrupt the binding of the antibody to the epitopic sequence.

The antibody preparation which is obtained is a monospecific suspension that recognises only one epitope.

Steps (ii) and (iii) may be repeated with the same device, to ensure complete removal of the sub-population of antibodies specific for the epitopic sequence.

Preferably, all the antibody preparation obtained after step (iii) is adjusted to the same protein level using a proprietary method for estimating protein levels e. g. Pierce's BCA system or Bradford reagent. The preparations may also be examined by ELISA, to ensure comparability with the complete (parent) antiserum pool in terms of reactivity (optical density).

In optional step (iv), the antibody preparation obtained after step (iii) may be assayed for its ability to neutralise the target organism. Any suitable assay known to the skilled person may be used. In general terms, the steps of such an assay may comprise the following: 1. The antibody is mixed with a predetermined optimal number of the target organism for an optimised time period. The mixture may or may not also contain a source of complement.

2. The antigen-antibody mixture, along with controls, is then seeded onto tissue culture cells, such as for example Syrian hamster kidney cells, HeLa cells, Buffalo Green Monkey or McCoy cells.

3. The tissue culture is then treated by incubation at 37 °C for 2 hours, preferably in a shaking water bath. Further tissue culture medium, usually containing a compound such as cycloheximide, is then added.

4. The cultures are then fixed, stained by Giemsa using conventional methods or preferably by the fluorescent antibody staining technique, and examined for the number of inclusion bodies present. A significant reduction in the number of inclusion bodies compared with controls indicates that the antibody preparation tested had a neutralising effect.

The invention is now described with reference to non-limiting examples.

EXAMPLES METHODS Reactive peptides were identified using antisera from humans One pool of 50 human sera was used, these sera deriving from patients known to be positive for Chlamydia trachomatis, as tested using a commercial kit (Savyon Diagnostics). Equal volumes from all 50 samples were added together to generate the pool.

Chlamvdla trachomatis culture and harvest protocols : Chlamydia trachomatis serovars were obtained from the ATCC, details of which are as follows: Serovar Ba- (Apache-2) Serovar D (UW-3/Cx) Serovar E (BOUR) Serovar F (IC-Cal-3) Serovar G (UW-57/Cx) Serovar H (UW-43/Cx) Serovar I (UW-12/Ur) Serovar J (UW-36/Cx) All strains supplied were described as free from mycoplasmal contamination.

Cell culture growth in HaK, McCoy or BGM cells : Empty growth medium out of flask, wash cell sheet with prewarmed PBS then discard. Add trypsin/EDTA to cells, approximately 4 ml to a 75 cm2 flask and 2 ml to a 25 cm2 flask. Swirl the PBS around the cells, then place the flasks in the incubator for 2-5 minutes [BGM and HaK cells are more difficult to remove than McCoys

and require a longer incubation period]. The detached cells are diluted in growth medium at ratios of between 1 to 5 to 1 to 10 (depending on cell type and degree of confluence) to achieve monolayering in 3 days. The cell growth medium is pre-warmed in a water bath to 35-37°C before addition to the cells. The cell suspension is then dispensed into tissue culture flasks, 10 ml to a 25 cm2 flask and 20 ml to a 75 ci flask, then placed in an incubator at 37°C with 5% CO2.

Inoculation and culture of Chlamvdaa trachomatis in HaK, McCoy or BGM cells : Only use flasks once they have monolayered satisfactorily. Prepare chlamydia inoculum as follows: 'If inoculum removed from-85 °C, add 2-5 glass beads to the vial and vortex for 2 minutes. Make up the volume to 5 ml with chlamydia growth medium (prewarmed to 37°C) for each 25 cm2 flask inoculated.

If the inoculum is a"hot"pass, see below for details Discard growth medium in flask (s). Add chlamydia inoculum to flasks then place flasks on centrifuge plate carriers and centrifuge at 2000 x g/30 minutes/25°C.

Remove flasks and place in an incubator at 37°C for 90 minutes. Add further 5 ml of chlamydia growth medium to each 25 cm2 flask. Incubate at 37°C/5% CO2 for 3-4 days, examining daily for the presence of inclusion bodies within the cells using an inverted tissue culture microscope.

Protocol for harvesting chlamydiae from cell cultures.

Pour off supernatant from infected flasks into Universals.

Add 25-30 sterile glass beads to each flask.

Vortex flask for 2 min.

Add supernatant back into original flask and shake gently to suspend the cell debris. Pipette off supernatant plus cells back into the Universal.

Discard glass beads Centrifuge Universal at 1000 rpm for 10 minutes at 25°C.

Remove supernatant into Oakridge centrifuge tubes and centrifuge at 12000 rpm/45 minutes/25°C.

Remove and discard the supernatants.

Resuspend the deposits in chlamydia growth medium, pre-warmed to 37°C. Note: if hot pass intended, the resuspension volume is 5 ml per flask. If harvesting to-85 °C, then the suspension volume is usually 2 ml per harvested flask. If making a hot pass, add-20 glass beads to each Oakridge centrifuge tube and vortex for 2 minutes prior to adding the suspension to new flasks.

Cell growth medium: lx Eagles Minimal Essential Medium (Invitrogen; code no. 21090) 500 ml Fetal Bovine Serum 50 ml MEM nonessential amino acids (100x) 5 ml L-glutamine (100x) 5 ml Mix reagents well, place bottle at 37°C for 3 days for a sterility check and then store at 4°C until required.

Chlamydia growth medium: This comprises cell growth medium as described above with the addition of sterile filtered cycloheximide to a final concentration of 2 llg/ml. Preparation of mono-epitope-specific polyclonal antibody preparations (MESPAPs) Reagents:

1. Binding and washing buffer (B & W) (1M NaCl, 10 mM Tris-HCI, 1 mM EDTA in deionised water pH to 7.5) 2.100 mM glycine pH 2.5 (7.507 g glycine in deionised water pH adjusted to 2. 5 with I M HCI) 3.2M phosphate buffer pH 8 (1M NaH2PO4 and 1M Na HPQ in deionised water, adjusted to pH 8 with 5M NaOH) 4. Magnetic particle concentrators (MPC, Dynal UK Ltd, Product code 120.2 and 120.01) 5. Streptavidin-coated magnetic beads at 1 mg/ml (Dynal UK Ltd, Product code 602.1), 6. Antibody-positive human serum pool 7. Biotinylated peptides 8. PBS containing 40 Rg/ml Gentamycin, 5 pg/ml Fungizone Procedure: 1. Binding of peptides to beads The beads are prepared for binding the peptides by first washing with the B&W buffer. 1 ml of beads from the stock bottle (mixed thoroughly first to resuspend) are placed in an Eppendorf in the small MPC. Once all the beads have been attracted to the magnet the liquid will be clear and can be carefully removed using a pipette. Place the pipette tip furthest away from the magnet side of the tube and draw out the liquid. Then add 1 ml of B&W buffer, remove the tube from the MPC and place in the rotator. Rotate gently for 1-2 minutes until all the beads are resuspended, then place the tube back into the MPC. Again remove the medium carefully and repeat this wash step once more. The beads are now ready for binding.

Remove the stock biotinylated peptides from the freezer, defrost and dilute to a concentration of 20 pg/ml in B&W buffer. Add 1 ml to 1 mg of prewashed beads. Incubate the beads and peptides on the rotator for 30 minutes at room temperature to allow binding to take place.

Wash the beads twice by placing the Eppendorf in the MPC until the liquid clears, then removing the buffer and adding 1 ml of B&W buffer. Vortex the tube to thoroughly resuspend the beads then place back in the MPC. Remove the buffer then add a further 1 ml and repeat the wash. Following the removal of the second wash buffer resuspend the beads in 1 ml of B&W buffer and store at 4 °C for use, or if they are to be used immediately follow the protocol below.

Isolating peptide-specific antibodies from serum pools: Remove the serum pool from the freezer and thaw at 37 °C. Dilute the serum 1 : 1 in PBS containing 40 ug/ml Gentimycin, 5, ug/ml Fungizone in a Universal; 10 ml of diluted serum is required per peptide. Make up the appropriate number of separate serum pools. Gently resuspend the beads by shaking the vial, place in the MPC and remove the buffer, then add 1 set of beads to one serum pool by pipetting.

Incubate the beads with the serum for 30 minutes at room temperature on the rotator. Place the Universals containing the beads in the Universal MPC and leave for 1 minute, or until the serum clears and the beads are clearly visible next to the magnet. Carefully remove the serum into a clean Universal taking care not to disturb the beads.

Remove the Universals from the MPC and resuspend the beads in 1 ml of B&W buffer. Transfer to the original Eppendorf when fully resuspended. Add a further 500 Fl of B&W buffer to the universal to wash out any remaining beads and place this with the beads in

the Eppendorf. Place the Eppendorf in the rotator for 2 minutes to ensure the beads are washed then back into the Eppendorf MPC to isolate the beads again. Wash the beads twice more with the B&W buffer, using the vortex and MPC as described previously.

Elution of antibody from the peptides : To elute the antibody from the peptide, remove the wash buffer following the second wash then add 1 ml of 100 mM glycine, pH 2.5 to the Eppendorf and incubate with rotation for 30 minutes at room temperature. Place the tube in the Eppendorf MPC and collect the eluate into a bijou-this should contain the antibody. Add 0.75 ml of the IM phosphate buffer pH 8.0 to the bijou to neutralise the low pH elution buffer, after which the antibody can be stored.

Wash the beads 3 times using B&W buffer. For the last wash incubate the beads with rotation for 5 minutes then isolate the beads with the MPC to remove any residual traces of glycine. The beads are then ready to be used again. Following each round of elution the eluates should be pooled together in the bijou containing the neutralisation buffer.

The peptide-specific antibody"fishing"procedure should be repeated three times on the same pool of antiserum. Following this procedure, the reactivity by ELISA of the serum pool using the relevant peptide as antigen should have diminished to virtually undetectable levels; conversely, the eluate pool formed from the fishing procedure should give a good signal by ELISA.

All MESPAPs should be stored at 4°C and must be concentrated and purified within 1 week of production Concentration and purification of MESPAPs :

1. Bring the MESPAPs to room temperature for 1 hour prior to carrying out the filter procedure to ensure the buffers are fully in solution.

2. Assemble a centricon tube (Centricon 50 tubes; Millipore 4225) according the manufacturer's instructions by inserting the sample reservoir into the filtrate vial.

3. Add 1 ml of deionised water or PBS into the top of the tube taking care not to touch the filter with the pipette tip. Attach the spin cap.

4. Centrifuge at 1000 x g for 2 min until half the rinse has passed through the filter.

5. Remove the liquid that has passed through the filter and invert the spin device. Centrifuge at 1000 x g for 2 minutes to remove the remaining rinse. Discard all deionised water or PBS in the device.

6. Label each device with the MESPAP code number.

7. Add 2 ml of MESPAP solution to the sample reservoir of the appropriately labelled tube taking care not to touch the membrane with the pipette tip, and attach the spin cap. Place the assembly in the centrifuge rotor and counterbalance with a similar unit.

Centrifuge at 3000 x g for 30 minutes.

8. Remove the tube from the centrifuge and separate the filtrate vial from the reservoir. Collect the filtrate into a sterile bijou and re-attach the reservoir to the filtrate vial.

9. Add the remaining 1.75 ml of MESPAP to the sample reservoir, attach the spin cap and centrifuge again at 3000 x g for 30 minutes.

10. Remove the tube from the centrifuge and add the filtrate to that collected in step 8.

11. Add 1 ml of PBS to the sample reservoir, attach the spin cap and centrifuge at 3000 x g for 7 minutes to wash the membrane. Discard the buffer in the reservoir.

12. Repeat this wash step twice more, ensuring that all the liquid has passed through the filter at the last wash, if not centrifuge again for 3 minutes until this has happened.

13. Place the retentate vial over the sample reservoir, invert the assembly and centrifuge at 2000 x g for 4 minutes to transfer the concentrated MESPAP into the spin cap.

14. Remove the tube from the centrifuge and separate the spin cap. Using 1 ml of PBS or other appropriate diluent transfer the concentrated MESPAP from the spin cap into a sterile Eppendorf labelled appropriately.

15. Store the bijou and the Eppendorf at 4°C for evaluation of antibody content by ELISA against the original peptide. If the concentrated MESPAP is not to be used within 2 weeks, store aliquots at-20°C until required.

EXAMPLE 1 : Serum neutralisation assay for Chlamvia trachomatis in HaK cells Preparation of cells Prior to the test being carried out, the test plate was coated with HaK cells. This was accomplished using confluent monolayers of HaK cells grown in 25 cm2 flasks in a CO2 incubator. After removing the medium, the cells were washed with PBS, then with trypsin/versene. The flasks were returned to the incubator until the cells began to detach from the plastic, approximately 5 minutes. The cells were then fully detached from the plastic by repeated pipetting of the suspending medium. The cell suspension was then transferred to a Universal.

A 100 ul aliquot of the cells was removed and transferred to a bijou containing 900 ill of trypan blue and mixed. A sample of the stained cells was placed in a haemocytometer and the cells counted under the microscope. The cell suspension was then adjusted to 1 x 106 cells/ml and 100 pl pipetted into each well of a flat bottomed 96 well tissue culture treated plate. The plate was incubated overnight at 37°C and 5% CO2 to allow the cells to become confluent monolayers in the wells.

Performance of the serum neutralisation assay.

Aliquots of C. trachomatis stock were removed from the freezer and glass beads added prior to vortexing to release chlamydiae from the cells. The chlamydiae were then diluted to 5 x 104 ifu by dilution with SPG buffer (250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid; Spencer and Johnson, 1983) pH 7.2. Tubes were vortexed at each stage of the dilutions. 90 pl of chlamydial suspension was then added to each well of the preparation plate.

90 ul of undiluted MESPAP or 90 pl of SPG (as a control) were added to each well according to the plate plan. 90 ul of the C. trachomatis positive serum pool was added to the positive control wells. 9 pl of guinea pig serum was added to the mixture if required.

The mixtures were incubated in a 37°C shaking water bath for 30 minutes. 50 pl of each of the antibody/antigen preparations was then transferred to triplicate wells of the HaK coated 96 well tissue culture plate. Each well was washed with PBS prior to addition of the samples.

The plates were incubated for 2 hours at 37 °C on a rocker platform in a water bath. A further 150 al of EMEM containing 2 llg/ml cycloheximide was added to each plate, and the plates were then incubated at 37°C, 5% CO2in air.

Following incubation, the monolayers were washed with PBS. The cell sheets were fixed with absolute methanol (100 gl for 20 minutes) which was then washed off with PBS.

Serovar specific antibodies were diluted in stabilisation buffer (Dako, code number CD 210084) prior to being added to each well. The plates were then incubated for 30 minutes at 37°C wrapped in a damp towel, then all wells were washed three times with PBS.

The plates were dried with a towel, then to each well was added 50 ill of anti-mouse FITC conjugated antibody, diluted 1: 50 in Dako stabilisation buffer. The plates were incubated for 30 minutes at 37°C wrapped in a damp towel.

The stain was flicked from the wells, which were then washed three times with PBS and allowed to dry in air. The number of ifus per well was then counted under the fluorescent microscope before calculating as follows: Serum neutralising titres were expressed as a % reduction in ifus: (ifu control serum-if experimental serum)/ifu control serum x 100 A reduction in inclusion counts of more than 50% was considered positive neutralisation.

RESULTS: Peptide % neut % neut % neut % neut % neut % neut SEQ ID NO serovar Ba serovar D serovar E serovar F serovar H serovar K 1 4.25 <50% 46. 33 35. 37 2 0.69 <50% 63. 87 13. 43 3 94.67 81.2 56. 49 26. 3 4 48.14 71.2 45. 61 24. 6 5 0.00 <50% 43. 76 73.46 43.05 6 43.62 <50% 38. 39 63. 26 7 64. 6 55. 32 55. 44 8 27.07 56.6 9 71. 2 38.23 <50% 10 0 94.4 45. 22 22. 55 11 0 <50% 11.42 50 12. 3 12 5.62 71.2 30.07 76.28 13.6 13 0 <50% 21.91 66.83 90.47 14. 57 14-0 <50% 6.76 81.7 60.95 0 15 0.22 <50% 17.25 64.42 77. 55 6.68 16 1.12 <50% 21.91 65. 71 23. 69 17 0 36.6 27.74 60. 26 27. 11

18 16.11 60 11. 16 19 0 <50% 17.25 66.99 20 0 <50% 25.41 63.14 66.03 21 1.79 79.6 4. 43 0 22 9.62 <50% 0 49 73. 12 23 21.25 60.4 5.59 0 24 62.8 38. 38 84. 12 25 0 94. 8 44. 06 0 26 <50% 75 27 0 36.9 60. 09 0 28 0 <50% 44. 63 65. 37 16. 86 29 0 67.3 20. 75 32. 07 30 0 <50% 7. 93 0 51. 3 31 3.80 <50% 74. 36 0 64. 92 32 0 75.75 47. 94 0 45.02 33 12.08 <50% 49. 88 0 34 16.66 49 2. 10 0 0 35 <50% 86. 4 0 36 <50% 53. 96 0 37 0 <50% 96.89 0 38 0 <50% 6. 76 50. 64 39 <50% <50% 66.96 40 <50% <50% 67.29

References : Arno JN et al., Fertil Steril. 1995 Oct; 64 (4): 730-5; PMID: 7672143 Anttila T et al., JAMA. 2001 Jan 3; 285 (1) : 47-51 ; PMID: 11150108 Batteiger BE et al., J Infect Dis. 1989 Apr; 159 (4): 661-9; PMID: 2926160 Cerrone MC et al., Infect Immun. 1991 Jan; 59 (1) : 79-90; PMID: 1987066 Everett KD et al., Int J Syst Bacteriol. 1999 Apr; 49 Pt 2: 415-40 ; PMID: 10319462 Money DM et al., Am J Obstet Gynecol. 1997 Apr; 176 (4): 870-7; PMID: 9125613 Morrison RP et al., J Exp Med. 1989 Oct 1; 170 (4): 1271-83; PMID: 2571668 Neuer A et al., Hum Reprod. 1997 May; 12 (5) : 925-9; PMID: 9194641 Pearlman MD et al., Obstet Gynecol Surv. 1992 Ju1 ; 47 (7): 448-61; PMID: 1620525 Peeling RW et al., J Infect Dis. 1997 May; 175 (5): 1153-8 ; PMID: 9129079 Spencer WN et al., Vet Rec. 1983 Dec 3; 113 (23): 535-6; PMID: 6364542 Wagar EA et al., J Infect Dis. 1990 Oct; 162 (4) : 922-7 ; PMID: 2205652 Wang SP et al., Am J Ophthalmol. 1970 Sep; 70 (3): 367-74; PMID: 4915925 Wang SP et al., J Infect Dis. 1985 Oct; 152 (4): 791-800; PMID: 4045232 Witkin SS et al., Am J Obstet Gynecol. 1994 Aug; 171 (2): 455-60; PMID: 7520213 Witkin SS et al., J Clin Microbiol. 1997 Ju1 ; 35 (7): 1781-3; PMID: 9196193 Where"PMID :" reference numbers are given for publications, these are the PubMed identification numbers allocated to them by the US National Library of Medicine, from which full bibliographic information and abstract for each publication is available at www. ncbi. nlm. nih. gov.