WO1998018821A1

WO1998018821A1 - Vaccines against pathogens characterized by serological cross-reaction but lacking cross-protection

Info

Publication number: WO1998018821A1
Application number: PCT/IE1996/000070
Authority: WO
Inventors: Grace Veronica Therese Mulcahy
Original assignee: Forbairt Trading As Bioresearch Ireland; University College Dublin
Priority date: 1996-10-31
Filing date: 1996-10-31
Publication date: 1998-05-07

Abstract

A vaccine against pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection, such as parasites within the Phylum Apicomplexa. The vaccine comprises as an immunogen an amino acid sequence from a conserved, non-immunoreactive region of an antigen of the pathogen. The immunogenic sequence is identified by selecting a region of an antigen from the pathogenic organism, binding a series of overlapping sequences from said region to solid phase supports and probing said sequences for reactivity with test sera from an animal infected with said pathogen, and selecting one or more sequences on the basis of failure to bind antibody in test sera.

Description

VACCINES AGAINST PATHOGENS CHARACTERIZED BY SEROLOGICAL CROSS-REACTION BUT LACKING CROSS-PROTECTION

5 Technical Field

This invention relates to vaccines for use in conferring immunity against pathogens which are characterised by serological cross- reactivity accompanying lack of cross-protection and to a method for identifying amino acid sequences for use as the immunogen in such 10 vaccines.

Background Art

The invention described herein has broad applicability as regards pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection but the invention will be 15 illustrated for convenience with reference to parasites within the Phylum Apicomplexa.

Organisms within the Phylum Apicomplexa, which are pathogenic in domestic animals and man, include those in the genera Eimeria, Toxoplasma, Cryptosporidium, Babesia and Plasmodium. 20 The problems associated with developing conventional vaccines for the control of these organisms are typified in the case of the genus Eimeria.

The genus Eimeria contains at least seven species which cause the economically important disease of avian coccidiosis in domestic 25 chickens. Other species cause disease in other domestic poultry and in ruminants. Avian coccidiosis can affect the health and productivity of breeder birds and broilers in deep litter houses during the crucial 42- day production cycle. Infection spreads within the house via oocysts which are passed in the faeces of infected birds. The oocysts become infective in warm, humid conditions, and are extremely persistent in the environment, such that cross-infection between sequential batches of birds in the same house occurs. Modern poultry production is largely dependent on the use of in-feed medication (coccidiostatic drugs) to keep the level of coccidial infection in houses down below clinically and economically significant levels. Management procedures, including humidity control, are also important. While there are some live and modified live vaccines available, the cost of these renders them practicable for use only in more valuable breeder flocks, and not in broilers. The high cost of production of these vaccines is determined by the necessity to produce the constituent oocysts in chickens and the requirement of including several species in each vaccine preparation. Protective immunity following infection is largely species-specific.

Various vaccination protocols have been adopted which are referred to below and which involve using either virulent (viable oocysts) or attenuated "precocious" strains. However, these protocols, irrespective of the nature of the vaccine, have been limited by the species-restriction of the response.

Protective immunity following infections largely species-specific (Rose, M.E. (1973) Immunity: In: The Coccidia. D.M. Hammond and P.L Long eds., University Park Press, Baltimore U.S.A., 295-341). The basis of the protection is incompletely understood. However, correlations between protection and cytotoxic T-cell activation, but not antibody production, have been observed (Bumstead, J.M. et al., (1995) Parasitology 111, 143-151); Talebi, A. and Mulcahy, G. (1995) Avian Pathology 24, 485-495. Paradoxically, however, birds infected with one Eimeria species develop antibodies and T-lymphocytes which cross-react broadly with other species (Talebi, A. and Mulcahy, G. (1995) Avian Pathology 24, 533-544. The prevailing opinion has been, therefore, that cell-mediated responses are important in protection, with antibody playing only a minor role (Lillehoj, H.S. (1987) Veterinary Immunology and Immunopathology, 13, 321-330). The solidly protective immunity induced in chickens by small doses of viable oocysts (that is, by controlled infection), has been used as the basis of vaccination procedures. Such vaccines are usually administered as trickle infection, over the first few weeks of life, in feed, and provide immunity against those Eimeria species which are incorporated in the vaccine (Davis, P.J. et al., (1986) Immune response of chickens to oral immunization by "trickle" infections with Eimeria. In: Research in Avian Coccidiosis - Proceedings of the Georgia Coccidiosis Conference. Mc Dougald L.R., Joyner, J.P. and Long P.L., eds. Georgia University, Athens, Georgia, U.S.A., 618-633). More recently, vaccination protocols based on attenuated, "precocious" strains have been introduced (Shirley, M.W. and Long, P.L. (1990)

Control of coccidiosis in chickens: immunization with live vaccines, in: P.L. Long (Ed) Coccidiosis of Man and Domestic Animals, 321- 341 (Florida, Boca Raton, CRC Press Inc). While these vaccines deliver effective immunity without risk of causing disease, they, like the fully virulent vaccines above, are limited by species-restriction of the response, and hence the necessity of incorporating several species in a single vaccine. These products, therefore, which require the growth of the constituent oocysts in chickens, are expensive to produce, and currently, because of economic constraints, used only in breeder flocks, not broilers.

There have been many attempts to produce sub-unit or recombinant vaccines for use in the control of avian coccidiosis. These, for the most part, have used conventional techniques for the identification of putative immunogens. While several of these vaccines have produced partial protection against challenge, none has yet resulted in a commercial vaccine. Some, however, have shown that such sub-unit vaccines, unlike complete parasite vaccines, can induce a degree of cross-species protection (Crane, M.S.J., et al, (1991) Infection and Immunity, 59: No. 4. 1271-1277). Most of the attempts to produce a sub-unit vaccine for the control of avian coccidiosis have been focused on antigens identified in the invasive stages of the parasite, namely, sporozoite and merozoite. Only a very few of the antigens so far used in vaccination trials, have been identified by anything other than conventional methodology, involving usually the construction of cDNA libraries, and screening with monoclonal antibodies or sera from convalescent chickens. One exception which, uniquely, shows that antibody can mediate protection against Eimeria in chickens, is a transmission-blocking vaccine which uses gametocyte antigens, administered to hens, to provide maternal immunity to chicks, via yolk immunoglobulins (Wallach, M„ et al, (1992), Infection and Immunity 60: No. 5. 2036-2039). This latter approach, while demonstrating the premise that the mechanism of vaccine-induced immunity is not necessarily, or even desirably, the same as that induced by infection, has not yet succeeded in discriminating between protective, non-protective, and anti-protective elements in the immunogenic preparation.

The need for multivalency within the conventional vaccines for the control of avian coccidiosis currently available illustrates an important point about the nature of the immune response to Eimeria and like infections and the limitations of conventional vaccines. Although birds which recover from infection are immune, the resistance to re-infection applies only to the species which caused the infection. These cross-reactive responses, therefore, are not protective, and infection and conventional vaccination induce only species-specific immunity.

The characteristics of synthetic peptides as immunogens are well- recognised. Notably, they are capable of inducing much more broadly cross-reactive responses than native antigens. (Doel, J.R. et al., (1990) Journal of Virology, 64, No. 5, 2260-2264.

Patorroyo, M.E. et al., ((1987) Nature 328, 629-632)have shown that synthetic peptide immunogens can provide the basis for vaccines capable of inducing protective immune responses against apicomplexan pathogens, in this case the causal agents of human malaria.

Thus, there is a need for vaccines against pathogenic parasites in the Phylum Apicomplexa and in the wider domain against other pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection. Disclosure of Invention

The invention provides a vaccine against pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection, which vaccine comprises as an immunogen an amino acid sequence from a conserved, non-immunoreactive region of an antigen of said pathogen.

The vaccine according to the invention is applicable to parasites within the genus Eimeria, to other apicomplexan organisms, and in the wider domain, to other pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection. This seeming paradox is difficult to understand when it is considered that these classes of pathogens typically share common mechanisms, structures, and ligands, for gaining access to host cell targets (Taylor, D.W., et al., (1990) Journal of Protozoology 57:540-545). It can be resolved, however, by considering the possibility that such organisms have evolved strategies for camouflaging crucial functional residues with highly variable, but immunodominant regions. Infection with one variant, therefore, may allow for the development of a degree of protective immunity - for example through steric hindrance of important structures by antibody bound to adjacent regions, but will not affect the intra-host life cycle of another variant initiating a subsequent infection, because the flanking regions this time will be different.

The design of effective vaccines for the control of diseases refractory to conventional vaccine technology relies heavily on the strategic incorporation of appropriate B-cell and T-cell epitopes in an immunogen, and presentation to the target species via an appropriate adjuvant or delivery system. However, another important consideration, as illustrated by the present invention, is the exclusion of epitopes which do not contribute to protective immunity, and which may interfere with it through the induction of inappropriate responses, or masking of protective responses. As shown herein synthetic peptide immunogens according to the invention are capable of inducing responses to epitopes normally unrecognised after infection or conventional immunisation.

Preferably, the peptide immunogen according to the invention has the characteristics of both a B-cell epitope and a T-cell epitope and thus can stimulate both B-cells and T-cells.

However, in the case where the peptide does not have the ability to stimulate a T-cell response or an adequate T-cell response, then the vaccine can include a T-cell epitope which is known to elicit the requisite T-cell stimulation.

Suitably, the pathogen is a parasite within the Phylum Apicomplexa and the antigen is an antigen of an apicomplexan organism.

Preferably, the antigen is a surface antigen. However, the antigen is not necessarily a surface antigen. Thus, the vaccines can be used against secretory antigens.

By carrying out epitope mapping experiments it is found that certain regions of surface antigens of pathogens of the type defined herein are never recognised by the immune system of an infected host. However, one would expect these "non-recognised" areas or regions to be recognised, because they occur in hydrophilic regions which implies that they should be accessible and, therefore, should be good epitopes.

Furthermore, in the case of Eimeria species, some of these regions are common to several such species, which implies that they are conserved between species. Such conservation usually implies a functional importance. The fact that these conserved regions are not recognised suggests that they may be deliberately "hidden" so as to be shielded from the immune system of a host organism. By using a vaccine according to the invention one presents the immune system with antigens which are protective, but which are not recognised following infection or conventional vaccination as indicated above. By using conserved, non-immunoreactive regions of parasitic antigens, one can present the host with such antigens in a manner which enables the host to recognise previously unrecognised areas and, thereby, overcome the 'shielding' phenomenon observed with such regions when conventional vaccines are used.

The amino acid sequence preferably consists of at least ten amino acids.

To potentiate the immunogenic response, the vaccine preferably contains a number of copies of said amino acid sequence.

Alternatively, or additionally, the vaccine can contain more than one amino acid sequence from a conserved, non-immunoreactive region. When the vaccine contains more than one amino acid sequence, the sequences are preferably intercalated.

In order to potentiate the immune reaction, the or each amino acid sequence can be disposed in a lattice or branched structure. Typically, such branched amino acid structures will contain a peptide containing one or more lysine residues.

Other amino acid arrangements or structures that can be used to form the vaccines according to the invention are dimers which are formed by disulphide bridges involving cysteine residues. One can use looped structures where the amino acid sequence includes a terminal cysteine residue. Such arrangements or structures are found to potentiate the response to the vaccine according to the invention.

The peptides can also be synthesised by recombinant methodology, and purified before delivery to hosts, or can be inserted into vectors (bacterial or viral) which replicate, and express the appropriate antigen, within the host. Thus, in another embodiment, the DNA coding for the or each amino acid sequence is incorporated in a vector.

Suitably, the vector is a bacterial vector such as a salmonella vector.

DNA expressing appropriate antigenic sequences, and administered intramuscularly to hosts, is taken up and translated into peptide sequences in host cells. Methods known to those skilled in the art can be used to secure expression of immunogenic peptides, as described herein, to domestic poultry and to other host species.

The vaccine can consist of an amino acid sequence as such optionally in the presence of a T-cell epitope as hereinabove described or, alternatively, it can include an adjuvant such as an oil, Freund's Complete Adjuvant or Freund's Incomplete Adjuvant or a carrier such as ov albumin.

Where the parasite is an Apicomplexan organism, the organism is suitably selected from Eimeria, Toxoplasma, Crypto poridium, Babesia and Plasmodium, more especially a species of Eimeria.

The vaccine can also take the form of liposomes or ISCOMS.

For a particular immunisation protocol frequently different adjuvants will be used. For example, a particular adjuvant may be used for the primary immunisation and a different form of adjuvant or adjuvants for subsequent immunisation steps. Furthermore, a combination of adjuvants may be used at the different stages in an immunisation protocol.

The invention also provides a method for identifying conserved and non-immunoreactive amino acid sequences in a given pathogenic organism which are capable of inducing broad-spectrum immunity in an organism susceptible to infection by said pathogenic organism, which method comprises selecting a region of an antigen from said pathogenic organism, binding a series of overlapping sequences from said region to solid phase supports and probing said sequences for reactivity with test sera from an animal infected with said pathogen, and selecting one or more sequences on the basis of failure to bind antibody in test sera.

We have found (as hereinafter exemplified) that it is possible to obtain a good T-cell response with the peptides selected for use in the vaccine according to the invention as such based on the lymphoproliferative response of such peptides when used in a suitable lymphocyte proliferation assay.

Thus, additionally the ability of one or more of said selected sequences to cause lymphocyte proliferation is determined and selected for use as an immunogen on the basis of its ability to elicit a lymphoproliferative response.

Thus, the method for identifying the amino acid sequences to be used in the vaccines according to the invention involves epitope mapping experiments.

For B-cell epitope mapping a series of overlapping peptides covalently bound to solid phase supports are reacted with sera from a given host. Evidence of binding of antibody to the peptide can be detected (or visualised) in various ways, such as by an enzyme immunoassay, more particularly an enzyme linked immunosorbant assay (ELISA). Those peptides that fail to bind antibodies are selected as candidates for use as an immunogen and potentially for use in a vaccine according to the invention.

It is generally accepted that T-cell epitopes are essential for an effective vaccine and that a B-cell epitope alone will not be effective. As demonstrated herein the peptides for use in accordance with the invention can possess both types of epitopic characteristics. In assessing various peptides for their ability to serve as a T-cell epitope, soluble overlapping peptides from a selected region of a protein of the organism of interest are used in a conventional lymphocyte proliferation assay (LPA). In such an assay the soluble peptides are reacted with lymphocytes from an infected host and the degree of proliferation (stimulation) of the cells is measured. If the cells recognise a given peptide they begin to divide or proliferate. Such proliferation can be measured in different ways such as by measuring the uptake of a radio-labelled nucleotide or base, such as tritiated thymidine. T-lymphocytes can also be sorted by f ow- cytometry and the subset stimulated by particular peptides isolated.

Other methods of measuring proliferation include non- radioactive methods such as flow cytometry wherein the binding of cells is measured.

Those peptides which fail to elicit a proliferate response are candidates for use as immunogens and potentially vaccines in accordance with the invention.

Brief Description of Drawings

Fig. 1 is a plot of optical density (O.D.) at 450nm for an overlapping sequence of 90 hexapeptides used in an epitope- mapping experiment as described in Example 1 in which the recognition pattern of serum from a chicken recovered from an E. tenella infection was compared with that from a chicken immunised with peptides derived from said sequence;

Fig. 2 is a graph of antibody response for peptide-immunised chickens to the peptide itself, and to two species of Eimeria, as compared with control chickens; and

Fig 3 is a measure of lymphocyte response for peptide- immunised chickens to the peptide itself, and to two species of Eimeria, as compared with control chickens. The invention will be further illustrated by the following Examples.

Modes for Carrying Out the Invention

Preparatory Example A

Parasites

Pure cultures of oocysts of five Eimeria species, namely E. acervulina, E. maxima, E tenella... were used during the experimental period described herein. These were passaged in Cobb 500 chickens as required, and purified from faeces for antigen preparation or inoculation. The faeces were collected daily in 2.5% potassium dichromate solution from the 6th day post-inoculation and the collection was continued for another 3 days. Faeces from each chicken were pooled and counting of the oocysts was carried out using the McMaster technique (see Davis, P.J., et al., (1973) Techniques. In:

Coccidia D.M. Hammond and P.L. Long Eds. University Park Press, Baltimore, USA. 411-458) with some modifications as described below.

Briefly, the faeces of each chicken were homogenised for 5 minutes and made up to 1,500 ml with tap water. After mixing completely, 15 ml samples were withdrawn using a graduated pipette and serial dilutions (1/10, 1/100, and 1/1000) made up. A 15 ml sample was withdrawn from the last dilution, passed through a sieve, transferred into a 15 ml tube and then centrifuged for 3 minutes at 500 xg. The sediment was suspended in 15 ml saturated salt (specific gravity 1.2) and after inverting several times, a McMaster slide was filled from the tube and the oocysts were counted. The total number of oocysts produced by individual chickens was calculated by multiplying the McMaster slide count by 10^, The oocyst production rate of each chicken was then calculated according to the following formula: Total oocysts produced by each chicken Oocyst production rate = - - — —

No. of oocysts inoculated

Sporulated oocysts were prepared by aeration in 4% potassium dichromate at 30° C for 3 days, cleaned by suspension in 14% sodium hydrochlorite solution for 30 minutes, and separated from non- sporulated oocysts by centrifugation at 800xg for 15 minutes in 0.45M sucrose solution, after which only sporulated oocysts float.

Sera

Chicken anύ-Eimeria sera for use in the epitope mapping described in Example 1 were obtained from chickens inoculated with two doses of sporulated oocysts of a single Eimeria species at two-week intervals.

Serum samples from Eimeria-infected chickens for use in the epitope-mapping assays of Example 1 were obtained by puncture of the wing vein (cutanea ulnearis), using a 25 gauge 1/2 inch needle attached to a lml plastic syringe. After the blood was drawn it was placed in a glass bottle, incubated by 37 °C for 30 minutes and then left at 4°C for 12 hours. It was then spun at 2000 x g, the serum removed and stored at -20°C until required.

Preparatory Example B

Preparation of antigen

Water soluble oocyst antigen for use in Preparatory Example D was prepared by homogenising 2(?)xl0⁷/ml purified sporulated oocysts in phosphate buffered saline (PBS) for 20 minutes while cooling on ice. The solution was frozen at -20°C, then it was thawed and homogenised again for 10 minutes. This step was repeated twice more in order to break down all parts of the oocysts. The solution was centrifuged in a Sorvall superspeed refrigerated centrifuge using a SM-24 rotor at 5000xg for 10 minutes and the supernatant was collected as previously described (Rose, M.E. (1977) supra) and stored at -70°C until used. The protein concentration of the antigen was determined using the BCA technique (Pierce Chemical Company) and a standard curve employing bovine serum albumin (BSA) standards.

Preparatory Example C

Immunisation and challenge experiments

Chickens of the Cobb 500 strain were purchased at one day of age, and housed on wire-floored cages in a controlled environment house under coccidia-free conditions. They were fed on a non- medicated broiler diet (Lillico Ltd.) ad lib . The chickens were immunised between two and five weeks of age (see Examples 1-3) in all cases with peptide emulsified in oil adjuvants, administered subcutaneously. Primary immunisations were given using Freund's Complete Adjuvant (Sigma Chemical Co. Ltd, Fancy Road, Poole, Dorset), and subsequent doses given using Freund's Incomplete Adjuvant, from the same manufacturer. The vaccine emulsion was prepared by adding the required amount of peptide, dissolved in PBS, to the adjuvant, 0.1ml at a time, and homogenising the mixture in a high-speed blender between each addition. The blending was continued until all of the peptide had been added (in a volume of PBS equal to the volume of adjuvant used), and a stable emulsion had been formed as judged by the ability of the mixture to remain as a discrete drop when placed in a beaker of water at 4°C. Just prior to administration to the birds the emulsion was drawn into an oil-resistant syringe and injected (lml) via an 18 gauge needle under the skin in the pectoral region of each bird.

Preparatory Example D

Lymphocyte Proliferation Assay

Pre- and post-infection blood samples were taken using 1 ml sterile syringes containing 100 IU heparin sulphate. Roswell Park Memorial Institute medium (RPMI 1640, Life Technologies Ltd., Paisley, Scotland) containing L-glutamine and 25mM HEPES, supplemented with penicillin (100 IU/ml) and streptomycin (lOOμg/ml) was used for cultivation of the cells. In this assay, concanavalin A (Con A, Sigma Co., Poole Dorset, England) was used as a positive control for measurement of blastogenic responses. Optimal conditions for the assay were as previously described (Talebi et al. (1995) Veterinary Immunology and Immunopathology 46, 293-301). Briefly, 0.4μg/culture of Con A and 0.4μg/culture of E. maxima oocyst antigen reconstituted in PBS, pH 7.2 were dispensed in triplicate into wells of a 96 well flat-bottom tissue culture microplate (Nunc, Kamstrupuej 90, Roskilde, Denmark) and 200μl of 1/50 blood dilution in RPMI of each sample were added into the wells. The cultured plates were then incubated for 66 hours at 40°C in a 5% CO₂ humidified atmosphere. The cultures were pulsed with 0.2μCi/culture of [methyl-3H] thymidine (TRA-120, Amersham International Pic, Buckinghamshire, England) for 18 hours prior to harvesting the cells on filter mats (Skatron Instruments Ltd. Suffolk, England) using a cell harvester (Type 11025, Skatron, Tranby, Norway). The discs were punched out, placed into disposable scintillation tubes and mixed with 2ml scintillation fluid (Ultima Gold Cocktail, Packard Instrument BV Chemical Operation, Groiningen, The Netherlands). The radioactivity of individual tubes was measured as disintegration per minute (DPM) using a liquid scintillation analyzer (Packard 1900 CA, Packard Instrument Co., Downers Grove, IL, U.S.A.). The stimulation index (SI) for each sample was calculated according to the following formula:

Mean DPM of stimulated cultures SI = — - ---

Mean DPM of unstimulated cultures Preparatory Example E

Assessment of antibody titre

Serum samples were taken from the chickens on day 0 (pre- inoculation) and weekly post-inoculation (pi) using 2ml vacutainer tubes with 20 gauge needles (Becton Dickinson Co. , Plymouth, U.K.). The blood samples were incubated for 30 minutes at 37°C in a water bath and after storage overnight at 4°C, were centrifuged for 10 minutes at 500xg. The sera were collected and stored at -20°C until used. An antibody capture immunoassay (ELISA) was used for determination of the antibody titre of the sera. In outline, 96 well Nunc II immunoplates (Nunc, Kamstrupuej 90, Roskilde, Denmark) were coated with 5μg/ml of oocyst antigen in 0.1 M carbonate/ bicarbonate buffer pH 9.6 (lOOμl/well). After incubation overnight at room temperature, the coated plates were washed five times with PBS containing 0.1% Tween 20 (washing buffer) and dried on tissue paper. Serial three-fold dilutions of the sera were made in dilution buffer (1% sodium caseinate, 10% sheep serum and 0.1% Tween 20 in PBS). Starting at a serum dilution of 1/30, volumes of lOOμl/well were used. The plates were incubated for 1 hour at 37°C and then washed five times in order to remove all non-specific binding. Specific binding was detected by adding 100 μl/well of 1/400 rabbit anti -chicken horseradish peroxidase (R α HRPO) conjugate (SUN 157, Serotec Ltd., Oxford, England) an 3,5,5,5'-tetramethylbenzidine (TMB, Sigma Co., Poole, Dorset, England). The substrate reaction was stopped after 10 minutes by adding 100 μl/well of 10% H₂S0₄. The plates were read in an ELISA microplate reader (Bio-Rad model 3550, Hemel Hempstead, Herdfordshire HP2 7TD, U.K.) at 450nm with 429nm as the reference wavelength. The antibody titres of antisera were expressed as the optical density (O.D) at 450nm using 1/270 dilution of the sera. Example 1

Overlapping oligopeptides from an antigen found in E tenella sporozoites and merozoites (Bhogal, B.S. et al, (1992) Veterinary Immunology and Immunopathology, 31. 323-335) having the following sequence:

-N-A-E-E-L-P-G-E-E-G-G-A-G-A-G-G-A-E-G-E-T-G-L-P-G- G-E-E-G-G-A-G-G-A-G-E-G-A-G-G-E-G-G-E-V-Q-P-G-E-G- E-G-A-S-E-G-G-E-Q-V-P-E-T-P-E-T-P-E-P-E-T-P-E-A-E-R- P-E-E-Q-P-S-T-E-T-P-A-E-E-P-T-E-G

I

were probed for reactivity with various sera.

The oligopeptides were linked to polypropylene pins, which pins were used as the solid phase of an enzyme linked immunosorbant assay (ELISA) as hereinafter described. This was achieved by a custom- designed pin-plate provided by Cambridge Research Biochemicals. The methodology is well-established (Geyson, H.M., et al, (1984) P.N.A.S. 87, 3998-4002).

The peptides were synthesised by a standard solid phase peptide synthesis technique, using 9-fluorenyl-methoxycarbonyl(F-moc) chemistry. High performance liquid chromatography (HPLC) was used to monitor the purity of the synthesised peptides.

The results are represented in Fig. 1 which shows the reactivity of serum from a chicken post-infection with E. tenella, and the reactivity of serum from a chicken immunised with the peptide of the sequence I above. The epitope-mapping experiment performed with sequence I allows one to compare the recognition pattern of serum from a chicken recovered from an E. tenella infection with that from a chicken immunised with sequence I or a peptide derived therefrom. Peptides capable of inducing the altered recognition pattern shown in Fig. 1 include those spanning conserved motifs found in antigenic proteins of Eimeria and other Apicomplexans. Examples of these peptides are shown in Table 1.

Table 1

Sequence ID No.

LSNEQVERQLPPSEQVETC 1

CEQVPPEAERPEQVERQLSPPSEQVPC 2 CPEAERPEQVPEQVPLIQIQPPSVQEC 3

CEQVPPEAERPEQVERQLEQVPC 4 EQVPPEAERPEQVERQLEQVPC 5

CPEAERPEQVEWSPEVEREQVC 6

Peptides 1-6 in Table 1 contain residues from Eimeria antigenic sequences not recognised by infected chickens, but recognised by antibodies in the serum of chickens immunised with these peptides. More particularly peptides 1-6 contain conserved residues found in Eimeria surface antigens and surface antigens from other Apicomplexa and which are optimised for the induction of a protective humoral immune response when used to immunise poultry according to the procedure of Preparatory Example C.

Peptides 1-6 are also optimised for induction of cellular immune response when used to immunise poultry according to the procedure of Preparatory Example C.

Optimisation of the cellular immune response is achieved by the use of a lymphocyte proliferation assay according to the procedure of Preparatory Example D. Example 2

Peptides 1-6 listed in Table 1 of Example 1 were used as synthetic antigens to immunise chickens prior to challenge with viable Eimeria oocysts (as prepared according to Preparatory Example A), and confer protection against coccidiosis in the immunised birds. Prior to inoculation, the peptides were emulsified in either Freund's Complete Adjuvant (FCA) or Freund's Incomplete Adjuvant (FIA). The peptides were administered to the target species subcutaneously. Examples of their use in immunisation /challenge experiments are shown in Table 2.

Table 2

Experiment Primary Second Third Challenge

Immunisation Immunisation Immunisation

1 Peptide 1, Peptide 1, Nil. 10⁴ £. 200μg 20μg acervulin in FCA in FIA a oocysts

(week 3)

2 Peptide 1, Peptide 1, Peptide 1 , 10³ 200μg 20μg 20μg E. tenella in FCA in FIA in FIA (week oocysts (week 3) 5)

3 Peptide 2, Peptide 2, Peptide 2 10⁴ 200μg 20μg 20μg E. tenella in FCA in FIA in FIA (week oocysts (week 3) 3)

4 Peptides 2-6 Peptides 2-6 Peptides 2-6 10⁴ (equimolar ) (equimolar ) (equimolar ) E. tenella total total total oocysts

200μg in 20μg in FIA 20μg in FIA FCA Example 3

Peptides such as Peptide 1 used to immunise the bird whose serum reactivity is shown in Fig. 1 are also capable of conferring partial protection to chickens against challenge with two different Eimeria spp., as assessed by reduction in oocyst output and reduction in lesion scores. The results are shown in Table 3.

Table 3

Parameters Experiment 1 Experiment 2 Experiment 3 E. acervulina E. tenella E. tenella

10⁴ oocysts 10³ oocysts 10⁴ oocysts

No. of chickens/group 6 6 6

Mean oocyst outpu ot 8.23 +/- 1.0 0.58+/- 0.05 1.27+/-0.20 of vaccinates x 10

Mean oocyst output of 11.1 +/-1.1 0.81 +/- 0.09 2.20+/-0.50 controls x 10⁸

% Reduction 29% 27% 42% P value 0.03 0.015 0.039

Lesion scores of vaccinates 1.0 1.91+/- 0.35 1.91+/-0.39

Lesion scores of 1.0 3.16 +/- 0.33 3.08 +/- 0.38 controls

% Reduction 0 40% 38%

Weight gain of 59.6+/- 3.8 47.8+/- 2.1 56.25+Λ9.43 vaccinates (g/day)

Weight gain of 58.5+/- 3.2 44.8+/- 2.3 42.08+/-7.03 controls (g/day) Peptide 1 (Experiments 1 and 2) and Peptide 2 (Experiment 3) were used as vaccines to confer protection on chickens against subsequent infection with Eimeria. Protection was measurable in terms of reduced oocyst output and reduced lesion scores as shown in Table 3.

Birds vaccinated with the peptides mounted immune responses which were measurable in terms of specific serum antibody, and lymphocyte proliferation in vitro in response to stimulation with peptide and Eimeria sporulated oocyst antigens, as shown in Figs. 2 and 3.

Fig. 2 illustrates antibody responses of peptide-immunised chickens as compared with controls. Serum from the vaccinated chickens recognised peptide, together with sporulated oocyst antigen. Fig. 3 illustrates lymphocyte responses from the same birds.

Lymphocytes from these birds proliferated in vitro in response to both peptide and sporulated oocyst antigen.

Claims

Claims:-

1. A vaccine against pathogens which are characterised by serological cross-reactivity accompanying lack of cross-protection, which vaccine comprises as an immunogen an amino acid sequence from a conserved, non-immunoreactive region of an antigen of said pathogen.

2. A vaccine according to Claim 1, wherein the pathogen is a parasite within the Phylum Apicomplexa and the antigen is an antigen of an apicomplexan organism.

3. A vaccine according to Claim 1 or 2, wherein the antigen is a surface antigen.

4. A vaccine according to any preceding claim, wherein the amino acid sequence consists of at least 10 amino acids.

5. A vaccine according to any preceding claim, wherein the vaccine contains a number of copies of said amino acid sequence.

6. A vaccine according to Claim 5, wherein the vaccine contains more than one amino acid sequence from a conserved, non- immunoreactive region.

7. A vaccine according to Claim 6, wherein the different sequences are intercalated.

8. A vaccine according to any one of Claims 1-6, wherein the or each amino sequence is disposed in a lattice structure.

9. A vaccine according to any preceding claim, wherein DNA coding for the or each amino acid sequence is incorporated in a vector.

10. A vaccine according to Claim 9, wherein the vector is a bacterial vector.

11. A vaccine according to any one of Claims 2-10, wherein the apicomplexan organism is selected from Eimeria, Toxoplasma, Cryptosporidium, Babesia and Plasmodium.

12. A vaccine according to Claim 11, wherein the apicomplexan organism is a species of Eimeria.

13. A method for identifying conserved and non- immunoreactive amino acid sequences in a given pathogenic organism which are capable of inducing broad-spectrum immunity in an organism susceptible to infection by said pathogenic organism, which method comprises selecting a region of an antigen from said pathogenic organism, binding a series of overlapping sequences from said region to solid phase supports and probing said sequences for reactivity with test sera from an animal infected with said pathogen, and selecting one or more sequences on the basis of failure to bind antibody in test sera.

14. A method according to Claim 13, wherein additionally the ability of one or more of said selected sequences to cause lymphocyte proliferation is determined and selected for use as an immunogen on the basis of its ability to elicit a lymphoproliferative response.

15. A method according to Claim 13 or 14, wherein the pathogenic organism is a parasite within the Phylum Apicomplexa and the organism susceptible to infection by said parasite is an avian organism.

16. A vaccine according to Claim 1, substantially as hereinbefore described and exemplified.

17. A method according to Claim 13 substantially as hereinbefore described and exemplified.