WO1998018821A1 - Vaccins contre des pathogenes caracterises par une reaction serologique croisee mais ne presentant pas de protection croisee - Google Patents

Vaccins contre des pathogenes caracterises par une reaction serologique croisee mais ne presentant pas de protection croisee Download PDF

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Publication number
WO1998018821A1
WO1998018821A1 PCT/IE1996/000070 IE9600070W WO9818821A1 WO 1998018821 A1 WO1998018821 A1 WO 1998018821A1 IE 9600070 W IE9600070 W IE 9600070W WO 9818821 A1 WO9818821 A1 WO 9818821A1
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Prior art keywords
vaccine
antigen
vaccine according
organism
sequences
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PCT/IE1996/000070
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English (en)
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Grace Veronica Therese Mulcahy
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Forbairt Trading As Bioresearch Ireland
University College Dublin
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Priority to PCT/IE1996/000070 priority Critical patent/WO1998018821A1/fr
Publication of WO1998018821A1 publication Critical patent/WO1998018821A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/455—Eimeria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • 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.
  • 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.
  • 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.
  • 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 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.
  • the vaccine can include a T-cell epitope which is known to elicit the requisite T-cell stimulation.
  • the pathogen is a parasite within the Phylum Apicomplexa and the antigen is an antigen of an apicomplexan organism.
  • the antigen is a surface antigen.
  • the antigen is not necessarily a surface antigen.
  • the vaccines can be used against secretory antigens.
  • the amino acid sequence preferably consists of at least ten amino acids.
  • the vaccine preferably contains a number of copies of said amino acid sequence.
  • the vaccine can contain more than one amino acid sequence from a conserved, non-immunoreactive region.
  • the sequences are preferably intercalated.
  • the or each amino acid sequence can be disposed in a lattice or branched structure.
  • such branched amino acid structures will contain a peptide containing one or more lysine residues.
  • 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.
  • vectors bacterial or viral
  • the DNA coding for the or each amino acid sequence is incorporated in a vector.
  • 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.
  • an adjuvant such as an oil, Freund's Complete Adjuvant or Freund's Incomplete Adjuvant or a carrier such as ov albumin.
  • 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.
  • adjuvants 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.
  • 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.
  • the method for identifying the amino acid sequences to be used in the vaccines according to the invention involves epitope mapping experiments.
  • 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).
  • ELISA enzyme linked immunosorbant assay
  • T-cell epitopes are essential for an effective vaccine and that a B-cell epitope alone will not be effective.
  • the peptides for use in accordance with the invention can possess both types of epitopic characteristics.
  • soluble overlapping peptides from a selected region of a protein of the organism of interest are used in a conventional lymphocyte proliferation assay (LPA).
  • LPA lymphocyte proliferation 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.
  • 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.
  • 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;
  • O.D. optical density
  • 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;
  • 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.
  • 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.
  • 15 ml saturated salt specific gravity 1.2
  • 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.
  • Chicken an ⁇ -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.
  • Water soluble oocyst antigen for use in Preparatory Example D was prepared by homogenising 2(?)xl0 7 /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.
  • BSA bovine serum albumin
  • 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.
  • 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 (lOO ⁇ g/ml) was used for cultivation of the cells.
  • concanavalin A Con A, Sigma Co., Poole Dorset, England
  • Optimal conditions for the assay were as previously described (Talebi et al.
  • the cultures were pulsed with 0.2 ⁇ Ci/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:
  • 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. , Madison, 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.
  • ELISA antibody capture immunoassay
  • 96 well Nunc II immunoplates (Nunc, Kamstrupuej 90, Roskilde, Denmark) were coated with 5 ⁇ g/ml of oocyst antigen in 0.1 M carbonate/ bicarbonate buffer pH 9.6 (lOO ⁇ l/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 lOO ⁇ l/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 ⁇ l/well of 1/400 rabbit anti -chicken horseradish peroxidase (R ⁇ 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 ⁇ l/well of 10% H 2 S0 4 .
  • R ⁇ HRPO rabbit anti -chicken horseradish peroxidase conjugate
  • TMB 3,5,5,5'-tetramethylbenzidine
  • 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.
  • O.D optical density
  • 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.
  • Fig. 1 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.
  • 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.
  • 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.
  • Peptide 2 Peptide 2 10 4 200 ⁇ g 20 ⁇ g 20 ⁇ g E. tenella in FCA in FIA in FIA (week oocysts (week 3) 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.
  • 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.

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Abstract

Vaccin contre des pathogènes caractérisés par une réaction sérologique croisée s'accompagnant d'une absence de protection croisée, tels que les parasites appartenant au phylum Apicomplexa. Le vaccin comprend comme immunogène une séquence d'acides aminés tirée d'une région non immunoréactive conservée d'un antigène du pathogène. Pour identifier la séquence immunogène, on sélectionne une région d'un antigène tiré de l'organisme pathogène, on fixe une série de séquences chevauchantes tirées de ladite région sur des supports en phase solide et on sonde lesdites séquences, afin de tester leur réactivité, avec des sérums tests provenant d'un animal infecté par ledit pathogène, et on sélectionne une ou plusieurs séquences sur la base de l'incapacité à fixer l'anticorps dans les sérums tests.
PCT/IE1996/000070 1996-10-31 1996-10-31 Vaccins contre des pathogenes caracterises par une reaction serologique croisee mais ne presentant pas de protection croisee WO1998018821A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016627A1 (fr) * 1991-03-14 1992-10-01 British Technology Group Usa, Inc. Vaccin de recombinaison anti-coccidiose
EP0519547A2 (fr) * 1991-06-18 1992-12-23 Akzo Nobel N.V. Vaccin contre la coccidiose aviaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016627A1 (fr) * 1991-03-14 1992-10-01 British Technology Group Usa, Inc. Vaccin de recombinaison anti-coccidiose
EP0519547A2 (fr) * 1991-06-18 1992-12-23 Akzo Nobel N.V. Vaccin contre la coccidiose aviaire

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BHOGAL B.S. ET AL.: "Potential of a recombinant antigen as a prophylactic vaccine for day-old broiler chickens against Eimeria acervulina and Eimeria tenella infections", VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, vol. 31, 1992, AMSTERDAM, NL, pages 323 - 335, XP000197593 *
CRANE M.S.J. ET AL.: "Cross-protection against four species of chicken coccidia with a single recombinant antigen", INFECTION AND IMMUNITY, vol. 59, no. 4, April 1991 (1991-04-01), WASHINGTON US, pages 1271 - 1277, XP000676511 *
TALEBI A. AND MULCAHY G.: "correlation between immune response and oocyst production in chickens monospecifically infected with Eimeria maxima", AVIAN PATHOLOGY, vol. 24, 1995, HOUGHTON, GB, pages 485 - 495, XP000197610 *
TALEBI A. AND MULCAHY G.: "High-resolution mapping of B-cell epitopes within an antigenic sequence from Eimeria tenella", INFECTION AND IMMUNITY, vol. 62, no. 10, October 1994 (1994-10-01), WASHINGTON US, pages 4202 - 4207, XP000676512 *
TALEBY A. AND MULCAHY G.: "cross-reactivity among antisera raised against five avian Eimeria species in the natural host and in rabbits", AVIAN PATHOLOGY, vol. 24, 1995, HOUGHTON, GB, pages 533 - 544, XP000197611 *

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