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EP1294396A2 - Vaccin contraceptif pour controler la peste - Google Patents

Vaccin contraceptif pour controler la peste

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Publication number
EP1294396A2
EP1294396A2 EP01943659A EP01943659A EP1294396A2 EP 1294396 A2 EP1294396 A2 EP 1294396A2 EP 01943659 A EP01943659 A EP 01943659A EP 01943659 A EP01943659 A EP 01943659A EP 1294396 A2 EP1294396 A2 EP 1294396A2
Authority
EP
European Patent Office
Prior art keywords
vaccine
polypeptide
vaccine according
peptide
antigen
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01943659A
Other languages
German (de)
English (en)
Inventor
Harry Univ. of Sheffield MOORE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Sheffield
Original Assignee
University of Sheffield
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Filing date
Publication date
Application filed by University of Sheffield filed Critical University of Sheffield
Publication of EP1294396A2 publication Critical patent/EP1294396A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0006—Contraceptive vaccins; Vaccines against sex hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/16—Masculine contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/18—Feminine contraceptives
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/542—Mucosal route oral/gastrointestinal

Definitions

  • This invention relates to contraceptive vaccines for the control of pest species.
  • New Zealand has experienced an explosion in its possum population.
  • the animals have become a daunting pest, chewing their way through thousands of tonnes of foliage a night, gorging themselves on birds' eggs and spreading bovine tuberculosis.
  • the cost of controlling New Zealand's possum population which currently stands at 60 million, is an estimated 100 million dollars per annum.
  • pest species in the UK include rabbits, rats, mice, badgers, foxes and all vermin injurious to livestock, game, crops or human health.
  • the control of seal populations poses a problem in the light of severely threatened fish populations.
  • GMO Genetically Modified Organism
  • Any approach must reduce the fecundity of the target population. It should involve an effective delivery system and be humane, cost effective and environmentally benign. There is a real need for target-specific action. Due to the non species-specificity of the egg antigen, any antigen should be based on sperm proteins that are species-specific.
  • Proteins and peptides isolated from tissues, or produced synthetically for use as contraceptive vaccines are often only weakly antigenic, especially if autologous, and require immunostimulants to elicit an effective response.
  • the mode bf delivery and presentation of an antigen is crucial for its effectiveness.
  • Oral vaccination has an advantage of preferentially inducing an immune response at mucosal surfaces such as the reproductive tract and is the most practical route of administration for wild animals. However, oral vaccinations may produce weak immunity which may not be effective.
  • an immunocontraceptive vaccine is to induce a strong local mucosal immune response in the reproductive tracts.
  • the vaccine must evoke an appropriate immune response that blocks one of the steps in the reproduction process.
  • Antibodies generated from the immunisation should bind to the appropriate reproductive proteins preventing essential biological interactions and hence fertility.
  • the vaccine should be able to induce a strong mucosal immune response that is sustained over a period of time.
  • a number of delivery systems have been reported to induce both a systemic and mucosal responses in mammals
  • Mucosa-associated lymphoid tissue is present throughout the intestine either as isolated lymphoid follicles or as lymphoid follicle aggregates such as Peyer's patches in the small intestine (Bienenstock and Befus, 1984).
  • the lymphoid tissue is separated from the intestinal milieu by the Follicle-associated epithelium (FAE) which contains highly specialised microfold cells or M cells.
  • FAE Follicle-associated epithelium
  • M cells facilitate the trans-epithelial transport of an antigen.
  • the FAE represents a highly dynamic tissue, which is able to modify its epithelial and lymphoid cells in response to antigenic stimuli.
  • a previous study (Borghesi et al., 1996) demonstrated that one hour exposure to living pneumococci induces dramatic alterations of the architecture and structure of the FAE. Transport of antigens through the follicle-associated epithelium (FAE) of Peyer's patch (PP) is the critical first step in the induction of mucosal immune responses. Cross-species gamete binding experiments have been carried out to confirm the specificity of fertilisation mechanisms in the grey and red squirrel.
  • a vaccine comprising a polypeptide wherein the polypeptide is a germ cell polypeptide.
  • the polypeptide is a cell surface polypeptide. More preferably the polypeptide is a receptor polypeptide. Preferably the peptide corresponds to at least part of the extracellular domain of a germ cell polypeptide. Most preferably the polypeptide is a sperm cell or sperm progenitor cell surface polypeptide
  • the receptor is a 95-kDa phosphoprotein (95PP) located on the plasma membrane of the anterior acrosome in spermatozoa.
  • 95PP 95-kDa phosphoprotein
  • sperm antigens use different sperm antigens as vaccine antigen candidates.
  • these proteins include Acrosin, FA-1 (Naz et al, 184), DE protein (Cuasnicu et al., 1884; Biol. Reprod. 55, 200 206), Fertilin (Blobel et al, 1990), PH-20 (Primakoff et al, 1988), SP17 (Richardson et al., 1994), LDH-C4 (Goldberg) and Sp56 (Bleil et al, 1990).
  • Antigen presenting cells are responsible for antigen processing and their subsequent presentation to lymphocytes. The antigens must be presented in such a way that the lymphocytes recognise and react to the antigen. Efficient antigen presentation is largely dependent upon the size of the antigen and peptides of 9 to 18 amino acids in length are most commonly used in the art.
  • the peptide of the invention is at least nine amino acids in length and preferably from 9 to 18 amino acids in length. More preferably the peptide is 9, 12 or 13 amino acids in length.
  • the peptide comprises the amino acid sequence AVTLGGVGFSDPVC (pep 1), or variant thereof.
  • the variant may bind to antibodies to the cell surface polypeptide with increased affinity and may be characterised by conservative or other amino acid substitutions at any position in the sequence of pep 1.
  • a variant may be characterised by amino acid substitution, deletion, insertion, etc., at any position in the peptide, it will be understood that the nature of the amino acids at the third and ninth position (i.e., the anchoring amino acids) will be particularly important.
  • the peptide is species specific. This feature is preferable because it eliminates possible interference with the fertility of non-target and especially endangered species.
  • Various conserved and varying domains exist within 95PP from different species. Because the invention relies on the production of antibodies to that region of 95PP which is specific to a particular target species, non target organisms remain unaffected.
  • the peptide is conjugated, associated or cross-linked to a suitable adjuvant and/or carrier protein.
  • a suitable adjuvant and/or carrier protein Preferably the cross-link is a covalent linkage.
  • adjuvant- and carrier are construed in the following manner.
  • Some polypeptide or peptide antigens contain B-cell epitopes but no T cell epitopes. Immune responses can be greatly enhanced by the inclusion of a T cell epitope in the polypeptide/peptide or by the conjugation of the polypeptide/peptide to an immunogenic carrier protein such as key hole limpet haemocyanin or tetanus toxoid which contain multiple T cell epitopes.
  • the conjugate is taken up by antigen presenting cells, processed and presented by human leukocyte antigens (HLA's) class II molecules.
  • HLA's human leukocyte antigens
  • T cell help to be given by T cell's specific for carrier derived epitopes to the B cell which is specific for the original antigenic polypeptide/peptide. This can lead to increase in antibody production, secretion and isotype switching.
  • carrier proteins include Keyhole Limpet Haemocyanin (KLH), ovalbumin, Bovine Serum Albumin and cholera toxin B.
  • KLH Keyhole Limpet Haemocyanin
  • ovalbumin ovalbumin
  • Bovine Serum Albumin cholera toxin B.
  • An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells.
  • adjuvants include, by example only, agonsitic antibodies to co-stimulatory molecules, Freunds adjuvant, muramyl dipeptides, liposomes.
  • An adjuvant is therefore an immunomodulator.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • the peptide is conjugated, associated or cross-linked to cholera toxin B, preferably using a cross-linker compound.
  • Chemical cross-linkers are well known ' in the art and are commercially available from Sigma Co. or Pierce and Warner.
  • the cross-linker compound is succinimidyl 3-(2- pyridyldithio) propionate (SPDP).
  • cross-linker compounds include AMAS (N-[(X- Maleimidoacetoxyjsuccinimide ester), BMPS (N- ⁇ -Maleimidoproploxy]succinimide ester) and EMCS (N- ⁇ -Maleimidocaproyloxy]succinimide ester).
  • AMAS N-[(X- Maleimidoacetoxyjsuccinimide ester)
  • BMPS N- ⁇ -Maleimidoproploxy]succinimide ester
  • EMCS N- ⁇ -Maleimidocaproyloxy]succinimide ester
  • the peptide is conjugated, associated or cross-linked with an adjuvant such as Freund's adjuvant, alum (aluminium hydroxide) or Immune stimulatory complexes (ISCOMS).
  • an adjuvant is a reagent that augments the natural immune response, therefore acting as an immunopotentiator.
  • Adjuvants can also act as immunomodulators.
  • An adjuvant may stimulate the proliferation of antibody secreting B-cells, isotype switching and/or secretion of antibodies to increase serum antibody titre.
  • the peptide is incorporated within a suitable delivery system.
  • Suitable delivery systems include, by way of example only, liposomes, Immune Stimulatory complexes (ISCOMS) and microparticles.
  • ISCOMS Immune Stimulatory complexes
  • Liposomes are lipid based vesicles which encapsulate a selected therapeutic agent which is then introduced into an organism.
  • the liposome is manufactured either from pure phospholipid or a mixture of phospholipid and phosphoglyceride.
  • liposomes can be manufactured with diameters of less than 200nm. This enables their intravenous injection and ability to pass through the pulmonary capillary bed. They are permeable across blood vessel membranes and can gain access to selected tissues.
  • ISCOMS are colloidal spherical particles consisting of saponin Quil A, cholesterol and phosholipids such as phosphatidylcholine or phosphatidylethanolamine. They have a negative charge and a cage-like structure, typically an icosahedral structure with a 40 nm diameter in which antigen can be entrapped.
  • ISCOMs are extremely stable and remain intact after lyophilisation. Once antigen is added, the ISCOM complex can be stored at sub zero temperatures, (e.g., -70 °C), depending on the stability of the added antigen.
  • Microparticles composed of, by way of example only, poly (D,L -lactic-co-glycolide) can be also be used as an effective vaccine delivery system (see Challacombe, 1995).
  • the technique for the preparation of the biodegradable microparticles is known, (Rafati, et al. 1997).
  • Recent developments in microparticle technology have shown that using the polymer Eudragit (R) LI 00-55 as stabiliser gives improved protection against gastric digestion of antigens as compared with the conventional polyvinyl alcohol. This method may increase the effective concentration of the antigen and may help to further retain immunogenicity of the antigen complex.
  • the peptide preferably conjugated, associated or crosslinked with a carrier protein, is incorporated within a biodegradable microparticle.
  • suitable materials which might be used in the formation of a microparticle include Poly-lactiyl-glycolide (PLG), Dichloromethane and Polyvinyl alcohol.
  • the microparticle is comprised of poly(D,L-lactic-co-glycolide), or variant thereof.
  • the antigen concentration varies from 0.5 to 1.5 mg/dose.
  • the antigen concentration is 1 mg/dose.
  • the size of a microparticle and antigen complex varies from 0.2 to 5.0 ⁇ m.
  • the size of a microparticle and antigen complex is 1 ⁇ m.
  • the microparticle includes a stabiliser to increase the effective concentration of the antigen and help to retain immunogenicity of the antigen complex.
  • a stabiliser may take the form of an enteric coating which resists degradation in the stomach of the target organism. This is important in oral administration of the vaccine.
  • the stabiliser of use is Eudragit (R) LI 00-55.
  • Other possible stabilisers which may be used include, by no means of limitation, carboxymethylcellulose.
  • molecules actively released or induced by certain bacteria are used as antigen delivery proteins whereby they increase transport capability and enhance absorption of the orally delivered biologically active compound.
  • the antigen delivery protein is Mucosal Antigen Delivery Protein (MADP).
  • the antigen delivery protein is incorporated separately into the delivery system either as crude supernatant or purified material.
  • antigen delivery protein is conjugated directly to the peptide antigen in addition to the carrier protein.
  • antigen delivery protein is not incorporated within the delivery complex and is administered as a separate entity. Recombinant antigen delivery protein may also be used.
  • the vaccine is formulated for oral delivery.
  • oral baiting is a feasible route for administration of the vaccine to wild animals.
  • a problem associated with oral vaccinations however is that they have been found in some cases to produce only weak immunity and this may not be effective.
  • the need for a safe and effective delivery system which can both be administered orally and produce an effective immune response is apparent.
  • Antibodies (IgG + A) were detected on rabbit sperm over the anterior acrosome in five out of eight treated females at 6 and 12 weeks after immunisation.
  • composition suitable for use as a contraceptive vaccine comprising a peptide corresponding to at least part of the extracellular domain of a sperm surface polypeptide wherein said peptide is formed synthetically using, by way of example only, an oligosynthesiser.
  • the peptide is formed using recombinant techniques.
  • the invention therefore provides for a method of manufacture of a peptide corresponding to at least part of the extracellular domain of a sperm surface polypeptide wherein said method includes peptide synthesis and/or recombinant techniques.
  • a method of preparing a composition for use as a contraceptive vaccine comprises:
  • composition comprising a polypeptide or peptide with a cross-linker, carrier protein and/or adjuvant and/ or an antigen delivery protein;
  • the invention includes a method in which the peptide (e.g., in the form of a peptide- complex) is incorporated within a suitable delivery system.
  • the peptide e.g., in the form of a peptide- complex
  • the method includes the step of incorporating the peptide or the peptide-complex into a microparticle, wherein said microparticle optionally comprises a stabiliser.
  • the method comprises one or a combination of any of the following steps;
  • the invention provides for use of the vaccine of the present invention as a contraceptive.
  • the invention provides for a method of contraception comprising administration of the vaccine of the invention in an amount effective to reduce the fecundity of a mammalian species.
  • the invention also provides an oral bait or inoculum comprising the vaccine of the present invention.
  • a vaccine according to the present invention is used as an immunogen.
  • an antibody which is produced by the immune response that is induced by the vaccine of the invention.
  • the antibody is a monoclonal antibody.
  • the invention provides for a method of testing the species specificity of a vaccine comprising: (1) obtaining a sperm sample from a pest species of interest;
  • microparticles consisted of lactide-glycolide polymer in the ratio 75: 25 and were prepared by high speed emulsification to produce particles of 0.5 - 2.0 ⁇ m as determined by scanning electron microscopy and fluorescent sorting techniques Rafati H et al. (1997).
  • Protein from (1) was emulsified with 10 ml of 6% solution of PLG in DCM using a silverson SL2 for 2 minutes at full speed (11600 rev). NB.
  • the speed of homogenisation is important, therefore the speed of the homogenisation was switched to maximum quickly.
  • the position of the blade in the emulsion was also standardised.
  • microparticles were centrifuged at 16 000 (31 000 x g) for 20 min at 4°C (Beckman, JA20 rotar).
  • Vaccines were prepared using sperm peptide 1 and the carrier protein keyhole limpet haemocyanin or cholera toxin B. The latter is effective in enhancing oral immunity in several species, Jackson et al. (1993) and had been used successfully in rabbit vaccination studies. The following schedule was used:
  • Substance Amount No. females microparticles (control) 2ml microparticles + KLH + peptide 1 (pepl+KLH) 2ml 8 microparticles + Cholera toxin-B + peptide 1 (pepl+CTB) 2ml 8
  • Progesterone concentration was determined by ELISA assay (OVUCHECK plasma
  • Samples from treated and control animals were compared with known control samples; i.e. samples from known non-pregnant females and samples from two stages of pregnancy taken from a captive squirrel.
  • Gametes and oocytes were collected and prepared. Experiments were carried out with a matrix design as shown in table 5. Typical binding patterns are shown in figure 1 Table 5 Inter-species binding of gametes
  • the microparticle encapsulated vaccine inhibits fertility in treated squirrels compared with controls. A significant immune response was observed in some animals correlating with inhibition of fertility. Both KLH and CTB were effective apparently as carriers of the peptide vaccine. In the phase II trial there was a significant inhibition of fertility (pregnancy rate) in the combined treatment groups (15.4 %) compared with controls (50%).
  • Vaccinations were timed to give an optimal immune response during a breeding season. As it was necessary to re-capture animals for blood samples further vaccinations were performed. Whether such repeated application of a vaccine is practical in the long-term is unclear. Because of the timing of the breeding season it is likely that only the first three vaccinations would have had an effect on fertility. Exactly when and how often an oral vaccine would have to be administered will require further investigation. The phase II trial covered the summer breeding period of the squirrels and additional trials should be performed that cover the winter breeding period.
  • the Cross-species in vitro fertilisation experiments between gametes of red and grey squirrels provide convincing evidence for differences in the binding affinities of spermatozoa with homologous and heterologous zona pellucida. These findings strongly support the contention of differences in the egg receptor on sperm from the two species.
  • the acrosome reaction involves vesiculation of the membranes over the anterior sperm head, which is essential for sperm penetration and fertilisation of the egg.
  • the in vitro assay assessed secondary binding of spermatozoa to the zona pellucida, after the induction of the acrosome reaction, Brewis, I and Moore, H.D.M (1997). Where spermatozoa had bound to a heterologous zona, few had undergone the acrosome reaction.
  • the antigen used in the contraceptive vaccine is to a region of the receptor involved in the induction of the acrosome reaction and one would therefore expect functional and structural differences in the receptor between the two species.
  • Anti-rat IgA antibody (which cross-reacts with squirrel IgA) was previously used in the assays to measure immunological response. This is not ideal and a homologous assay was devised.
  • a squirrel IgA fraction was obtained from serum and milk by affinity chromatography. The purified fraction was used to immunise a rabbit and the antiserum tested for specificity and titre.
  • the vaccine uses CTB carrier in preference to KLH.
  • the rabbit studies indicate greater microparticle uptake with this molecule compared to KLH.
  • CTB is toxic in the rabbit at least.
  • the potential use of KLH should not however be excluded.
  • Costs of the vaccine was also considered.
  • CTB is currently obtained in relatively small quantities and the costs are high. While there will be economy of scale when producing many doses of a vaccine, a cost-effective method of producing the immunogen will be required. In this regard there may be an opportunity to exploit existing techniques for producing a cholera vaccine for human use which is based on CTB subunit and which has been shown to be safe.
  • contraceptive vaccines antigen conjugated to carrier protein
  • Vaccine encapsulated in poly lactide-glycolide microparticles induced a significant increase in IgA in the reproductive tract and serum and reduction in fertility.
  • the ability of the rabbit alimentary canal to absorb and process microparticles was examined in detail by developing an in vivo mucosal assay using the isolated intestinal loop technique.
  • the uptake of particles (latex microspheres) across specialised regions (follicle-associated epithelium (FAE) via specialised M cells) was demonstrated initially along with a dramatic increase in particle uptake following short-term exposure to the bacteria, streptococcus pneumoniae.
  • CTB cholera toxin B
  • IgA immune response
  • a vaccine based on sperm peptide may be more effective than uteroglobin peptide.
  • Liposomes Liposomes with the composition of distearoylphosphatidylcholine (DSPC), phosphotidyl serine and cholesterol are stable in the gastrointestinal tract and taken up by Peyer's patches ([Fujii et al, 1993). Studies have demonstrated that liposome vaccines can induce both mucosal and systemic immune response against, for example, hepatitis B ([Diminsky et al, 1993); and influenza (Ben Ahemeida et al, 1993).
  • DSPC distearoylphosphatidylcholine
  • phosphotidyl serine phosphotidyl serine
  • cholesterol Liposomes with the composition of distearoylphosphatidylcholine (DSPC), phosphotidyl serine and cholesterol are stable in the gastrointestinal tract and taken up by Peyer's patches ([Fujii et al, 1993). Studies have demonstrated that liposome vaccines can induce both mucosal and
  • liposome composition was those prepared by (Fujii et al, 1993).
  • Phospholipids consisting of a 1:1:2 molar ratio of phosphatidylserine, distearoyl L- ⁇ - phosphatidylcholine (DSPC) and cholesterol in chloroform/methanol (9:1 v/v) were evaporated to dryness in a 250 ml round bottomed rotary evaporation flask.
  • the preparation was adjusted to 40% sucrose by addition of an equal volume of 80% sucrose. This was layered onto 1 ml of 75% sucrose in PBS in polycarbonate centrifuge tubes, followed by 3 ml of 30% and 3 ml of 15% sucrose. These tubes were balanced with PBS. The preparation was then centrifuged at 26 000 rpm for 24 hrs. The liposome bands were pooled and dialysed against 8 changes of 500 ml PBS for 48 hrs. It was estimated that the antigen concentration per immunisation was ⁇ 500 ⁇ g/ml.
  • New Zealand White adult female rabbits (8) were immunised (x2 at 2 week intervals) with 2 ml of liposomes carrying 500 ⁇ g/ml of antigen preparation (or control) in 1 M Na 2 CO3 buffer pH8.0 and delivered to the back of the throat using a 5 ml syringe. Liposome preparations were examined by electron microscopy and liposome vesicles were identified.
  • Rabbits were assessed in serum and vaginal secretions for immune responses by enzyme-linked immunosorbent assay (ELISA) up to 20 weeks after immunisation. ELISA plates were coated with 5 ⁇ g of BSA-peptide. Rabbit sera IgGs were detected with anti-rabbit IgG alkaline phosphatase conjugate, while vaginal IgAs were detected with goat anti-rabbit IgA followed by mice anti-goat IgG alkaline phosphatase conjugate.
  • ELISA enzyme-linked immunosorbent assay
  • Immune stimulating complexes ISCOMs carrying antigens were shown to stimulate both B and T cell response by intramuscular, intranasal, oral and parenteral routes (reviewed by McGhee, 1993).
  • Antigen concentration was estimated at 0.5 - 1 mg /ml. Rabbits (8 +4 controls) were immunised and assessed as described above.
  • Biodegradable microparticles composed of poly (D,L -lactic-co- glycolide) can be used as a vaccine delivery system (see Challacombe, 1995).
  • the technique for the preparation of the biodegradable microparticles was developed by Davis, SS., School of Pharmaceutical Sciences, University of Nottingham. (Rafati, et al. 1997) and is sensitive to minor alterations to the protocol.
  • Sperm antigen (as above) was emulsified into 10 ml of 6% poly lactide glycolide (75:25) in dichloromethane using a Silverson SL2 homogeniser at 11600 revs for 2 minutes. 40 ml of 10% poly vinyl alcohol were added to the emulsified polymer and homogenised for a further 4 minutes. The dichloromethane was evaporated from the emulsion overnight at ambient temperature.
  • Microparticles were recovered, washed twice with distilled water by centrifugation at
  • microparticles 31 000 g for 20 minutes at 4 C and resuspended in 5 ml of distilled water.
  • the microparticles were examined with a Fluorescence Activated Cell Sorter (FACS) and their mean size was determined to be 0.5 - 2.0 ⁇ m (figure 1).
  • the particle size was confirmed by scanning electron microscopy (figure 2).
  • the microparticles were then freeze-dried which increases their shelve life for up to 1 year. Antigen concentration was estimated at 0.5 - 1 mg /ml.
  • Rabbits (8 +4 controls) were immunised and assessed as described above. Compared with controls, 4/8 females immunised with sperm antigen had significant titres of IgA antibodies in vaginal secretion (1/400 - 1/1500 titre) at 6 and 12 weeks after immunisation and 3/8 females at 20 weeks. Serum IgG responses were significantly elevated at 6 and 12 weeks in 3 of these females.
  • Cholera toxin enterotoxin produced and secreted by the bacterium Vibrio cholerae which infects the mucosal membranes of the gastrointestinal tracts and activates adenylate cyclase/cAMP signal transduction pathway of cells.
  • Cholera toxin comprises of two subunits, A (CTA) and B (CTB).
  • CTB is a pentameric protein and mediates the binding of the toxin to a glycosylated lipid (ganglioside G ml ) on the surface of mucosal cells, and is the non-toxic component of CT (Jackson, et al. 1993, Holmgren et al. 1994).
  • the property of CTB is thought to play a useful role in the development of a oral vaccine as it can assist in the uptake of the vaccine and display to the GALT. Both CTA and CTB have been fully characterised and cloned.
  • CTB was conjugated to sperm peptide 1 (AVTLGGVGFSDPVC) using the cross- linker succinimidyl 3-(2-pyridyldithio)propionate (SPDP).
  • CTB 4 mg CTB was dialysed into 20 mM phosphate buffer pH 7.5 and mixed with 25 ⁇ l of 25 mM SPDP for 60 min at room temperature. Excess SPDP was removed using a G-10 column and the CTB-SPDP complex was mixed with peptide for 18 hrs. The CTB-peptide was dialysed in PBS and then encapsulated in microparticles as described previously.
  • Booster immunisations were given at 4 and 8 weeks. Rabbits were monitored (test bled and vaginal swab) at 8 and 18 weeks and mated at 20 weeks (vaginal swab taken). One rabbit in the complete vaccine group was culled at 16 weeks due to infection after fighting.
  • a vaccine was prepared that incorporated uteroglobulin peptide (T4) conjugated to Keyhole limpet haemocyanain (KLH) with biological solutions containing MADP activity.
  • T4 uteroglobulin peptide conjugated to Keyhole limpet haemocyanain
  • KLH Keyhole limpet haemocyanain
  • Vaccines incorporating immunostimulatory bacterial protein (CTB or MADP) and specific reproductive antigen sperm pepl or uteroglobulin T4 caused a decrease in fertility. With sperm-based vaccine there was significant decrease in the fertility as measured by the total number of foetuses produced divided by the number of corpora lutea (indication of oocytes ovulated).
  • microparticle encapsulation of antigen was the most efficient method to evoke a significant immune response after oral administration in the rabbit.
  • a parenteral (injection) approach leads primarily to a humoral IgG immune response
  • oral vaccination with microparticles induced an IgA response in both serum and vaginal secretion. This would be critical for the efficacy of an immunocontraceptive vaccine where immune inhibition of fertility in the reproductive tract is required.
  • Microparticle vaccines containing bacterial protein were constructed also to enhance immune response and inhibition of fertility.
  • sperm-based vaccines pep 1 -CTB and microparticles
  • the research findings may be adapted for other oral immunisation procedures in any mammalian species.

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Abstract

L'invention porte sur des vaccins contraceptifs antiparasitaires et sur leurs procédés de préparation. Lesdits vaccins comportent un peptide produisant chez le parasite un anticorps se fixant à un polypeptide de cellule germinale et réduisant par là la fécondité.
EP01943659A 2000-06-30 2001-06-29 Vaccin contraceptif pour controler la peste Withdrawn EP1294396A2 (fr)

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GBGB0015935.0A GB0015935D0 (en) 2000-06-30 2000-06-30 Vaccine
GB0015935 2000-06-30
PCT/GB2001/002877 WO2002002137A2 (fr) 2000-06-30 2001-06-29 Vaccin

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