Classifications

• • 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
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
  • C07K14/43531—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from mites
• • 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
  • A61K39/0003—Invertebrate antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/14—Ectoparasiticides, e.g. scabicides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • 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
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine

Definitions

• the present invention relates to antigens capable of raising host immune responses to parasites.
• the invention provides vaccines for use in protecting against and/or reducing instances of parasite infection in avian hosts.
• Poultry red mite infestation is a major health and welfare issue for laying hens as a result of anaemia, increased irritation and restlessness, feather-pecking and an increased incidence of cannibalism [1]. Poultry mites have also been implicated as intermediate hosts for a number of important diseases [3].
• D. gallinae candidate antigens have been identified through either a "pragmatic" approach of fractionating native protein extracts of the mites and using these as vaccines [9] or a “rational-type” approach of selecting suitable D. gallinae antigens based on assumed orthology with protective antigens from other species of ectoparasite [8].
• the identification of potential vaccine candidates by a “rational” approach has been advocated in the field of ectoparasite vaccines for several years, with the two caveats that, to be practically useful, we must: i) understand what molecules are truly essential to ectoparasite survival, and ii) demonstrate that these molecules are accessible to the host immune system [6].
• the present invention seeks to obviate the problems associated with the prior art.
• the present invention is based on the finding that specific antigens derived from Dermanyssus gallinae (D. gallinae: poultry red mite) can be used to raise immune responses in avian species, prone or susceptible to, infection or infestation with/by the same. Furthermore, the inventors have discovered that the immune response raised is protective and may prevent or facilitate the clearance or eradication of a D. gallinae infection/infestation, in or from, an avian host. Furthermore, given the significant health problems associated with D. gallinae infections/infestations, the present invention may not only be used to reduce populations of D. gallinae infection/infestation in avian hosts, but it may also be used as a means of indirectly addressing the numerous secondary diseases and/or conditions associated with D. gallinae infections/infestations .
• D. gallinae poultry red mite
• the invention relates to one or more of the D. gallinae antigens listed as (i)-(xi) below:
• An exemplary Deg-SRP-1 antigen is at least partly encoded by the following sequence (SEQ ID NO: 1)
• Deg-SRP-1 SEQ ID NO : 1
• An exemplary Deg-SRP-1 antigen may comprise the following amino acid sequence (SEQ ID NO: 2)
• Deg-SRP-1 SEQ ID NO : 2
• An exemplary Deg-HGP-1 antigen is at least partly encoded by the following sequence (SEQ ID NO: 3)
• An exemplary Deg-VIT-1 antigen is at least partly encoded by the following sequence (SEQ ID NO: 5)
• An exemplary Deg-VIT-1 antigen may comprise by the following amino acid sequence (SEQ ID NO: 6)
• Deg-PUF-1 full length sequence: SEQ ID NO: 7
• An exemplary Deg-PUF-1 antigen may comprise the following amino acid sequence (SEQ ID NO: 8)
• Deg-PUF-1 full length sequence: SEQ ID NO: 8
• An exemplary Deg-ASP-1 antigen is at least partly encoded by the following sequence (SEQ ID NO: 9)
• Deg-ASP-1 SEQ ID NO : 9
• An exemplary Deg-ASP-1 antigen may comprise the following amino acid sequence (SEQ ID NO: 10)
• the exemplary variant 1 Deg-PUF-2 antigen may comprise the following amino acid sequence (SEQ ID NO: 12)
• An exemplary variant 2 Deg-PUF-2 antigen is at least partly encoded by the following sequence (SEQ ID NO: 13)
• An exemplary variant 2 Deg-PUF-2 antigen may comprise the following amino acid sequence (SEQ ID NO: 14)
• Deg-PUF-2-Variant 2 SEQ ID NO: 14
• An exemplary Deg-SRP-2 antigen is at least partly encoded by the following sequence (SEQ ID NO:
• An exemplary Deg-SRP-2 antigen may comprise the following amino acid sequence (SEQ ID NO: 16)
• An exemplary pepCIA- 13094 antigen may comprise the following amino acid sequence (SEQ ID NO: 18)
• An exemplary Deg-GPD-1 antigen is at least partly encoded by the following sequence (SEQ ID NO: 19)
• An exemplary Deg-GPD-1 antigen may comprise the following amino acid sequence (SEQ ID NO: 20)
• MSAALQIKKVLI SDSCDARCAEILREAGCDVTVKTDFTKEQLVEAIKDFDALVVRSATKVTADVINAATNLKVIGRAGTG VDNIDCDVATARGVLVINAPGGNTLAAAEMTCAMII SLSRDVAAACASLKAGRWDRKTFMGTELNGKTLGIVGLGRIGRE VAIRMQAFGMTTIGYDPI IPAEQAAKFNVKAMSLDELWPQCDYITVHTPLLPETKNLI SAGTLARCKKGVKVVNCARGGI VNENDLLAALESGQASGAGFDVFEDEPPKNTAFIAHPKVICTPHLGANTKEAQSKVAIEIAEQFVALKKGERAWGAVNKP KPAN
• An exemplary Deg-PUF-3 antigen is at least partly encoded by the following sequence (SEQ ID NO:
• Deg-PUF-3 SEQ ID NO: 21
• An exemplary Deg-PUF-3 antigen may comprise the following amino acid sequence (SEQ ID NO: 22)
• Deg-PUF-3 SEQ ID NO: 22
• the invention relates to and/or exploits one or more D. gallinae antigens at least partly encoded by, or comprising, any one of the sequences provided by SEQ ID NOS: 1-22 above.
• D. gallinae antigen encompass the whole or native antigens named as (i)-(x) above; antigens comprising or encoded by any of the SEQ ID NOS: 1-22 and/or immunogenic/antigenic fragments, variants, recombinant forms and/or derivatives of any of these.
• An immunogenic/antigenic fragment may be any D. gallinae antigen fragment (for example a fragment of any of the D. gallinae antigens described herein) capable of eliciting an immune response when administered to an animal - in particular an avian species.
• An "immune response" may be regarded as any response which elicits antibody (for example IgY, IgA, IgM and/or IgG or any other relevant isotype) responses and/or cytokine or cell mediated immune responses.
• the various D. gallinae antigen fragments provided by this invention may be capable of eliciting an immune response which is substantially identical or similar to, an immune response elicited by the complete antigen from which the fragment is derived.
• the antigen fragments provided by this invention are capable of providing protective immune responses in avian species.
• D. gallinae antigens of this invention define one or more epitopes and as such, the term "antigen" may also embrace proteins or peptides which comprise one or more D. gallinae antigen epitopes.
• the term “antigen” embraces recombinant forms of any of the antigens described herein.
• the term “antigen” also includes recombinantly prepared immunogenic fragments of any of the D. gallinae antigens of this invention. Recombinant forms of the antigens of this invention and methods for obtaining the same are described later.
• the term “antigen” or “antigen fragment” may encompass variants or derivatives of any of the antigen(s) described herein - such antigens being referred to as "variant” or “derivative” antigens. Again, it should be understood that these terms include variants/derivatives of any of the D.
• any variant or derivative antigen may also be immunogenic/antigenic in that it elicits an immune response which is similar or substantially identical to an immune response elicited by the corresponding complete or native antigen in the same host - such variants/derivatives may be referred to as "immunogenic variants/derivatives".
• An immunogenic variant/derivative may comprise or be encoded by, a protein/peptide sequence or nucleic acid or amino acid sequence which comprises one or more nucleobase and/or amino acid substitutions, inversions, additions and/or deletions relative to a reference sequence (for example sequences of or encoding the D. gallinae antigens of this invention).
• substitution may encompass one or more conservative substitution(s).
• conservative substitution is intended to embrace the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physico-chemical properties and/or structure or function of the native (or wild type) protein.
• a variant/derivative antigen may comprise or be encoded by a mutant sequence which when compared to a reference sequence (such as for example a wild type sequence (including sequences of or encoding any of the specific D. gallinae antigens given as (i)- (x) above) or antigens comprising or encoded by any of SEQ ID NOS: 1-22 (or fragments thereof) above), is found to contain one or more amino acid/nucleotide substitutions, additions, deletions and/or inversions.
• An antigen which may be regarded as a derivative may further comprise one or more features of a fragment or variant described herein optionally in combination with one or more modifications to the structure of the antigen or one or more of the amino acid residues thereof.
• the degeneracy of the genetic code permits substitution of one or more bases in a codon without alteration to the primary amino acid sequence.
• genetic degeneracy may be exploited in order to yield variant sequences which encode peptide or protein sequences of any of the D. gallinae antigens described herein.
• useful fragments, variants, mutants and/or derivatives may comprise anything from about 5 to about 10 residues (amino acids and/or nucleic acids) of the complete amino acid or nucleic acid sequence (n) of (or encoding) the relevant complete wild-type or native D. gallinae antigen, to about n-1 residues.
• the fragments, variants and/or derivatives provided by this invention comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, 5400, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400,1500, 2000, 3000, 4000 or 5000 residues - the upper limit (n-1) depending upon the size (n) of the nucleic acid encoding the complete antigen or the number (n) of amino acid residues comprising the primary sequence of the antigen.
• the antigenic fragments, variants and/or derivatives provided by this invention comprise sequences or are at least partially encoded by sequences, which are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous or identical to the sequences (encoding or primary amino acid) of the various reference (exemplary) antigens disclosed herein.
• the degree of (or percentage) "homology" between two or more (amino acid or nucleic acid) sequences may be determined by aligning two or more sequences and determining the number of aligned residues which are identical or which are not identical but which differ by redundant nucleotide substitutions (the redundant nucleotide substitution having no effect upon the amino acid encoded by a particular codon, or conservative amino acid substitutions.
• a degree (or percentage) "identity" between two or more (amino acid or nucleic acid) sequences may also be determined by aligning the sequences and ascertaining the number of exact residue matches between the aligned sequences and dividing this number by the number of total residues compared - multiplying the resultant figure by 100 would yield the percentage identity between the sequences.
• this invention relates to:
• antigens comprising or at least partially encoded by one or more of the sequences provided as SEQ ID NOS: 1-22;
• the invention may further relate to any one of the antigens defined by (a) - (d) for use raising immune responses in animals - in particular avian species.
• compositions may embrace any form of immunogenic or vaccine composition.
• an immunogenic composition may be any composition comprising one or more of the antigens of this invention which, when administered to an avian species, elicits an immune response.
• a vaccine or vaccine composition of this invention may comprise one or more of the D. gallinae antigens of this invention and, when administered to an avian species, elicits an immune response which may comprise a protective immune response.
• an induced immune response may comprise a humoral (or antibody mediated) and/or cell mediated component.
• An induced immune response may comprise a protective immune response and one of skill will appreciate that a protective immune response may contribute to an animal's ability to resolve an infection/infestation and/or which helps reduce the symptoms associated with an infection/infestation.
• a protective immune response may contribute to an animal's ability to resolve an infection/infestation and/or which helps reduce the symptoms associated with an infection/infestation.
• the immune responses raised through exploitation of the antigens described herein may be referred to as "protective" immune responses.
• the term "protective" immune response may embrace any immune response which: (i) facilitates or effects a reduction in host pathogen burden; (ii) reduces one or more of the effects or symptoms of an infection/infestation; and/or (iii) prevents, reduces or limits the occurrence of further (subsequent/secondary) infections.
• a protective immune response may prevent an animal from becoming infected/infested with a particular pathogen and/or from developing a particular disease or condition.
• a protective immune response may prevent an animal from becoming infected/infested with D. gallinae; limit the extent of a D. gallinae infection/infestation; and/or may prevent an animal from developing a disease or condition associated with a D. gallinae infection/infestation and/or one or more symptoms associated therewith.
• the symptoms associated with a D. gallinae infection/infestation in, for example, poultry (including domestic chickens) are pain, and irritation - particularly around the breast and leg area; and a decrease in egg production.
• Other symptoms may include the formation or development of pustules, scabs and hyperpigmentation and the loss of feathers. Severe infections/infestations in avians can lead to anaemia.
• compositions are to be understood as including either immunogenic compositions and/or vaccine composition.
• references to the raising of "immune responses” should be understood as encompassing not only humoral and/or cell mediated immune responses, but also any form of "protective immune response”.
• An aspect of this invention provides one or more Dermanyssus gallinae (D. gallinae) antigens, for use in raising an immune response in an avian species, wherein the one or more D. gallinae antigens are selected from the group consisting of:
• Protein of unknown function 2 (Deg-PUF-2): variants 1 and/or 2;
• the antigens for use according to the first aspect of this invention are provided in the form of a composition.
• the composition may comprise one or more excipient or diluents.
• the composition may be an immunogenic composition or a vaccine composition.
• the compositions may further comprise an adjuvant.
• the invention provides the use of one or more of the Dermanyssus gallinae (D. gallinae) antigens listed as (i) to (xi) above, in the manufacture of a medicament for raising an immune response in an avian species.
• the invention provides a method of raising an immune response in an avian species, said method comprising administering one or more of the Dermanyssus gallinae (D. gallinae) antigens listed as (i) to (ix) above to said avian species.
• avian avian species
• avian host as used herein are intended to encompass all avian species prone or susceptible to D. gallinae infection or infestation.
• the "avian species” encompassed by this invention include, for example, those collectively known as poultry or fowl. In other embodiments these terms extend to include domesticated or game bird species such as, for example, chicken, pheasant, grouse, turkey, guineafowl and/or duck species. In one embodiment, the terms “avian”, “avian species” or “avian host” extend to commercially important or farmed bird species.
• the antigens and compositions for use, uses, medicaments and methods provided by this invention may be exploited in order to raise immune responses, for example protective immune responses, in chickens (Gallus gallus domesticus).
• the invention provides antigens and compositions for use, medicaments and methods which may be exploited to prevent, reduce and/or treat the occurrence of D. gallinae infections/infestations in chickens.
• D. gallinae is a blood feeding avian ectoparasite and is exposed to host immunoglobulin when taking blood meals.
• D. gallinae parasites are exposed to the anti- D. gallinae antigen antibodies which adversely affect, debilitate, destroy, kill or inactivate the D. gallinae parasites.
• gallinae parasites may involve antibody mediated cell cytotoxicity processes and/or complement pathways.
• antibodies and “specific” or “selective” antibodies, these terms encompass antibodies (and active or epitope binding fragments thereof) which bind to the antigens described herein (or epitopes thereof) and/or antibodies (and active or epitope binding fragments thereof) which exhibit a degree of selectivity, specificity and/or affinity therefor.
• antibody may relate to polyclonal antibodies or monoclonal antibodies. Antibodies of this type are discussed later.
• the invention relates to vaccines or vaccine compositions comprising one or more the D. gallinae antigens described herein for raising immune responses in avian species.
• Vaccines may be used prophylactically to prevent the establishment of a D. gallinae infection/infestation on an avian host or to reduce, ameliorate or eradicate an established D. gallinae infection/infestation.
• vaccines or vaccine compositions comprising any of the D. gallinae antigens described herein may be used as a means to indirectly ameliorate, reduce the symptoms of or eradicate, a secondary complication, disease or condition associated with a D. gallinae infection/infestation.
• any reduction in the population of a D. gallinae infection/infestation affected by the vaccine described herein may, in turn, affect a reduction in instances of secondary complications associated with D. gallinae infections/infestations.
• These secondary complications may be associated with, for example bacterial pathogens including Salmonella, Campylobacter or E. coli, mycobacterial species such as M. gallisepticum, or viruses including avian influenza.
• pathogens are often carried on or within the mites [3] affecting the avian host, or may be the result of the detrimental health effects on the avian host or on humans within the mites' environment, of allergens produced by the mites.
• avian species administered the antigens, compositions, immunogenic composition, vaccines or vaccine compositions according to this invention may produce protective anti-D. gallinae antigen antibodies serving to debilitate, destroy, kill or inactivate D. gallinae.
• One or more of the antigens described herein may directly and/or indirectly be involved in, for example, mating, fertilisation, embryogenesis, vitellogenesis, sequestration of nutrients for embryonic development, embryonic development, gender-specific reproductive and developmental processes, sexual differentiation and maturation, sexually- differentiated somatic and germ cell processes, oogenesis, spermatogenesis, oocyte maturation and/or ovulation.
• mating, fertilisation, embryogenesis, vitellogenesis sequestration of nutrients for embryonic development, embryonic development, gender-specific reproductive and developmental processes, sexual differentiation and maturation, sexually- differentiated somatic and germ cell processes, oogenesis, spermatogenesis, oocyte maturation and/or ovulation.
• compositions provided by this invention may be formulated as sterile pharmaceutical compositions comprising one or more of the antigens described herein and a pharmaceutical excipient, carrier or diluent. These composition may be formulated for oral, topical (including dermal and sublingual), parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and/or mucosal administration.
• compositions described herein may comprise a discrete dosage unit and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association one or more of the D. gallinae antigens described herein with liquid carriers or finely divided solid carriers or both.
• compositions suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of one or more of the D. gallinae antigens of this invention.
• a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
• Compressed tablets may be prepared by compressing in a suitable machine an active compound (for example one or more D. gallinae antigen(s)) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent.
• Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored.
• Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope.
• An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet.
• Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion.
• compositions suitable for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound (for example one or more D. gallinae antigen(s)) is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film.
• an active compound for example one or more D. gallinae antigen(s)
• Such compositions may be particularly convenient for prophylactic use.
• Composition formulated for parenteral administration include sterile solutions or suspensions of an active compound (for example one or more D. gallinae antigens) in aqueous or oleaginous vehicles.
• an active compound for example one or more D. gallinae antigens
• compositions may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use.
• an active compound for example one or more D. gallinae antigen(s)
• a suitable vehicle such as sterile, pyrogen-free water or PBS before use.
• compositions comprising one or more of the D. gallinae antigen(s) of this may be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly.
• Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. They may also include preparations or adjuvants known to enhance the affinity and/or longevity of the avian immune response, such as single or double emulsions of oil in water.
• Such long-acting compositions are particularly convenient for prophylactic use.
• compositions suitable (or formulated) for mucosal administration include compositions comprising particles for aerosol dispersion, or dispensed in drinking water. When dispensed such compositions should desirably have a particle diameter in the range 10 to 200 microns to enable retention in, for example, the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve.
• Other suitable compositions include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.
• compositions described herein may include, an appropriate one or more additional (pharmaceutically acceptable) carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
• additional (pharmaceutically acceptable) carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.
• Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, mineral oils and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
• compositions suitable for topical formulation may be provided for example as gels, creams or ointments.
• compositions for veterinary use may conveniently be in either powder or liquid concentrate form.
• conventional water-soluble excipients such as lactose or sucrose, may be incorporated in the powders to improve their physical properties.
• suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) (for example one or more D. gallinae antigens) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients.
• active ingredient(s) for example one or more D. gallinae antigens
• These powders may either be added to, for example, avian feed - perhaps by way of an intermediate premix, or diluted in animal drinking water.
• Liquid concentrates of this invention suitably contain one or more D. gallinae antigens and may optionally further include an acceptable water-miscible solvent for veterinary use, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol.
• the liquid concentrates may be administered to the drinking water of animals.
• a suitable dose of the one or more D. gallinae antigens provided by this invention may be in the range of about 10 to about 1000 ⁇ g per bird.
• the one or more antigens described herein may be administered on about 2 to about 5 occasions over a period of about 1 to about 10 weeks.
• each bird may be administered about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ⁇ g of the one or more antigens described herein.
• each bird may be administered the antigen(s) on 2, 3, 4 or 5 occasions over a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 week period. It should be understood that each bird may receive the same or a different dose of the D. gallinae antigen(s) on each administration occasion.
• compositions, immunogenic compositions and vaccines of this invention may comprise one or more of the antigens described herein.
• the various compositions may comprise two or more of the antigens described herein.
• An immunisation protocol may comprise the use (or administration) of one or more different compositions which together ensure that an animal (for example an avian subject) has been administered one or more of the antigens of this invention.
• the antigen(s) that are to be administered may be administered alone or together. Where two or more antigens are to be administered, the may be administered separately and at different times, together or concurrently.
• Transdermal administration may be achieved with the use of impregnated coverings dressings, bandages or the like or via the use of some form of transdermal delivery device.
• antigens for use in this invention may be obtained from whole or fragmented parasites harvested from donor animals and/or from the environment or habitat of the avian.
• Donor animals may be naturally infected/infested animals or animals which have been deliberately (or experimentally) infected with D. gallinae.
• D. gallinae from which antigens may be derived may be obtained from a number of different donor avian subjects - those avian subjects inhabiting the same or different environments and/or habitats.
• the D. gallinae may be obtained from one or more sites within a farm (for example a particular pen, poultry house, cage or shed within the farm) and/or from one or more of the infected or infested avian hosts within said site.
• a vaccine or composition intended for use on a specific population of avian subjects infected/infested with D. gallinae may comprise one or more D. gallinae antigens derived from (i) one or more D. gallinae collected or harvested from the environment, habitat or locale of the specific population to be administered a composition of this invention and/or vaccinated and/or (ii) one or more D. gallinae collected or harvested from an environment, habitat or locale linked or associated with the specific population to be administered a composition of this invention and/or vaccinated.
• the invention may exploit D. gallinae antigen(s) obtained or harvested from one or more of the sites of the complex - these antigens could be used to provide compositions and/or vaccine for use on those avian subjects farmed at each site of the complex.
• compositions and/or vaccines of the type described in the two paragraphs above as “autologous compositions” or “autologous vaccines”.
• autologous compositions or “autologous vaccines”.
• the invention provides autologous composition and vaccines comprising one or more of the D. gallinae antigens disclosed herein, for use in raising immune responses in avian species.
• D. gallinae antigens for use, harvested or collected D. gallinae may be subjected to a homogenisation protocol to yield a homogenised suspension of D. gallinae components.
• the resulting homogenised D. gallinae suspension may then be subjected to one or more size/density separation techniques (such as centrifugation) so as to remove unwanted D. gallinae debris from fractions containing one or more of the antigens of this invention.
• the antigen containing fractions may then be subject to sterilisation procedures to render them suitable for use in this invention.
• Antigen containing fractions prepared in this way may be further processed in order to yield fractions comprising fewer and more highly purified (or cleaner)/concentrated antigens or specific or select antigens - the ultimate aim being to produce one or more fractions free of all but antigens corresponding to those described herein and/or any fragments thereof.
• techniques such as, for example, anion exchange, gel filtration and/or affinity chromatography may be used to prepare antigen fractions for use, and/or to extract one or more specific antigens.
• antibodies extracted from avian subjects administered one or more of the antigens of this invention may be immobilised on to a suitable substrate and brought into contact with a solution comprising one or more of the antigens of this invention under conditions which permit binding between the antibodies and their target antigens.
• the solution may comprise a pool of different antigens. In this way, one or two specific antigens can be extracted or enriched from a mixed population of antigens.
• the antigens of this invention may be used to raise yolk (IgY) immune responses.
• Yolk IgY may be immobilised on to a suitable substrate.
• D. gallinae antigens for use in this invention may be further prepared for cold or freeze storage by the addition of one or more cryopreservative/cryoprotectant agents.
• the antigens and compositions for use, uses, medicaments and methods provided by this invention comprise one or more D. gallinae antigens comprising or encoded by a sequence at selected from the group consisting of:
• (c) a sequence at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous or identical to an immunogenic fragment of any one of (a), (b) or (c).
• the invention in addition to providing D. gallinae antigen amino acid sequences (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22), the invention further relates to the various nucleic acid sequences encoding the same (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21) and or fragments (preferably fragments encoding immunogenic peptides) thereof.
• nucleic acid sequences may be modified to replace one or more of the naturally occurring residues with corresponding or equivalent synthetic or chemically or enzymatically modified nucleotides.
• the nucleic acid sequences disclosed in this specification may be further subjected to modification to allow for the degeneracy of the nucleic acid code. That is to say, any given wild type sequence may be modified such that one or more of the codon sequences is modified without having any effect on the primary amino acid sequence.
• nucleic acid sequences that are substantially complementary to all or part of any of the sequences provided by SEQ ID NOS: 1-22 - in particular the nucleic acid sequences.
• Such complementary sequences may be useful as primers or probes.
• the complementary sequences may be complementary to sequences which represent 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more (for example up to 100, 200 or more) continuous or contiguous bases of any given sequence disclosed herein.
• nucleic acid sequence having a level of identity or homology with a sequence complementary to any of SEQ ID NO: 1-22 may exhibit at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or homology with the full length complementary sequence or the relevant portion thereof.
• the invention may further relate to natural or artificially created variants or analogues of any of the protein/nucleic acid sequences described herein.
• Such variants may exhibit one or more amino acid/nucleic acid deletions, additions, substitutions and/or inversions, relative to, for example a reference sequence (such as for example sequences disclosed herein including those disclosed as SEQ ID NOS: 1-22).
• the substitutions may represent conservative substitutions.
• a conservative substitution involves replacing one or more amino acids of a protein or peptide sequence with an alternate amino acid having similar properties and which does not substantially alter the physio-chemical properties and/or structure or function of the native (or wild type) protein.
• Nucleic acid molecules provided by this invention may take the form of nucleic acid constructs or vectors such as, for example a cloning or expression cassettes/vectors including phage vectors for use as described, for example in EP1370284.
• the term "nucleic acid constructs" or “vector” may further encompass constructs intended for use as DNA vaccines.
• Vectors provided by this invention may be capable of directing the expression of nucleic acid sequences encoding D. gallinae antigens in, for example, bacterial, fungal, animal (including Protozoa, avian species and mammalian) and/or insect cells.
• this invention provides a vector, preferably an expression vector, comprising a nucleic acid sequence encoding a D. gallinae antigen of this invention or having or encoded by a sequence selected from the group consisting of:
• Expression vectors suitable for use in this aspect of the invention may further comprise one or more promoter sequences capable of directing expression in prokaryotic or eukaryotic cells such as, for example, Protozoan, avian, mammalian, fungal, bacterial, plant and/or insect cells.
• a vector provided by this invention may be circular or linear, single stranded or double stranded and can include DNA, RNA or a combination or modification thereof.
• vectors of this invention may be, for example, plasmids, cosmids or viral vectors (for example retroviral or bacteriophage vectors).
• Vectors provided by this invention may further comprise selection or marker elements, for example antibiotic resistance genes and/or optically detectable tags.
• selection or marker elements for example antibiotic resistance genes and/or optically detectable tags.
• a large number of suitable vectors are known and further information may be obtained from Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and supplements), Elsevier, N.Y.; and Rodriquez, et al. (ads.) Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988) - both of which are incorporated herein by reference.
• an expression vector comprising one or more nucleic acid sequences encoding the relevant D. gallinae antigen (such as any of those described herein) may be used to produce one or more recombinant D. gallinae antigens.
• the present invention provides host cells transfected or transformed with a vector as described herein.
• Eukaryotic or prokaryotic cells such as, for example Protozoan, avian, plant, insect, mammalian, fungal and/or bacterial cells, may be transfected with one or more of the vectors described herein.
• One of skill in this field will be familiar with the techniques used to introduce heterologous or foreign nucleic acid sequences, such as expression vectors, into cells and these may include, for example, heat-shock treatment, use of one or more chemicals (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information regarding transformation/transfection techniques may be found in Current Protocols in Molecular Biology, Ausuble, F.M., ea., John Wiley & Sons, N.Y. (1989) which is incorporated herein by reference.
• the present invention further provides a process for the production of a
• D. gallinae antigen comprising or encoded by a sequence of SEQ ID NO: 1-22 (or an immunogenic fragment thereof) and for use in raising an immune response in an avian, said method comprising the step of (a) transforming a host cell with a nucleic acid sequence according to this invention or transfecting a host cell with a nucleic acid construct of the invention; (b) culturing the cells obtained in (a) under conditions in which expression of the nucleic acid (or rather a protein encoded thereby) takes place; and (c) isolating the expressed recombinant protein or peptide from the cell culture and/or the culture supernatant.
• Recombinant proteins/peptides produced according to the method described above may be partially purified from the host cell before being used in a vaccine or vaccine composition.
• the cells may be separated from the media by centrifugation.
• the supernatant, which contains the secreted polypeptide may be used directly as a vaccine, or in a vaccine composition.
• the polypeptide may be partially purified from this supernatant, for example using affinity chromatography.
• D. gallinae antigens described herein may be admixed with another component, such as another polypeptide and/or an adjuvant, diluent or excipient.
• compositions, immunogenic compositions and/or vaccines/vaccine compositions provided by this invention may, for example, contain viral, fungal, bacterial or other parasite antigens used to control other avian diseases/infections or infestations.
• the vaccine or vaccine composition may be included within a multivalent vaccine which includes antigens against other avian (for example chicken) diseases.
• These vaccines may include, for example, the vaccines and target organisms listed in Table 1, below.
• HIPRA S.A Egg drop syndrome virus [EDS virus, Avian adenovirus],
• NDV Avian paramyxovirus
• Eimeria praecox Eimeria tenella
• Eimeria mitis Eimeria mitis
• Chevivac-P12 Ceva Animal Pigeon paramyxovirus 1 [PPMV-1 ]
• An adjuvant for use in this invention or for addition to a composition described herein may comprise for example, aluminum salts (alum), oil emulsions, saponins, immune- stimulating complexes (ISCOMs), liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, and/or a form of bacterial derivative.
• alum aluminum salts
• ISCOMs immune- stimulating complexes
• liposomes e.g., liposomes, microparticles, nonionic block copolymers, derivatized polysaccharides, cytokines, and/or a form of bacterial derivative.
• the present invention provides an avian population, for example a farmed population of chickens, treated, vaccinated or immunised with a vaccine or composition described herein, and/or a vaccine or composition comprising one or more of the D. gallinae antigens described herein.
• the vaccines described in this invention may take the form of subunit-type vaccines whereby one or more D. gallinae antigens are used to inoculate an animal.
• the vaccine may comprise a nucleic acid molecule (known as a DNA vaccine) encoding one or more antigens provided by this invention or an immunogenic fragment thereof, to be expressed by the cells of an animal to be vaccinated.
• constitutive expression of D. gallinae antigens in a vaccinated host such as, for example a vaccinated chicken
• a constitutive protective immune response may be elicit a constitutive protective immune response.
• the present invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have affinity or specificity for) any of the D. gallinae antigens described.
• Production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example "Basic methods in Antibody production and characterisation” Howard & Bethell, 2000, Taylor & Francis Ltd.
• Such antibodies may be used in diagnostic procedures, to, for example detect or diagnose D. gallinae infection/infestations in avian species, as well as for passive immunisation.
• the term "antibody” includes egg yolk antibody (IgY).
• the present invention further provides a vaccine for use in preventing or controlling D. gallinae infection/infestation and associated diseases in avian hosts.
• the vaccine may be a polypeptide or polynucleotide vaccine.
• the invention further provides a method for immunising an avian against D. gallinae infection/infestation and associated disease (for example secondary infections etc.), said method comprising the step of administering a vaccine of the invention to the avian.
• the invention further provides methods of diagnosing or detecting D gallinae infections, infestations and/or contamination in samples.
• the methods may exploit one or more of the D.
• gallinae antigens of this invention and/or antibodies with specificity and/or affinity for any of the antigens of this invention.
• the antibodies and/or antigens for use may be immobilised (perhaps via some form of binding moiety, for example an antibody) to a substrate.
• Suitable substrates may include those comprising nitrocellulose, glass and/or plastic - for example a microtitre plate or the like.
• the antibody and/or antigen(s) for use may be immobilised as an array comprising multiple discrete spots. Any given spot may independently comprise a single type of antibody/antigen or antibodies with a single antigen specificity/affinity. Additionally or alternatively, any given spot may comprise different types of antibody or antigen or antibodies with specificity/affinity for different antigens.
• a method of diagnosis or detection may comprise bringing a sample into contact with an (optionally immobilised) antigen or antibody of this invention.
• the sample and the (optionally immobilised) antigen or antibody of this invention may be brought into contact under conditions which permit binding between any antibody and/or antigens in the sample and the (optionally immobilised) antibodies and/or antigens of this invention.
• samples may be probed for the presence of antibodies reactive to (or with specificity and/or affinity for) a D.
• gallinae antigen and/or D. gallinae antigens may be indicative of a D. gallinae infection, infestation and/or contamination.
• the presence of antibodies reactive to (or with specificity and/or affinity for) a D. gallinae antigen and/or D. gallinae antigens may be confirmed by detection of antibody/antigen complexes.
• the sample may be contacted with any of the (optionally immobilised) antigens of this invention under conditions suitable to permit binding between any antibodies in the sample and the antigens to which the sample has been contacted.
• antibody/antigen complexes may be detected by the use of, for example, labelled (for example fluorescent or chemiluminescently labelled) secondary antibodies (or other binding moieties) with affinity for the antibodies present in the sample.
• labelled for example fluorescent or chemiluminescently labelled
• secondary antibodies or other binding moieties
• affinity for the antibodies present in the sample.
• a similar method can be practiced in order to detect the presence of D. gallinae antigens in a sample.
• the antigens may be any of those described herein and the sample may be contacted with (optionally immobilised) antibodies with specificity and/or affinity for the antigen(s).
• a sample may comprise a biological sample, for example a sample of blood (for example avian/chicken blood, whole blood, plasma or serum), yolk, faeces, urine, sweat, tissue, skin, scrapings, secretions and the like.
• a sample may comprise an environmental or field sample, for example a sample of bedding material, straw, soil, grass, food, fluid for drinking and the like.
• kits optionally for use in methods of detecting and/or diagnosing D. gallinae infections, infestations and/or contamination, said kits comprising one or more of the antigens described herein and/or antibodies with specificity and/or affinity to/for any of the antigens of this invention.
• the antigens and/or antibodies for use in the kit may be immobilised on to suitable substrates.
• the kits may further provide instructions for use, receptacles for reactions and diluents, buffers and/or washing solutions for use in methods of detection and/or diagnosis. DETAILED DESCRIPTION
• Figure 1 The in vitro feeding device used to deliver a defined blood meal to Dermanyssus gallinae.
• the device is a modification of that described by Wright et al., 2009 [9]. Specific modifications include: the replacement of a chick-skin feeding membrane with pre-stretched parafilm"M" (Bemis® Flexible packaging) and the placement of a lamb rubber castration ring (NetTex) around the pastette bulb to create a tight seal to prevent blood-meal leakage.
• M pre-stretched parafilm
• NetTex lamb rubber castration ring
• Mites were fed with whole heparinised blood from hens vaccinated with the IEX-pools [Group (Gp) 1-5], soluble mite extract (Group 6) or adjuvant only (Group 7).
• a survival analysis was conducted using Cox's proportional hazards model with frailty function. The mean proportions of surviving mites at 24, 48, 72 and 96 hours post-feeding, with corresponding 95 % confidence intervals (shaded), are plotted.
• IEX Group 1 proteins were separated on replicate gels with a pi range of 7-11.
• One gel was stained with SimplyBlueTM SafeStain (Life Technologies) (panel C) and two replicate gels were immunoblotted and screened with either 100 ⁇ g/ml yolk-IgY from hens vaccinated with Group 1 proteins (panel A) or with a 100 ⁇ g/ml yolk-IgY from control (Group 7) hens (panel B).
• Bound IgY was detected with rabbit anti-IgY-peroxidase IgG and visualised with chemiluminscent substrate. Spots for excision and identification following LC-ESI-MS/MS are circled and numbered.
• Purified yolk-IgY generated against IEX Group 4 proteins was cross-linked to a HiTrap NHS- activated column and IEX Group 4 proteins were selectively enriched by immunoaffinity chromatography.
• the Group 4 proteins prior to affinity purification (panel A, lanes 1 and 3) and the eluted affinity-enriched material (panel B, lanes 2 and 4) were separated on a 12% Bis-Tris Novex gel (Invitrogen) and transferred to nitrocellulose.
• Lanes 1 and 2 of the immunoblot were probed with a 1 :200 dilution of sera pooled from the Group 7 (adjuvant-only control) hens and lanes 3 and 4 were probed with a 1 :200 dilution of post-vaccination sera pooled from the Group 4 hens.
• Bound IgY was detected with rabbit anti-IgY-peroxidase (Sigma) and visualized with SIGMA FASTTM 3,3'-diaminobenzidine substrate (Sigma).
• SIGMA FASTTM 3,3'-diaminobenzidine substrate Sigma.
• the eluted immunoaffinity enriched material was concentrated, electrophoretically separated and stained with SimplyBlueTM SafeStain (panel B, lane 5).
• the stained enriched protein profile depicted in lane 5 was cut into 24 equally-sized horizontal slices and the proteins extracted and subjected to LC-ESI- MS/MS.
• Deg-SRP-1 is annotated as "Serp-02564"; Deg-HGP-1 is annotated as "HemeLGP- 13207"; Deg-VIT-1 is annotated as “Vit-12013”; Deg-PUF-1 is annotated as "UK-11549"; Deg-ASP-1 is annotated as "Asp-00293”; Deg-PUF-2 is annotated as "UK-13089”; Deg-SRP-2 is annotated as "Serp-01514"; Deg-CPR-1 is annotated as "PepClA-13094"; Deg-GPD-1 is annotated as "PGDH-00877” and Deg-PUF-3 is annotated as "UK-00
• Soluble mite proteins were extracted from snap-frozen mites as follows: lg of frozen mites was suspended in 10ml ice-cold phosphate buffered saline (PBS) and homogenized, on ice, for 2x 30 second pulses (Ultra Turrex® T 25 D-S2 with a S25N-8G dispersing element, IKA). Insoluble material and debris were removed by centrifugation at 25,000 xg for 20 minutes at 4°C. The soluble material was decanted and centrifuged for a second time to further clarify the SMPs. The SMPs were immediately snap-frozen in liquid nitrogen and stored at -80°C. The concentration of SMPs was estimated using a bicinchoninic acid (BCA) assay (Peirce), following the manufacturer's protocol.
• BCA bicinchoninic acid
• IEX Prior to ion-exchange chromatography (IEX), 20 mg SMP were desalted using a PD-10 desalting column (GE healthcare). IEX was performed at room temperature using a 1ml HiTRAP Q HP anion column (GE healthcare) in conjunction with an AKTA fast liquid protein chromatography (FPLC) system (Amersham Biosciences) eluting with a linear gradient of 0 to 0.5 M NaCl concentration in 20 mM Tris-HCL, pH 7.4, followed by an isocratic step (20 mM Tris-HCL, 1 M NaCl, pH 7.4). Eluted proteins were collected in 1ml fractions and immediately snap frozen in liquid nitrogen and stored at -80 °C.
• FPLC AKTA fast liquid protein chromatography
• Immunoblots of IEX-SMP fractions were probed with IgY from hens immunised with whole SMP in adjuvant or adjuvant/PBS alone (control group), as described below, and pooled into 5 distinct pools (Groups 1-5) based on their immunogenic profiles.
• Each of the IEX-SMP pools and unfractionated SMP were concentrated using Ultracel®-10K Amicon® Ultra- 15 centrifugal filter units (Merck Millipore Ltd), filter sterilised through a 0.22 ⁇ Millex-GV 13 mm PVDF unit (Merck Millipore Ltd) and their concentration determined (BCA kit, Pierce) prior to immunisation of hens.
• the immobilised proteins were probed with 80-100 ug/ml yolk-derived IgY (yolk-IgY) in PBS or with serum diluted 1 :50 to 1:200 in PBS.
• IgY and sera were derived from hens immunised with SMP in Quil A adjuvant or PBS/adjuvant only as described in [9]. Blots were washed and bound IgY was detected by incubating in rabbit-anti-IgY-peroxidase conjugate (diluted at 1:20,000 in PBS, Sigma).
• Blots were washed 5 times in PBST prior to developing in either the colorimetric substrate: SIGMA FASTTM 3,3'-diaminobenzidine (Sigma) or in SuperSignal West Pico Chemilurmnescent Substrate (Thermo Scientific). Blot images were captured using an ImageQuant LAS4000 luminescent image analyzer (GE healthcare).
• ISA-Warrens hens were randomly assigned into 7 groups; 5 IEX- pools (Groups 1-5), whole SMP (Group 6) and QuilA adjuvant only (control, Group 7) and were immunised 3 times, at fortnightly intervals with the appropriate immunogen.
• Each 200 ⁇ vaccine dose was formulated to contain 200 ⁇ g of the appropriate proteins and 200 ⁇ g QuilA adjuvant (Brenntag Bioserve) (Groups 1 to 6), or 200 ⁇ g QuilA alone (Group 7).
• Vaccines were administered intramuscularly into alternate breast muscles. Eggs were collected prior to vaccination and throughout the study as a source of yolk-IgY.
• IgY was purified from yolk using an IgY purification kit according to manufacturer's instructions (Pierce). Whole blood was withdrawn from the brachial wing vein prior to each vaccination and at 3, 5 and 6 weeks post final vaccination into heparinised tubes (final concentration 36 USP/ml blood). In order to assess the effect of ingesting blood from vaccinated hens on mite survival, heparinised blood was pooled from the two birds in each group and was fed to conditioned D. gallinae using a modified version of the in vitro feeding apparatus described previously [9] ( Figure 1). Ten feeding chambers were set up for each group and the mites allowed feed for a 24 hour period at 39 °C at 75 % relative humidity.
• Fed mites were removed from the feeding chamber and isolated in a 96 well microtitre plate, where they were immediately scored for mortality and again at 48, 72 and 96 hours post feeding.
• the feeding assays were performed at 3, 5 and 6 weeks after final immunisation.
• Proteins in Groups 1 and 4 from the IEX-enrichment were separated using 2-dimensional (2-D) electrophoresis and the constituent immunoreactive proteins identified using Western blotting coupled with proteomic analysis. All kits, materials and equipment used in the 2-D procedure were obtained from GE healthcare and all procedures, unless otherwise stated, were performed according to detailed methodology given in the manufacturer's handbook (GE healthcare, 2004).
• the focussed proteins were denatured in 75mM Tris-HCl pH 8.8, 6 M urea, 30 % (v/v) glycerol, 2 % (w/v) SDS, 0.002 % (w/v) bromophenol blue supplemented with 10 mg/ml DTT for 15 mins, followed by incubation for 15 mins in equilibration buffer containing 25 mg/ml iodoacetamide, then separated in the second dimension using Excel SDS Gel 2-D homogenous 12.5% (25 cm x 11 cm) gels in a Multiphor II Electrophoresis System. One replicate of the 2 n dimension gels was excised and the proteins stained with SimplyBlueTM SafeStain (Invitrogen).
• yolk-IgY from hens immunised with IEX Group 4 proteins was cross-linked to a 1ml HiTrap NHS-activated HP column (GE healthcare). Briefly, buffer exchange was performed on purified yolk-IgY using a PD-10 column (GE healthcare) equilibrated with coupling buffer (0.2 M NaHC0 3 , 0.5 M NaCl, pH 8.3) following the manufacturer's spin procedure.
• the HiTrap NHS-activated HP column was primed with 6ml ice-cold 1 mM HCl and 3.75 mg of yolk-IgY in coupling buffer was re-circulated through the column for 3 hours at 4°C.
• the yolk-IgY was cross-linked to the sepharose matrix and the column deactivated by 3 rounds of sequential washes with 2 ml buffer A (0.5 M ethanolamine, 0.5 M NaCl, pH 8.3) and 2ml buffer B (0.1 M Sodium acetate, 0.5 M NaCl, pH 4) followed by equilibration with 12 ml PBS.
• IEX Group 4 proteins (1.4 mg) were re-circulated through the column for 16 hours at 4°C.
• CDS Coding sequences for the open reading frames (ORFs) of the transcripts encoding each of the proteins were either inferred from bioinformatic analyses of their respective contigs or derived by rapid amplification of cDNA ends (RACE) in the case of Deg-SRP-2.
• RACE rapid amplification of cDNA ends
• the CDS for both of these Deg-PUF-2 variants were synthesized (MWG Operon) whereas the CDS for all remaining vaccine candidates were amplified by RT-PCR from total RNA as described previously [14].
• each of the recombinant proteins was assessed as follows: Hens, at point of lay, were injected intramuscularly (breast), 3 times at 2 week intervals, with 50 ⁇ g of each of the proteins described in Table 3, with 200 ⁇ g Quil A as adjuvant. An adjuvant-only control group was also included. From each hen, blood was withdrawn from the brachial wing vein prior to each vaccination and at weekly intervals commencing 2 weeks after the 3rd vaccination to monitor serum antibody levels and for in vitro feeding assays. Mites were fed with blood from immunised hens (blood taken 2, 3 and 4 weeks after 3 immunisation) using the modified in vitro feeding device described above and in Figure 1, and mortality was measured at 24h intervals over 5 days.
• the feeding assays were carried out in triplicate. The mean percentage mortality increase in the vaccine groups compared to the relevant adjuvant-only control groups was calculated.
• Mite mortality data were analysed using two different statistical approaches.
• a survival analysis incorporating the treatment group as an explanatory variable, was conducted based on Cox's proportional hazards model with frailty function. Mites that survived after 96 h were treated as censored data.
• the frailty function incorporated the replicate within each experiment as a random effect, and the estimate of random effect obtained using the restricted maximum likelihood method, assuming a Gaussian distribution.
• the cumulative mortality rates of mites in each treatment group at 24, 48 and 96 hours were analysed with separate generalised linear mixed models (GLMM) in order to examine differences in the mean mortality rates among groups at those time points.
• the model included the treatment group as a fixed effect and the replicate within each experiment as a random effect. The over-dispersion in the data was considered by including an individual-level random effect.
• the generalised linear mixed model was fitted using a Binomial distribution and logit link function.
• IEX was successful in separating the complex mix of proteins contained in the SMP into a "flow- through” (FT) fraction (Group 1 pool) containing unbound proteins and sequentially eluted fractions which could be pooled into 4 Groups (Groups 2-5) with minimum overlap in protein and immunoreactive profile ( Figure 2).
• FT flow- through
• Figure 2 Minimal immunoreactivity was detected when pooled IEX- fractions or SMP were incubated with IgY from an unimmunised hen, except for 2 proteins with molecular masses of approximately 25 and a 65 KDa, which correspond with IgY light and heavy chains.
• the IgY present in the IEX- fractions and SMP is derived from the blood meal present in the mite's guts and is detected by the anti-IgY-peroxidase conjugate.
• the 5 IEX-pools (Groups 1-5), whole SMP (Group 6) and QuilA adjuvant only (control, Group 7) were used to immunise hens and blood from these immunised hens was fed to mites. Mites that had ingested the blood meal were scored for mortality at 24 hours post-feeding and daily thereafter. The mean survival proportions of mites in all immunised groups, except group 2, were lower (/? ⁇ 0.001) than the control group (Table2, Figure 3). Mites in Groups 1 and 4 had the highest risk of mortality at any time during the experiment, and were 3.06 and 3.72 times more likely to die than the control group (/? ⁇ 0.001).
• Recombinant proteins were produced for the 10 selected candidate antigens (Table 3) identified from the IEX-Group 1 and Group 4 proteins by their immunoreactivity and putative function. These recombinant antigens were successfully expressed in E. coli, affinity purified using nickel- affinity chromatography and refolded prior to immunization of hens. Mites were fed on blood from the immunized hens in vitro and all data derived from the feeding assays were analysed using Cox's proportional hazards model applied over the 4 days post feeding period (Table 4, Figure 6). In a further analysis, a generalised linear mixed model (GLMM) was applied to mortality data 24 hour and 120 hours post feeding (Table 4). There was strong statistical evidence to indicate that immunisation with Deg-SRP-1, Deg-PUF-1, Deg-HGP-1 and Deg-VIT-1 produced the greatest increases (/? ⁇ 0.05) in mite mortality when analysed using both tests.
• Table 3 Summary of the 10 proteins selective as vaccine antigen candidates. Selection was based on proteomic identification using 2-D electrophoresis with immunoblotting and/or immunoaffinity protein enrichment. 7 antigens have inferred functions based on homology with known proteins of other species, 3 are of unknown function.

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