JP2018512152A - Avian vaccine - Google Patents

Abstract

The present invention uses a specific antigen derived from D. maninae (D. galinae, poultry duck) to elicit an immune response in an avian species that is prone to infection or prevalence or is susceptible to the above Based on the discovery that you can. The immune response elicited is protective and can prevent or promote the removal or eradication of D. Galinae infection / spread in avian hosts. Furthermore, if there are significant health problems associated with D. galinae infection / spread, the present invention can be used not only to reduce D. galinae infection / spread population in avian hosts, The present invention can also be used as a means of indirectly dealing with a number of secondary diseases and / or symptoms associated with infection / spread. [Selection] Figure 1

Description

  The present invention relates to an antigen capable of eliciting a host immune response against parasites. In particular, the present invention provides a vaccine used to protect and / or reduce cases of parasitic infections in avian hosts.

  The spread of poultry duck spiders (Dermanyssus gallinae De Geer) in the laying hens farm has resulted in an estimated 130,000,000 euros of damage per year to the EU poultry industry (approximately 0.43 euros / chicken) The spread of this pest has important implications for animal well-being [1,2]. The spread of poultry spiders is a major health and well-being problem for laying hens as a result of anemia, increased irritation and emotional anxiety, increased feathering and cannibalism [1]. Poultry mites are also implicated as intermediate hosts for a number of important diseases [3]. Control of tick populations is currently a major problem for the egg industry, with most acaricides resulting in limited or non-lasting reductions in tick populations. Furthermore, with the emergence of resistance to previously effective acaricides, the removal of traditional effective acaricides (eg, organophosphorus fenitrothion) exacerbates these problems related to tick control [eg 4 Please refer]. In a 2004 survey of UK poultry units, 87.5% of responding farms reported the spread of D. Galinae [5]. In addition, EU legislation requiring replacement of regular cages with “enriched” cages (including nesting areas, perches and liter / scratch pads) will allow more parasites to be accommodated by the parasite. It may have exacerbated the tick problem. As another control strategy, vaccination offers benefits including long-term efficacy, release from chemical residues / environmental pollution, and reduced risk of resistance. It is now recognized that vaccines against blood-sucking ectoparasites can provide effective and lasting control [6,7]. These include effective commercial tic vaccines developed using protective Bm86 immunogens [6]. Importantly, our previous study [8,9] in conjunction with studies published by other research groups [10,11] (with natural and recombinant antigens) on poultry buns It clearly established that vaccination is a goal that can be realized and submitted. The study described here is a vaccine candidate against poultry scabs through the screening of tick molecules present in the mite extract that have been shown to induce protection in immunized birds. For the first time reveals the use of combined immunology, proteomics and genomics approaches to discover

So far, for D. gallinae, the candidate antigen is the “practical” approach [9], which uses fractions of intact protein extracts of mites and uses them as vaccines, or other ectoparasite species. It was identified by a “reasonable type” approach [8] that screens the appropriate D. Galinae antigens based on a hypothetical orthology for protective antigens. The identification of potential vaccine candidates with a “rational” approach has been supported for several years in the ectoparasite field, but in order to be useful, it follows two warnings: i) which molecules Must be understood to be truly essential for the survival of ectoparasites; further ii) it must be demonstrated that these molecules have access to the host immune system [6]. A third approach, the use of protective antigens from other ectoparasite species, has also been used to elucidate cross-protection [10]. However, when hens were immunized with a validated protective tic antigen (Bm86), high antibody titers were achieved but no effective protection against D. Galinae was observed [10]. This indicates the risk of simply estimating vaccine targets from other ectoparasites in D. Galinae, which has relatively little research.
However, despite many studies in this area, no suitable vaccine against D. Galinae has yet been identified. Furthermore, there are no vaccines that provide cost-effective and intense protective immunity, including specific or single antigen candidates.
The present invention seeks to eliminate these problems associated with the prior art.

The present invention uses a specific antigen derived from D. maninae (D. galinae, poultry duck) to elicit an immune response in an avian species that is prone to infection or prevalence or is susceptible to the above Based on the discovery that you can. Furthermore, the inventors have found that the immune response elicited is protective and can prevent or promote the removal or eradication of D. Galinae infection / spread in avian hosts. Furthermore, if there are significant health problems associated with D. galinae infection / spread, the present invention can be used not only to reduce D. galinae infection / spread population in avian hosts, The present invention can also be used as a means of indirectly dealing with a number of secondary diseases and / or symptoms associated with infection / spread.
Throughout this specification, the term “comprising” is used to indicate that an embodiment of the invention includes a gist of indication (“comprise”), and thus may also encompass other gist. It should be noted. However, in the context of the present invention, the term “comprising” means “consist essentially of” the feature to which the present invention pertains or “consist of” of the feature to which the present invention pertains. ) "Examples can also be included.

The present invention relates to one or more of the D. Galinae antigens listed below as (i)-(xi):
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
(X) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x).

An exemplary Deg-SRP-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 1).
Deg-SRP-1: SEQ ID NO: 1
ATGGCCGACCAAGACTTAAAGGTGGCTTCGCCCAACGAGAAACCGGGCGGAAAGTATGCCCTTGGGATGAGCTTTCTACAAAAGTTGTGCCGGGATCCGGGGGAGAACTTTGCTTTCTCACCGCTCAGCCTTGGGATCGCGTTCTCGATGCTAGTGGCCGGAGTCAAAGGTGACACGAAGAAGCAACTTCTCGATCTGCTTGGCTTTGCTAACGAGGCAGATCTTCACGCAATGTACGCCGAGCTCATGAAAGACAAGGAACTGCCCATTAAAATTGCTAATAAATACGTGGTCCAGAACAAACTCAAAATTCAGAAGAATTTTGAAACGCTCGCTAAGGAAAAATACCAGTCAGAGGTTGAGTCGGTCGACTTCGTTAAGGATGGTCGAAAACTCGAGGCTTCGGTCAATGCGTGGGTGGCATCAAAAACAAACGACATGATAAAACAGCTTATCCAGCCTGGAACTTTTACAGCGGACACCATTCTTGTGTTACTTAACGCAGTCTACTTTAAAGGTACGTGGGTGAACGAGTTCGATCCTGTTCCGTATGAGATGGACTTCAAATTACGAAACGGCTCTACTGTAAAGAAGAACTTCATGACTCAAAAGTCATCCGACTTTAAATACTTAGAAACGGACAAACTTAAAATGGTTAAGATTCCGTACAAGGAGGCAGGATGCTACATGGTTGTCGCTCTTCCTAAGGATGATGGTAAACACATTGATGAAGTTCTGGTAACGATGACTGCCGCCGAGATGCACTCCGCTGTGGAGAAGCTAAACGCAACACGTAGCCCGCAGGTGCTGTTGACGATGCCGAAGTTCAAGATTGATTACAAATATGGCAACCTCGTCGAACACATGAAGGCTCTAAGTGTGACCAAGATTTTCGCAGGGGGCGACTTTGGCGACCTCTTTGAGGAGATGGGCGATGCCGTGGAAGTCTCCTCCGTGGTTCATAAAACTGTAGTTGAAGTGGATGAAAAAGGCACCAAAG CAGCGGCTGCTACTGCTATGGTGGTGTCGCTTCGTTGTTCAAGAGGAGCCGTGGAAGAACCTATTCAGCTGATTCTAAATCGAGCTTTTTTCTTTTCCATTTATATTGGCGAACATCATATTTCCGCCTTCAAGGGATTGTGTTTCAGTCCTTAAC

An exemplary Deg-SRP-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 2).
Deg-SRP-1: SEQ ID NO: 2
MADQDLKVASPNEKPGGKYALGMSFLQKLCRDPGENFAFSPLSLGIAFSMLVAGVKGDTKKQLLDLLGFANEADLHAMYAELMKDKELPIKIANKYVVQNKLKIQKNFETLAKEKYQSEVESVDFVKDGRKLEASVNAWVASKTNDMIKQLIQPGTFTADTILVLLNAVYFKGTWVNEFDPVPYEMDFKLRNGSTVKKNFMTQKSSDFKYLETDKLKMVKIPYKEAGCYMVVALPKDDGKHIDEVLVTMTAAEMHSAVEKLNATRSPQVLLTMPKFKIDYKYGNLVEHMKALSVTKIFAGGDFGDLFEEMGDAVEVSSVVHKTVVEVDEKGTKAAAATAMVVSLRCSRGAVEEPIQLILNRAFFFSIYIGEHHISAFKGLCFSP

An exemplary Deg-HGP-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 3).
Deg-HGP-1 (excluding signal peptide): SEQ ID NO: 3

CGAGAACGGCTTCGTGACGCTCGTCATCCTCAACAAGGGACACGGCCTTATGCAAATTGATGCGCCGATCTACGGCATCTATATGCTCGCCATGGACTCGGCGGCCTTCATcCAGGTGGCGCCCTACTATCgTgGCAAACTGTGCGGCCTTTGCGGCAACTTCAATCTGGACCGTCAGCACGAGTTCCACACGACCGACGGCTGTCACCACAGAACGCCCTGGTCATTCGCCAGGAATTTCGTGATTCCCAGCGATCAGTGCGCGCCCGTGCCCGAGGCCACTGATGCCGGCCAACCATACTGCAGACAAGCTTAG

An exemplary Deg-HGP-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 4).
Deg-HGP-1 (excluding signal peptide): SEQ ID NO: 4
HTQKPGMAFRSNVRVQRIVDNTYGLKIEDFEVATANERETDWSETEKMAFVKNERLSSMVERPFLVHLERTENLSTYEFKSLETHSDDPVWSTNIKKGLVSLITLHVPFDNDEQIYSSVEPTVYGKCNVHWHRQSKPHWTRPDSKVLNISRAIDHENCETVANRVYGSVMGTPCMDGSCQRHHTYPVSTSSQAKYSLVAASGDTNNWILETARDNSVFTHAPHTENGNVLKLKTAQRMRLIDTVDATEKLEIVGETQVDDQLMMHFTKRWRLERSVDLDKADDFVLDWDIRGCSNGTIALLQKLRSLRNADDNQLGMFDNNVAETFSAAIEMMYLLDREQLAVVYDRIVKAAPEAELEQAQRLFVEVASAAGSTTAMKFVMDKFQAGDISVRFMRPFLSGITRGLFDRTSGALEIFENFCTSDQMKALPEERRQCLLAYTALVYDTYRLEHFNVQKHERKDTRETIFKRIVPEYSVAAEEGREAFKTYMLIAGNLKTASSVEFLARAVLDEKLDRLTAMDAMRTLIMTADNSEVARKAALTVYADSSKPAELRMLAAIVIMRSNPPLSVFYSMADRLLHEKSDQVRSYVVSMFKELSQTTHPFFRHLANKAHYVAPKLEARLPSEWKRKDYLTSHTKLASGYHPKYDHGGHSIISMIMSDSYVPRDLYVNFGDFFAGFFFDNFGMSISQQGMERLIDHFNNPRTFGNKFGRNLWNMAGRRRQTRDAASVEHAMDEIDSTLNIHTKKYDPMRLDMTFSAFGENIHTVSLNESFFLPLLSPDYKPGTLLNKILSTERDMHSFKNLRDMTFMIPTAIGMPAWFDVQLPTVLSCRRKESSFDFGNEGALKLKLDQRIVMDAQLEESLRFGVPGLQVSLGVGFKRRLALNLPIKLDVDANVATGKVVMNQDFVLPRDIMRYKFEPYTIEDNFKEPEKTTYLALFKDEELVEFKSQPLKDLLGIDFFLEGKQLPKSVLSWDFSQWLKSDIRQKLYYAIVNPKWRPR SLSLSAAPAKEHPTTGRVLTFKHKMTPPEATERPGSRFEEFEKDLPESFVHVIQVSNELTSSKKRRADLEVRYSYTPNRVEHWIQLFYDRTPLSSQDTDHTKLCMVAKVKQTTTDWEKLTKEQVMHVNDGQRMDVLMNLQYGKSCKTGDEPDASSLQMLARATYEHSDEQKRWLSELTTGETRSRVHGLENPYMKFYTKCIKYLEKGLLMPFACHKFIVHTSQLNNMTLDVEVNDRAHVLTNPCMSTLRAYLAAQYDANLRMFAPQRNVSRPGHWHMNSVVREPCINQRLADIAIVGPYHSSFWESVPVSWGAHWAPRTATHLGSTNMQSYSRKWFHRYCDIQLNSVATFDNVVYELPETNCWKVLAKDCSEMKVFTLLGRANADKKKEIRLLVDKYEVHLKNVDGKIVLTLNGAEEVLAERQPKLVKNENGFVTLVILNKGHGLMQIDAPIYGIYMLAMDSAAFIQVAPYYRGKLCGLCGNFNLDRQHEFHTTDGCHHRTPWSFARNFVIPSDQCAPVPEATDAGQPYCRQA

An exemplary Deg-VIT-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 5).
Deg-VIT-1 (excluding signal peptide): SEQ ID NO: 5
AGCGCCGAAGTGTTCCACTTTGTGGGTCAACATGGCCAGGGTTCAACCGTTTACGGTGTCCGTGGCGCAGTGACTGTCGGCGCTCACCAGCTGACAGCTGAGAAGACTGCCCTCGAGTACAATGGCACGTTGGCCGTTGAGCAGATCCGTGAGGGCGAGTTTCTCACTAAATTCACTCACTTCACCGTTGGCAAGTACAACAAGCTCCAGAGGAGCGTCCAAGATGAGACTTTTGATGACCTCACGCCCGAAGAACAGCGCGTGGTCAAGACGCTCCGTGAGCCGGCTGTTTATGAACCTCATATGCAGAGACCCGTCAGGTTCTTTGTGAAGGAGGGCCAGATTGTGCGCATGGAGGCTGAAAAGGAGCACCCGCAGTGGTCCCTTAACATCTTCCGTAGCGTGCTCACCCTTTTCCAGAGCCAAGTTAGCAAACCCGCTACACTTGCTGTGCCCCATGTCGAGTACAAATATGAGGATGGCATCACTGGCAACTGCAAGGTCCAATACGAGGTCTTCTCTCTGCCCGAGGACGTGACCGTACAGGGCGTGTTCAACCTGACTAAGACCAAGAACTACAAGGACTGTCTCGGCCGCCCTGTCTACCTTCATCTTAAGGATACTCAGCGTGGCTGCGCTGGAGTATGCGACAACCACCGCCCCGAGAACTTCCTTGCCGGATATGAAGAGGAAATCACCGACTACGAGCTGAAGCCCACACCTGGCTGCCCGGTCAACCAGCAACGCAAGGATACCCTCGTAACTGTCCAAACTGTTACTAAGTACAATGTCTCCAACGGTTACCTCGATGAAGTTCGTTCGGAGCATACTGACATCTACCGTCTCTATGGTGGAAAGCTCCACGTTTTCACTACTCTTCAGCTGCGTCTTTATGGCGTTGCTGGCCCTAAAATCGAAGAGCCAAAGACCGTCGAAATTTACAAGACACTTCAACTGCGTCTCCCGCACGAGGAAGATGAGCTTGACATTCCCGTCTATG CTCTTCTGAGGGAACACACTACGCAGCAGCAATACGGTCAGCACTTCCAAAAGTACTTTGAGGCTGTTGTTCAGGAGCTTCTTCAACTTAAGGATACCCAGAAGCAGGGCCAACCCGAAAAGCAACACTACCACTCTACCGCCTATCTTGTCGAGCTGGTGCAAGCTGTCTCCTCTATGACTGAGAAGGAGCTTAAGAGTATCATACCGACCATTGTTCACCAAGCTCAGCCTAAGCAGCTCACTGAGGAGGAGCACGTCCGCCGCCAGCTCTGGGTTGAACTCTTGGGTAAAGCCGGTTCTAAGTCTGCTGTCAAGATCATTGTTGAGCTCGTCAAGGGCAAGCTTCTTACCCCCACCGAAATTCGTCGTGTTCTTCAGGATGTTGCCGCCTTCCAGTCCTATCCTGACACTGAAATGGTTGAGCAAATTCTTGCTCTTTGTGTTAAGGAGCAGGGTCTTACCGCCACCGGTAAAGCTACAGCTTGTGTCGCTGCCGGTAAGGTTCTTTCCAAAGCCTGCAACTCAAAGGTTTACCAGTTGGCTCAGAAGCACGAGCAACACAAGAAGACAATCAACGGCAAGTACCAATCTATTGTTCAGATGCAAGAACAGAAGTACACCCCCGAGACCGAACCTGAGGCTGAGGAGTACCGTGTGACTTTTGGCCACTTGCCTGTAGACCCTAAATTGGTCTGTACACCTGAAAAGCTCCAGAAATACGTTtCTGACCTTtCGCACGCTCTtCACCAaGCtACCGACTTCAAGCATGTCGTGGCCTacaTCAaTGGTTTGGCTCATGTTCAGAAGCCCGAGGTTCTTCCTGAGCTGCTGGGTTACGTAAACGGCACTGCTTCCAACTTGGTACATATCCATGAGCAAGGTGAGGATATCAAGGAGGCCGTTGAGTTTGCGCGACACGTCGCTATTGTATCCCTCCAACATGTTGCCGTCAAGTACCCGAAAGAGGTTAACCCGATTGTACGCGTTGTCTTCGAAAA TACAACTGAGAAGGTTCAGACCCGTATTTTGGCCTTCGACGTCTGGATGGACACTCAGCCCGCCCAGTGGGAGGTCGAGAAAGTAATGCAAATTGCCAATAAGGATTCCTCGCTTGAGTTGACGCACTACGTCTACACTGCTCTCAAGACCGCCATGAAGGCTGAGGAGCCTTGCTACCAATTGCTGGCTCAACGTGTCCGCGCCGCCTGGACCCAGCTCCGTCCCTTCGACCTTGGCTCCGAATTCTCTCACCTCCGCTCCAAATTCTACTATGACACTGTTGAGAACTACGGTATTCGCGGAGTTTGGAAAGTGATTGCCTCCAACACCACCATTCTGCCCTTCTATACCGAAGCTAAGGTTAACCAGGTCCGCGGCCCTTACAAGACGACTCTTTTCGGCGCTAAGTTGCTCGTGAAGGGTGGTGACAAGGTTTTGGAAGAGCTGGTTGGTAAGGATGGTCTCCTTGAGCGCATTGCTTACGCCCTTGTCGGTCAGATCAAGACGGGACCGCGCCAACAGAACACTGAGCAGCTCCTTAAGGATATCGCCCAGGGTATGGGTCTGAAACGCGAGAAGGACGAAACCCCCAAGGCTGTGCTCTTCTGGAAGCTTTTCTCTGGAGACGCTGTTATCCCTCTGGACTCCCACTACATCAATGAACTGAAGCAAGAGCTGCTTCAGACCGTAACGAAATTCGGCAAAGATGGAGTTACTGGCCACATCGTCCGGGTGCTTGTGCCCACCAAGGCTTTCCACGTCGAGCCCTCTACCATCGGTCTTCCGATTGTCCACTCGACGATCCACCCTGTCGTTCTCTCTGTTCGCTACGAAAACATCAAGATTCACTACGGTAACCAGGAAAGTCGTGTGGCCCCTAAGACGTTGGAGATCTCTGGTACTGTTCAACCTACCATTCTCTCCTTCCGCCAATCTCGTGTCTTTGTTTCTGACAAAGTAGGCCAGAAAAACCCCACCGTAAAGACCACTGATATCAAG GAGTTCAACGTGCGCCTCGCCTTCCGTGTAGTCTATGAGCACACTCCCAAGCGGTTCCGTGTTCACGTAAAACCAGTCTTTGACCGCGTGTTCCACTCCGGCCACTGCACTGAGCTTAAACTCGAGTCCGCCGTTCTCCTTAAAGAGGAACTCGCTGCTAAGACTGTTGAGTATGATAAGTGTATCAAGTCTCTTTATCAACCCATCCGCCGTAACCACCAGATTGCCGGTGAATGGTCTGGAATGATGCTTCGCCTTACCGGCGAGTCTCACCAGCCTTGGTCTGGCCTTCCCATGTTTGCCCCAAGCGTTGTAAGCAGCGAAGGCATTCTCGGCGCCATTATTAACCGTCTTTCTAACAAGGGTATGAAGCACCACACTGTTTCACTCTACCTTGAGACCAACAACCAACAACCGATTACCGAATGGGTCGCCACTATTGACGTCGACTCTAACGTCGAGCGTCTCGCCAAGGTGCCTCTTAGCCAGCAGATTACGAAAGTTCAGAAACTCAAGGTGCAGTACGCAAACCGTGCTCAGCCTCTTTACCCCGAACTGGAGCCGCTTGTTCGCAAGGTTGAAAGCCTTCTTGAGAAATTCGAAACCCTCGATGAAACTACCGTAGAGAAACTGATGCTGGTAAAGATTGAAGGTCTTTATCAAGGTCAACCTAAAAGTACTCTTAAAATCGCAATGAAGAAAATCTATAACCTtGAGAAGACTgAGCAGCAATACGCtCTaGCCGCtATGCaTCAGGAATCTCACAAGGGTCTTGAACTCTCTACCAACGTCTCTTACCCCAAGATCGGATCCCCATTCCGCTACGACCCTACCTTCTACGCTGAGGATGAGCGCATGAACGGCACCCTCATCGTCAAGCTCCAGAGCCCACAGGAGCAGgTCTTCCACGTCAAGTTTCAgGCTACGAAGTCCGAGGAACAGCTCAAGGAGACCGAGTATGAGTGGTTTGAAGTTCGTTGTCTCGCGGAGCAAAAGGCTG GTAAAATCATGACCGACGCGTGCAGGAAGGCCGTTCTCAAGGACAACTCCCTGGACCAATTGAAAATCGCCGTCACGGTTCCCCGAAATGTTCACCCCAAGATCCAAACGCTTGCCTACAAAACACTTGACCTAATGAAGTACATGTGGTACCCGAAGATGCAGACTGAAGTTGCTGGTCTCAAGCAACGCGAAGTGCTGCAGGCCCTTCAGCACACCGAACGGGAAGTACGCATCTCCGTTAACGCTACTCGTGAATCTCTCTGGCATCTTCTATATGATGTCCGCGTCGAGATGCCATTCGAGAACGTTACTTTCTCCAAGGTTAACATTCCTGGTGTTCGTCCCGCACACATGCAGCTCACAACAAAAGAGCAGCTTGAACATGTGTATTACCGTGGCCAAAAAGATAACGTCTGTGTTCTTGGCGACAAGTCTGTACGCACGTACGACAACGTCACCTTCGGCCTTGATGTTAAGACTGGCTGCGAATACGTGCTCACCCGTGATACAAGCTCGGGTACCCCCGACTTCACTGTCACCTTCCAGGTTGTTAAGCCTGACACCTTCGCCAAGAAGATTCGGGTCCAGCTTGAGAACACTCTGGTAGAGTTGGAGCCCTTCACTACCACCGATCGCTACATCACTGTTGTGGTGAACGGCACCCAGTACCAAATCACCTTCGAGAAGCCTGTGGTATTTGAGTACGCTGCCGGTAAGCGTGTGTTCCTTAACGTGGTAGACACTTCCAACGTTCACCACGCTCCCGTCATCACCCTCTACACTGAACCGAAGGAGGTCCGCGTCTTCTTCGATGGCCACTCAGCCAAGGTCTTTGTTGTCAACAAGTACAAGGGTAACACAAAGGGCGTATGCGGCAACAACGACAACGAGCAGGCCCACGAGTTTATTGGGCCCAACGGAAAGGAGTACCAACATGCCAATGAGTTTATCGCTTCCTATGGCATCGGACAGGCTTGCAAGGTGCCCGCCGAGAACAC GCGCGAGAAGCTCATGGAGACCTTGAAGAAAGAGGTCGAACAGATCCGCCGCCAGGAGCTAATCAAGAAGGAAAAGCTTCGCAAAGAGATGCAAGAACTCGAACGTGCGCGCAACCCGCAGTGGATGGAGCAGCAGGAAGAACAGTTCTGGGGCGAGCCACTAAGCACAGTCAGCAACGAGGAGTGGACTACAGACTCCGTTGAGGAACAACAGCTGCAGCGCCAGGTACTAAAGACGGCCATGTCTATCGAGAATGGCCACATTTGTTTCTCCGCTAGACCGATTGCCACCTGCAAGCAAGGTTACaAGAACCACGGCGTTCTCCGCACCGAGCGTGTCGAATCGATTTGCCTCGAAAAGAACGAGGAGGCCGCGATTCAAGCT

An exemplary Deg-VIT-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 6).
Deg-VIT-1 (excluding signal peptide): SEQ ID NO: 6
SAEVFHFVGQHGQGSTVYGVRGAVTVGAHQLTAEKTALEYNGTLAVEQIREGEFLTKFTHFTVGKYNKLQRSVQDETFDDLTPEEQRVVKTLREPAVYEPHMQRPVRFFVKEGQIVRMEAEKEHPQWSLNIFRSVLTLFQSQVSKPATLAVPHVEYKYEDGITGNCKVQYEVFSLPEDVTVQGVFNLTKTKNYKDCLGRPVYLHLKDTQRGCAGVCDNHRPENFLAGYEEEITDYELKPTPGCPVNQQRKDTLVTVQTVTKYNVSNGYLDEVRSEHTDIYRLYGGKLHVFTTLQLRLYGVAGPKIEEPKTVEIYKTLQLRLPHEEDELDIPVYALLREHTTQQQYGQHFQKYFEAVVQELLQLKDTQKQGQPEKQHYHSTAYLVELVQAVSSMTEKELKSIIPTIVHQAQPKQLTEEEHVRRQLWVELLGKAGSKSAVKIIVELVKGKLLTPTEIRRVLQDVAAFQSYPDTEMVEQILALCVKEQGLTATGKATACVAAGKVLSKACNSKVYQLAQKHEQHKKTINGKYQSIVQMQEQKYTPETEPEAEEYRVTFGHLPVDPKLVCTPEKLQKYVSDLSHALHQATDFKHVVAYINGLAHVQKPEVLPELLGYVNGTASNLVHIHEQGEDIKEAVEFARHVAIVSLQHVAVKYPKEVNPIVRVVFENTTEKVQTRILAFDVWMDTQPAQWEVEKVMQIANKDSSLELTHYVYTALKTAMKAEEPCYQLLAQRVRAAWTQLRPFDLGSEFSHLRSKFYYDTVENYGIRGVWKVIASNTTILPFYTEAKVNQVRGPYKTTLFGAKLLVKGGDKVLEELVGKDGLLERIAYALVGQIKTGPRQQNTEQLLKDIAQGMGLKREKDETPKAVLFWKLFSGDAVIPLDSHYINELKQELLQTVTKFGKDGVTGHIVRVLVPTKAFHVEPSTIGLPIVHSTIHPVVLSVRYENIKIHYGNQESRVAPKTLEISGTVQPTILSFRQSRVFVSDKVGQKNPTVKTTDIK EFNVRLAFRVVYEHTPKRFRVHVKPVFDRVFHSGHCTELKLESAVLLKEELAAKTVEYDKCIKSLYQPIRRNHQIAGEWSGMMLRLTGESHQPWSGLPMFAPSVVSSEGILGAIINRLSNKGMKHHTVSLYLETNNQQPITEWVATIDVDSNVERLAKVPLSQQITKVQKLKVQYANRAQPLYPELEPLVRKVESLLEKFETLDETTVEKLMLVKIEGLYQGQPKSTLKIAMKKIYNLEKTEQQYALAAMHQESHKGLELSTNVSYPKIGSPFRYDPTFYAEDERMNGTLIVKLQSPQEQVFHVKFQATKSEEQLKETEYEWFEVRCLAEQKAGKIMTDACRKAVLKDNSLDQLKIAVTVPRNVHPKIQTLAYKTLDLMKYMWYPKMQTEVAGLKQREVLQALQHTEREVRISVNATRESLWHLLYDVRVEMPFENVTFSKVNIPGVRPAHMQLTTKEQLEHVYYRGQKDNVCVLGDKSVRTYDNVTFGLDVKTGCEYVLTRDTSSGTPDFTVTFQVVKPDTFAKKIRVQLENTLVELEPFTTTDRYITVVVNGTQYQITFEKPVVFEYAAGKRVFLNVVDTSNVHHAPVITLYTEPKEVRVFFDGHSAKVFVVNKYKGNTKGVCGNNDNEQAHEFIGPNGKEYQHANEFIASYGIGQACKVPAENTREKLMETLKKEVEQIRRQELIKKEKLRKEMQELERARNPQWMEQQEEQFWGEPLSTVSNEEWTTDSVEEQQLQRQVLKTAMSIENGHICFSARPIATCKQGYKNHGVLRTERVESICLEKNEEAAIQA

An exemplary Deg-PUF-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 7)
Deg-PUF-1 (full length sequence): SEQ ID NO: 7

An exemplary Deg-PUF-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 8).
Deg-PUF-1 (full length sequence): SEQ ID NO: 8
MLFKLLLVVGLTAAIASAGRGSDQRDQRGGRDHSHDSHHGSTEGGRRDRETTDPLFAKFMETTKKCVNDLLPQSGLEQRDQQALLEMIKPMHVLQREHSTDQSGRQHAQHTFTPPYKCSPQGELLTISPAEVYVTSMGKHLDATDKVSQRAKAQLS
An exemplary Deg-ASP-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 9).
Deg-ASP-1: SEQ ID NO: 9
ATGGCGACGCGCGTCGTCCTCGTACTGTGGGCGGCGTCATGTGCCGCCCAAGCCGGCCTCATGAGGGTACCCCTGCTCAAGATGGAGACCATTCGGTCGCAAATGATGTCGAAAAATACACCGCGTCAATTGTTGCACTCACAGTCAGCAGGCGTCAATGGAGTCAAAGGAAGCGTTGAGCCTATCAACAACTATATGGATGCACAATACTACGGCCCGATCTCAATCGGCAGCCCGCCACAGCCATTCCAGGTCGTTTTCGATACTGGCTCATCGGATCTTTGGGTGCCCTCTTCCAAATGTCCACTTACCAATATTGCATGCCTGCTGCATAACAAGTACCATTCGGACAAATCGTCGACATACGTCAAAAACGGAACTGAATTCAAAATTCAGTACGGCTCGGGCGCGGTGAGCGGCGTCCTGTCGGCGGACACGGTTGATCTGAATGGTATGCGCGTCACCAACCAGACGTTTGCCGAGATCATGCGCGAATCGGGCCTCGGCTTTATAGCTGGAAAGTTCGACGGCATCCTCGGTATGGGCTATCCGACGATCGCCAGGGGTGGCCTACCGGTCTTCGACCAGATGGTGGCGCAGAACGTCATCGACCAGGCCGTCTTTACCTTCTTCCTcACCCGCGACCCCAACCACCCCACAGGCAGCGAGCTCGTTTTAGGAGGCATCGACCCGAAGCACCACAAGGGAGAAATTACCTACACCCCGGTCACCCGCAAAGGCTACTGGCAGTTCGGCGTTGACAAGATTGCAGTGAGTGGACATTCAGATGAGTTGTGTAAGGGCGGCTGCCAAGCTATCGCCGATACGGGCACGTCGCTTATCGCCGGCCCCACAAAGGAGGTCACGAAGCTGAACGAGCTCATCGGCGCCGCGCCATTCATCGGCGGCGAGTACATTGTTAACTGCAAAAATCTGCCCAACATGCCCAATATCGAGTTCACGATTTCAAACAGGACGTTCGTTCTCACTCCAGACGAAT ACATCCTCAAGATGAGCCAGGGCAGCATGCCGGTGTGCCTATCAGGTTTCATCGGCCTTGATGTCCCGCGTGATCCCGTCTGGATCCTGGGCGACGTCTTCATCGGCCGATACTTTACGGtTTTCGACCGCCAAAATGATCAAGTTGGCTTCGCCGACGCAGCCTAG

An exemplary Deg-ASP-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 10).
Deg-ASP-1: SEQ ID NO: 10
MATRVVLVLWAASCAAQAGLMRVPLLKMETIRSQMMSKNTPRQLLHSQSAGVNGVKGSVEPINNYMDAQYYGPISIGSPPQPFQVVFDTGSSDLWVPSSKCPLTNIACLLHNKYHSDKSSTYVKNGTEFKIQYGSGAVSGVLSADTVDLNGMRVTNQTFAEIMRESGLGFIAGKFDGILGMGYPTIARGGLPVFDQMVAQNVIDQAVFTFFLTRDPNHPTGSELVLGGIDPKHHKGEITYTPVTRKGYWQFGVDKIAVSGHSDELCKGGCQAIADTGTSLIAGPTKEVTKLNELIGAAPFIGGEYIVNCKNLPNMPNIEFTISNRTFVLTPDEYILKMSQGSMPVCLSGFIGLDVPRDPVWILGDVFIGRYFTVFDRQNDQVGFADAA
An exemplary variant 1 Deg-PUF-2 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 11).
Deg-PUF-2-variant 1: SEQ ID NO: 11
ATGTTGTCCATCCGTGTCGCACTCGTGGCCCTTTGCGCCTCGGTGGCCATCGCCGCTCCGTCTGGCGGCATTTGCCCAACTGGCGGCGACTGCCCAAAATGTGGTGTGCCGGTACAGGACCTCGAGGAGGTCCTTCGTGATGCCAAGTCTGCTTTTATTGTTGCCACTGGTATCGAGCAAGACGGCAAATCCTGGATGTCTCCTCGCCGTTGGCAGGATATTCTTGGAGCAGAGGGTAACAATTACGCCGTGCAGAAGTATCCCGTGGACGAGCGCCAATGGGAAGTAAAGGATGGCAAGTGCAGGCAGTACTTTGTTAAGCCTGTTTATGAGATCACCCCAGGCCAAACTCCTCGCGTCATCCACAAGCCAACTGTCGCAGGGATCCATATCAATGAGGATTGCCGATGCCCTGAGCTGATCCGCGGACATGGTTATGCTGTTATCGTCTTCAAGGACCATAAAATCAACCACCTCGATGAGCTTGCTCGGGCCGCTCTTGATGAGCACGTTGTCGTCGTGCCGCTCCCTCATGGCCAGTGGACCATCCCTTCGTCGACTGAAAATGACAGCATGAGCGACGTTGTTGAGACGAAACTCCCCTCTGCGTTTCTGAATGACGACGTCAAGGAGGAGTGTCCTGAGGCTACCACGTGCCCCGCCTGCGAAGTTGTTCATAGTTCGGAACAATTGCAGGGCATTTGCCAATTCCAGCAGGCTCTTCTCGTAAAGCGCACGATGGATCATCGTACGCCCGCCGAGGAGGTCGCTCCCTGGAGCATGCCCACACTAAGCGTCTACATGGCCAAGGCTGATGAGAACGACTACCATCTTCGGCTCGAAATAACTCCTCGCGAGCAAGGCTCATGCGTGCGGGGTGTGCTCCGCATCCTTGATGCCTTCACCAGTGAGTTCGAAGGCCGACGCAATCAGAAGTTCCAACTGGGTAAAGATTGCGAGTGTGAGTACCTTCGCAATAACACCGGCTTCGCTATCCTTC AGTCCGAAGAAAAGATCAGCAAGAACGGTGTGCTCTCCCAAAAGGAAAAAATCCTTGTGCTTCCTGAAGGTCAGTATTTGCTTCCGCAGTGCCAGCCAGAGCAAGCTGAAGAGCGTTCAGTAGATGAAAACGAGAAGGCCGTCGAATGTGGCGAGCCCGACGAAACCTGTCCCGTGTGCGACATGATTACCCCCGAGGTGCAGGAGGCAATCGAGGCCGAGGATACCTATCTGCTTAAGATGCAAACGAACACTCACCTCAAGTCGTCTGAAGAACACGAGAACGCCAAGTGCGGCGCTGCTCGTCTCATTTCCGTCTTCAAGGGTAACAAGGAGAACGTCAAACCCGAGCTTTCTTTCACCCTTCCCGAGAAGTGTCACTGCAATGCCATGTCTGCCAATGGTCGGCAATTCTACGCTGTGATCAAGAAGGACGCCGTGGATAATGACAACCTCGACAAACTGGTTCTCAATGATCAAGTCTATATCATTGGCTATAACTACCGTTCCCATGGTCTCATTCAGAGGTGGCTGAATCAAGGAGAAAAGGAGCAGCTTGAAGAGGCCGCTGATGAGCGTGTTGACTACGACGGATACTTGTCGTACGCTCCCCCGATGTACGCGCCTCCGCCCCCTCATCCGCTGTACGCGCCTCCGCC

An exemplary variant 1 Deg-PUF-2 antigen can comprise the following amino acid sequence (SEQ ID NO: 12).
Deg-PUF-2-variant 1: SEQ ID NO: 12

An exemplary variant 2 Deg-PUF-2 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 13).
Deg-PUF-2-variant 2: SEQ ID NO: 13
ATGTTGTCCATCCGTGTCGCACTCGTGGCCCTTTGCGCCTCGGTGGCCATCGCCGCTCCGTCTGGCGGCATTTGCCCAACTGGCGGCGACTGCCCAAAATGTGGTGTGCCGGTACAGGACCTCGAGGAGGTCCTTCGTGATGCCAAGTCTGCTTTTATTGTTGCCACTGGTATTGAGCAAGACGGCAAATCCTGGATGTCTCCTCGCCGTTGGCAGGATATTCTTGGAGCAGAGGGTAACAATTACGCCGTGCAGAAGTATCCCGTGGACGAGCGCCAATGGGAAGTAAAGGATGGCAAGTGCAGGCAGTACTTTGTTAAGCCTGTTTATGAGATCACCCCAGGCCAAACTCCTCACGTCATCCACAAGCCAACTGTCGCAGGTGTCCATATCAATGAGGATTGCCGATGCCCTGAGCTGATCCGCGGACATGGTTATGCTGTTATCATCTTCAAGGACCATAAAATCAACCACCTCGATGAGCTTGCTCGGGCCGCTCTTGATGAGCACGTTGTGGTCGTGCCGCTCCCTCATGGCCAGTGGACCATCCCTTCGTCGACTGAAAATGACAGCATGCGCGACGTAACTGAGACGAAACTCCCCTCTGCGTTCCTGAACGACGACGTCAAGGAGGAGTGTCCTGAGGCTACCACGTGCCCCGCCTGCGAAGTTGTTCATAGTTCGAAACAATTGCAGGGCATTTGCCAATTCCAGCAGGCTCTTCTCGTAAAGCGCACGATGGATCATCGTACGCCCGCCGAGGAGGTCGCTCCCTGGAGCATGCCCACACTAAGCGTCTACATGGCCAAGGCTGACGAGAACGACTACCAGCTTCGGCTCGAAATAACTCCTCGCGAGCAAGGCTCATGCGTGCGGGGTGTGTTCCGCATCCTTGAAGCCTTCACCAATGACTTTGAAGGCCGACGAAATCAGAAGTTCCAACTGGGTAAAGATTGCGAGTGTGAGTACCTTCGCAATCACACCGGCTTCGCTATCCTTC AGTCCGAAGACAAGGTCACCAAGAACGGTGTACTCTCCGAAAAGGAAAAAATCCTTATGCTTCCTGAAGGTCAGTATTTGCTTCCGCAGTGCCAGCCAGAGCAAGCTGAAGAGCGTTCAGTAGAGGAAAATGCGCAGGTCGTCGAATGTGGCGAGCCCGACGAAACCTGTCCCGTCTGCGACATGCTTACCACTGAGGTGCAGGAAGCGATCGAGGCCGAGGATACCTATCTGCTTAAGATGCAAACGAAGACTCACCTCAAGTCGTCCGAAGAACACGATAATGCCAAGTGCGGCGCTGCTCGTCTCATTTCCGTCTTTAAGGGCAACAAAGAAAACGTCAAACCCGAGCTTTCTTTCACCCTTCCCGAGAAGTGTCACTGCAATGCCATGTCTGCCAATGGTCGCCAATTTTACGCTGTGATCAAGAAGGATGCCGTGGATAATGACAAGCTCGATAAACTTGTTCTGAATGAGCAAGTCTATATCATCGGCTATAACTACCGTTCCCATGGCCTCATCCAGAGGTGGCTAAATCAGGGAGAAAAGGAGCAGCTTGACGAGCCCGCCGATGAACGTGCCGACTACGATGGGTACATGTCGTACGCTCCTAAAGCGTACGCAGCCCCTCCGGCATACGCTCCCATGCCTGCTTATGGCGCCCCCGTATACGCGCCTCCGCCTGCGTACTCGCCGCCTCCGCCGGCATACGCACCCCCGCCACCGGCATACGCACCCCCGCCACCTCCGCCTG

An exemplary variant 2 Deg-PUF-2 antigen can comprise the following sequence (SEQ ID NO: 14).
Deg-PUF-2-variant 2: SEQ ID NO: 14

An exemplary Deg-SRP-2 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 15).
Deg-SRP-2: SEQ ID NO: 15
CTCTCGATGGCCTTCGAGGGTTCTCGCGGATCGACGAGAAATGAAATGCTCGAAGCGCTGaAACTTAAAGCGCTTGAACCGGAGGCGATCTGGCGGGCCTACTCTGCTCTAACGTCACGCAAGCATGAAAACAACACTAAACCCGGCGACTATTTCTCAGATGTGCAGTTTATCATGAGGCTTGCGAATCGTATGTACCTCAGCAAGGGCTATCCGATCGATCCGTCGTACGTCGACCAATTGAAGACCACATACCATGCCGATGCCGTCAATGTCGACTTCGCCGCCGAAGGCCCTGCCGTCCAAAAGCAAATCAACGAATTCGTTCGCGAACGAACTAACAATCTCATTCCACAGATTCTTCCTAGCCCGCTTGCTGCGAGCACGGTACTCGCGCTCGTCAACTCTGTATACTTTAAGGGCGATTGGACAGAAGCCATGACCGAGAAGAATCAGGTGATGCTCTTTAACAAGGATTCGAAAGTCCATCAGCAAGAGTACTCCAATTGGCTCAACACGGAAGACAATATGCCGTATCTGGAATCGGCAGCACTGCGCGCAAAACTCATTCGCATACCATATAAGTCGAGCAAATATTCGGCAAGCATGCTTATCATTGTACCTGAAGACCTTCTCYGTGACGGCAACCAGTGGCTTCACGACGTCAAGTGGACGGACATACAACAGGAACTCAGCAAAATGCGGATGACAAGAGTGAACCTGACAATGCCTCGTTTTGAGGTCGGTGCTCGTCTTCAACTAGAAAATACGTTGCCTTCAATGGGAATGCCCTTTGCCTTTGACACGGCGACATCGGATCTGTTTGGAATGGTGACTGACAAAGGCCAACGCCTTAGCATTGATCAGGTTATACACCAGACAAAGATGACCGTCACGAAATACGGCACAGAAGCTGCTGCGGCTACAGTTCTCACGGTGATGCTAACTTTGTATCAGATACCAACCGAACCCATCACTATGGTCGTTGATCGCGCGTTTT ACTTCGCAATCCTTTATGGCACGTATGATGCAGCCGACGTAATGCCGCTTTTCAACGGAGTCATTTATAAAAAGTAGT

An exemplary Deg-SRP-2 antigen can comprise the following amino acid sequence (SEQ ID NO: 16).
Deg-SRP-2: SEQ ID NO: 16
LSMAFEGSRGSTRNEMLEALKLKALEPEAIWRAYSALTSRKHENNTKPGDYFSDVQFIMRLANRMYLSKGYPIDPSYVDQLKTTYHADAVNVDFAAEGPAVQKQINEFVRERTNNLIPQILPSPLAASTVLALVNSVYFKGDWTEAMTEKNQVMLFNKDSKVHQQEYSNWLNTEDNMPYLESAALRAKLIRIPYKSSKYSASMLIIVPEDLLXDGNQWLHDVKWTDIQQELSKMRMTRVNLTMPRFEVGARLQLENTLPSMGMPFAFDTATSDLFGMVTDKGQRLSIDQVIHQTKMTVTKYGTEAAAATVLTVMLTLYQIPTEPITMVVDRAFYFAILYGTYDAADVMPLFNGVIYKK
An exemplary Deg-CPR-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 17).
Deg-CPR-1: SEQ ID NO: 17
ATGTTGATCCGCTGCGTTGTCACGGCTCTTGCAGCCGTGACGGTTGTCTCAGGAGTTTCGGTGCCCCGAGGTGCGCCAGAGTTCCCGCCGTCCTACACTGCGTCGGGCTACATCCTCCTGCCGTACTGTGAGCTGCGAGAGCCGTTCACCGCCTACTACGATGGTGAATCGGACCGATCGCGTATTGACTATTACGACGGCGAGATGAAGACATTCGTCGGAAAATCAGGCACGTTCAAGGTTGTCTGGTCGCCCAATGAGAAGACACATATACCGGAGCTGAACTGCTACGAGGCCGGACCGGCTAAAAGCCAGAGCATTTTGCCGGACCTCACCAACTTCACCTTCGTTCGGGTCGAGCCGTGCGAAACCGATTCGACGCACATTGTCAAGCCCCTGCTCCGAGGTGCTGACAAATGCTATCGCTATGAGAAGAAAGTGGATAACTTCGGACGCGTCTCCAAGTACACGTTCTGGGCCTCACAGGACGACGATAACACCCCAATTCCGGTACGCTACGTCATGATGGGTTACGACTCACTCTTGGGATCGCACTTCGACAAGTATGAGGTCGTGTACACCGACTACACACCCGGACCCGTCGAAGATGACCTCTTCCAAGTCAAGACTGTTATTGACAAGGAATGCACTTCGTTCCCGTCGCCGCCGGGCGTGTCCACTACCCACCTGTTCAACCCGATGGCGGAGTTCATTGACGAGAAGGATTCGCACGTGCACGAACACTTCGAGCACTTCAAATCGACACACGGCAAGGCATACGGCCACCAGGCCGAGGAGATCATCCGCAAGGACAATTTCCGCCACAACCAACGCTTCGTCAATTCGATGAACCGCCGCAACCTTTCGTACGCGCTGAAGCTCAACCACCGCGCCGACTGGAGCCAGGACGAGTTCAGGCTGCTCCGGGGCCGTCTACAGTTCACCAGCCAGAAGTCGATGGCCAGGGAGTTCCCCAAGGAACAGTACTCGGATCGCGTCG AGCCGGACTACGTCGACTGGCGACTCGAGGGAGCCGTCACGCCGGTCAAGGACCAGGCTGTGTGCGGGTCGTGCTGGAGCTTTGGCACGGTCGGCCACATCGAAGGCGCCTACTTCCGCAAGTTCGGCGAGCTGGTCCGTTTCTCCGAGCAGCAGCTCGTAGACTGTTCGTGGAATGCCGGCAACGATGCCTGCGACGGTGGTCTGGACTTTATCGCCTACCACTACATCCAGAAGTACGGACTGGCCAGCAACGACCAATACGGACCCTACCGCGGCATTGACGGCAAATGCAAGGACCTGGAGATTTCCAACAAGCCCATTAGCACGCTGAAAGGCTACCGAAACGTGACCACTGTGGAAGACCTCCGCAAGGCGCTCGCGTTTGTCGGCCCCATATCGGTGTCGATCGATGCATCAAGGCCGTCGCTCAGCTTCTATTCGCATGGAGTCTACAGCGATCCGGACTGCAGTTCGACGGAACTCGACCACTCCGTGCTCGCTGTTGGCTACGGCACGCTGCACGGTGAGCCGTACTGGCTCATCAAGAACTCGTGGTCCACGTACTGGGGCAACGACGGATACATTCTCATCTCGCAGAAGAACAACATGTGGGCGTTGCCTCGCAGGCAACCTACGTCGAGCTGTAGATAG

An exemplary pepC1A-13094 antigen can comprise the following amino acid sequence (SEQ ID NO: 18).
Deg-CPR-1: SEQ ID NO: 18

An exemplary Deg-GPD-1 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 19).
Deg-GPD-1: SEQ ID NO: 19

An exemplary Deg-GPD-1 antigen can comprise the following amino acid sequence (SEQ ID NO: 20).
Deg-GPD-1: SEQ ID NO: 20
MSAALQIKKVLISDSCDARCAEILREAGCDVTVKTDFTKEQLVEAIKDFDALVVRSATKVTADVINAATNLKVIGRAGTGVDNIDCDVATARGVLVINAPGGNTLAAAEMTCAMIISLSRDVAAACASLKAGRWDRKTFMGTELNGKTLGIVGLGRIGREVAIRMQAFGMTTIGYDPIIPAEQAAKFNVKAMSLDELWPQCDYITVHTPLLPETKNLISAGTLARCKKGVKVVNCARGGIVNENDLLAALESGQASGAGFDVFEDEPPKNTAFIAHPKVICTPHLGANTKEAQSKVAIEIAEQFVALKKGERAWGAVNKPKPAN

An exemplary Deg-PUF-3 antigen is encoded, at least in part, by the following sequence (SEQ ID NO: 21).
Deg-PUF-3: SEQ ID NO: 21

An exemplary Deg-PUF-3 antigen can comprise the following amino acid sequence (SEQ ID NO: 22).
Deg-PUF-3: SEQ ID NO: 22
MRGVLVIAALGLLAEVGLGSPFSNSECSEQXIQYILTDALGPKMLRLYKELLKPDTGYTSPVDVNGQTYEVSMPKKNLIQNAIRKGVKESIIEGMLDDAAIVPTGTASRTSPTSFRPNQQTIERPHVLEPVRERRPSSQSHYKIYTTRNGDINIDIKGQTLRVPDDLTRLPHIVDDPDTVVDIVRHLKELGVPVSRDGNRITIGSTTSPQGLTLRVSIKKIDDKPSSVTIKANDDEYTLPGDENRLNRYITREPEVAYHILRILSRHGVPVEYDDQTKTIKAKLTPEDDYIGSRTSVPSHSSNPSHAGSTSTSLVTPSASSPRIIRVGTTSYYLPEDWDRLKRDLQDGRISATSVLDILDSQHIAAPRDIISVIRTRISTSVEKVRVMRYGDEVTVELGSQSFKLPKDDRELEKALRENEFPIELIRDSLLRIGVDTRRVGSSLRVILPGGEAYLSGLPTTRQYRYDIVSKPGGVQITAGSETYNLPAEKTILERAIAEKKVLPRTLVDAFNDVGIPSEMDITKQEMTVDLTSGLLRIPVPLNIQQTKVGTRLRLNMRGPEGNKVYTLTIGESGNDKVELPADVHIVNNYIRSGKVDSNLLMQLLGRMGVSHYFDXAKDVHVVTLNGRPYDIQKSHVSERGSYPHSSHQQARVF

Accordingly, the present invention relates to one or more D. Galinae antigens that are at least partially encoded by or comprise any one of the sequences provided by SEQ ID NOs: 1-22 above. And / or utilizing said one or more D. gallinae antigens. Accordingly, as used herein, the expression “antigen” or “D. Galinae antigen” is used to refer to all or the natural antigens listed as (i)-(x) above, SEQ ID NO: 1- Antigens comprising and / or encoded by any of 22 and / or any immunogenic / antigenic fragment, variant, recombinant and / or derivative thereof.
An immunogenic / antigenic fragment is any D. Galinae antigen fragment that can elicit an immune response when administered to an animal (especially an avian species) (eg, a D. Galinae antigen described herein). Any fragment). An “immune response” can be considered an antibody (eg, IgY, IgA, IgM and / or IgG or any other relevant isotype) response and / or any response that elicits cytokines or a cell-mediated immune response. For example, the various D. galinae antigen fragments provided by the present invention could elicit an immune response that is substantially the same or similar to the immune response elicited by the complete antigen from which the fragment is derived. In certain embodiments, an antigen fragment provided by the present invention can provide a protective immune response in avian species.
Those skilled in the art will appreciate that the D. Galinae antigens of the present invention define one or more epitopes, and thus the term “antigen” can also encompass a protein or peptide comprising one or more D. Galinae antigen epitopes. Will be done.

Furthermore, the term “antigen” encompasses recombinant forms of any of the antigens described herein. The term “antigen” also includes recombinantly prepared immunogenic fragments of any of the D. Galinae antigens of the present invention. Recombinant forms of the antigens of the invention and methods of obtaining the same will be described later.
In other embodiments, the term “antigen” or “antigen fragment” can include variants or derivatives of any of the antigens described herein (such antigens can be “variant” antigens or “antigens”). Induced "referred to as antigen). Again, it is understood that these terms include any variant / derivative of the D. Galinae antigen of the present invention, or variants / derivatives comprising or encoded by any of SEQ ID NOs: 1-22. It should be. Any variant or derived antigen is immunogenic / antigenic in that it elicits an immune response that is similar or substantially identical to the immune response elicited in the same host by the corresponding intact or intact antigen. It will be appreciated by those skilled in the art that such variants / derivatives may be referred to as “immunogenic variants / derivatives”. An immunogenic variant / derivative is a substitution, inversion of one or more nucleobases and / or amino acid residues relative to a reference sequence (eg the sequence of the D. Galinae antigen of the invention or the sequence encoding the D. Galinae antigen). May comprise or be encoded by a protein / peptide sequence or nucleic acid or amino acid sequence comprising additions and / or deletions.
It will be appreciated by those skilled in the art that the term “substitution” can include one or more conservative substitutions. It will be understood by those skilled in the art that the term “conservative substitution” is intended to encompass the replacement of one or more amino acids of a protein or peptide with alternative amino acids having similar properties ( The surrogate amino acid does not substantially alter the physicochemical properties and / or structure or function of the native (or wild-type) protein).

In the context of the present invention, the variant / derivative antigen is a sequence encoded by a reference sequence (eg, any or any of the specific D. Galinae antigens provided as wild type sequences (above (i)-(x) Or an antigen comprising or encoded by any of SEQ ID NOs: 1-22 (or fragments thereof) when compared to one or more amino acid / nucleotide substitutions, additions, deletions and / or Alternatively, it may contain or be encoded by a mutated sequence found to have inversion.
An antigen that can be considered a derivative can further include one or more features of the fragments or variants described herein in combination with one or more modifications to the structure of the antigen or its amino acid residues. .
As is well known in the art, the degeneracy of the genetic code allows substitution of one or more bases within a codon without changing the primary amino acid sequence. Thus, gene degeneracy can be used to obtain variant sequences encoding any peptide or protein sequence of the D. Galinae antigen described herein.
In addition to being immunogenic (as described above), useful fragments, variants, mutants and / or derivatives may be derived from the corresponding fully wild-type or native D. Galinae antigen (or above). The encoding) complete amino acid or any of about 5 to about 10 residues (amino acids and / or nucleic acids) of the nucleic acid sequence (n) can be included in about n-1 residues. Fragments, variants and / or derivatives provided by the present invention are at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 400, 500 , 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 3000, 4000 or 5000 residues, the upper limit (n-1) encodes the complete antigen Depending on the size of the nucleic acid (n) or the number of amino acid residues (n) containing the primary sequence of the antigen.
In addition or alternatively, antigen fragments, variants and / or derivatives provided by the present invention may comprise at least 30 sequences of various reference (exemplary) antigens (coding sequences or primary amino acid sequences) disclosed herein. %, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, It contains a sequence that is 99% homologous or identical, or is at least partially encoded by the foregoing.

The degree (or percentage) of “homology” between two or more (amino acid or nucleic acid) sequences aligns two or more sequences and is identical or not identical, but redundant nucleotide substitutions (redundant nucleotide substitutions are individual Can be determined by determining the number of alignment residues that differ due to conservative amino acid substitutions.
The degree (or percentage) of “identity” between two or more (amino acid or nucleic acid) sequences can also be used to align the sequences to confirm the exact number of residue matches between the alignment sequences and to compare this number It can be determined by dividing by the number of residues. Multiplying the resulting number by 100 will give the percentage identity between the sequences.
Accordingly, the present invention relates to:
(A) one or more D. Galinae antigens selected from the group consisting of (i)-(x) above;
(B) an antigen comprising or at least partially encoded by one or more of the sequences provided as SEQ ID NOs: 1-22;
(C) any immunogenic fragment, variant or derivative of any antigen provided by (a) or (b) above; and / or (d) any of the antigens provided by (a)-(c) above Recombinant type.
The invention can further relate to any one of the antigens defined by (a)-(d) used to elicit an immune response in an animal (especially an avian species).

The present invention provides medicaments, methods and compositions comprising (utilizing) one or more of the D. Galinae antigens described herein. As used herein, the term “composition” encompasses any form of immunogenic or vaccine composition. It will be appreciated by those skilled in the art that an immunogenic composition can be any composition comprising one or more of the antigens of the invention that elicit an immune response when administered to an avian species. Similarly, a vaccine or vaccine composition of the invention may comprise one or more of the D. Galinae antigens of the invention that elicit an immune response (which can include a protective immune response) when administered to an avian species. it can. One skilled in the art will appreciate that the immune response elicited can include humoral (or antibody-mediated) and / or cell-mediated components. The immune response elicited can include a protective immune response, and those skilled in the art will recognize that protective immunity can help resolve the infection / spread of an animal and / or alleviate the symptoms associated with the infection / spread. It will be understood that this can contribute to In the context of the present invention, an immune response elicited through the use of an antigen described herein can be referred to as a “protective” immune response. The term “protective” immune response can encompass any of the following immune responses: (i) promote or achieve a reduction in the amount of host pathogens; (ii) one of the effects or signs of infection / spread And / or (iii) prevent, reduce or limit the occurrence of further (subsequent / secondary) infections.
Thus, a protective immune response can prevent infection / spread by a particular pathogen and / or progression of a particular disease or condition in an animal. In the context of the present invention, a protective immune response can prevent infection / spread by D. Galinae in animals, limit the extent of D. Galinae infection / spread, and / or D. Galinae infection / The progression of the disease or symptom associated with the spread and / or one or more symptoms associated with the same can be prevented. Symptoms of eg poultry (including chickens) associated with D. Galinae infection / spread include pain and irritation (especially breast and leg areas) and decreased egg production. Other signs may include the formation or progression of pustules, scabs and excessive pigmentation and the disappearance of feathers. Severe infection / spread of birds can lead to anemia.
Thus, as long as the present invention relates to the provision of “compositions”, it can be understood that these compositions include immunogenic compositions and / or vaccine compositions. Furthermore, it should be understood that the term “initiating an“ immune response ”encompasses not only humoral and / or cell-mediated immune responses, but also any form of“ protective immune response ”.

A feature of the present invention is to provide one or more Dermanius Galinae (D. Galinae) antigens for use in raising an immune response in an avian species, wherein the one or more D. Galinae antigens are: Selected from the group consisting of:
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
(X) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x).
In certain embodiments, the antigen used of the first aspect of the invention is provided in the form of a composition. The composition can include one or more excipients or diluents. The composition can be an immunogenic composition or a vaccine composition. The composition can further comprise an adjuvant.
In yet another aspect, the invention provides for the manufacture of a medicament that elicits an immune response in an avian species, wherein one or more of the Delmanius Galinae (D. Galinae) antigens listed as (i) to (xi) above. Provide the use of.
In yet another aspect, the present invention provides a method for eliciting an immune response in an avian species, said method comprising the Delmanius Galinae (D. Galinae) antigen listed as (i) to (ix) above. Administering one or more of:

As used herein, the term “bird”, “bird species” or “bird host” includes, for example, what are generally known as poultry or domestic birds. In other embodiments, these terms broaden to include domesticated or hunting bird species, such as chicken, pheasant, grouse, turkey, guinea fowl and duck species. In certain embodiments, the terms “bird”, “bird species” or “bird host” are expanded to market-important or farm-raised bird species.
Advantageously, the antigens and compositions for uses, medicaments and methods provided in the present invention can be used to elicit an immune response (eg, a protective immune response) in chickens (Galls galus domesticus). . Thus, the present invention provides antigens and compositions for uses, medicaments and methods that can be utilized to prevent, reduce and / or treat the occurrence of D. Galinae infection / spread in chickens.
D. Galinae is a blood-sucking avian ectoparasite that is exposed to host immunoglobulins when sucking blood. Without being bound by theory, the present inventors have administered one or more D. Galinae antigens to birds, whereby one (or more) antibody specific for one or more D. Galinae antigens is avian blood. Hypothesize that it is generated in. In this way, upon blood sucking, the D. galinae parasite is exposed to anti-D. Galinae antigen antibodies that adversely affect, deplete, annihilate, kill and / or inactivate the parasite. Again, without being bound by theory, the depletion, eradication, killing and / or inactivation of D. Galinae parasites mediated by antibodies can involve antibody-mediated cytotoxic processes and / or complement pathways.

As generally referred to herein by “antibodies” and “specific” or “selective” antibodies, these terms refer to antibodies (and active or epitope-binding fragments thereof) that bind to the antigens described herein. And / or antibodies (and activity or epitope binding fragments thereof) that exhibit some selectivity, specificity and / or affinity.
Furthermore, it is to be understood that the term “antibody” may also relate to polyclonal or monoclonal antibodies. This type of antibody will be discussed later.
In certain embodiments, the invention relates to a vaccine or vaccine composition comprising one or more D. gallinae antigens described herein that elicit an immune response in an avian species. Vaccines can be used prophylactically to prevent the establishment of D. Galinae infection / spread in avian hosts or to reduce, alleviate or eradicate established D. Galinae infection / spread. Furthermore, as a means for indirectly alleviating, alleviating or eradicating secondary complications, diseases or symptoms associated with D. Galinae infection / spread, the D. A vaccine or vaccine composition comprising either can be used. The term “indirectly” means that the vaccine provided by the present invention may not have a direct impact on the secondary complications associated with D. Galinae infection / spread, but the vaccines described herein Those of ordinary skill in the art will be able to understand that reducing the D. Galinae infection / spread population affected by the same may affect the reduction of secondary complications associated with D. Galinae infection / spread as well. Will understand. These secondary complications include, for example, bacterial pathogens (eg, Salmonella, Campylobacter or E. coli, mycobacterial species (eg, M. gallisepticum)) or viruses May be closely related to (including avian influenza). These pathogens are often carried in or on the body of ticks that affect avian hosts [3], or these pathogens are the result of adverse health effects on avian hosts or humans surrounded by ticks Or the result of an allergen produced by a tick.

Without being bound by theory, an avian species administered an antigen, composition, immunogenic composition, vaccine or vaccine composition of the present invention can produce a protective anti-D. Galinae antigen antibody, said antibody D. Galinae can function to deplete or eradicate, kill and / or inactivate. One or more antigens described herein include, for example, mating, fertilization, embryogenesis, yolk formation, sequestration of nutrients for embryonic development, embryonic development, sex-specific reproduction and development processes, sex differentiation and It may be required directly or indirectly for maturation, somatic and embryonic sex differentiation processes, oogenesis, spermatogenesis, oocyte maturation and / or ovulation. Thus, and further without being bound by any particular theory, we have determined that at least some of the protective antibodies raised in avian hosts in response to the specific D. Galinae antigen utilized by the present invention It is hypothesized that can have anti-reproductive effects (limit parasite egg production and / or larval development).
The various compositions provided by the present invention can be formulated as sterile pharmaceutical compositions comprising one or more of the antigens described herein and a pharmaceutical excipient, carrier or diluent. These compositions can be formulated for oral, topical (including skin and sublingual), parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and / or mucosal administration.
The compositions described herein can include separate dosage units and can be prepared by any method well known in the pharmaceutical industry. The method typically includes binding one or more of the D. Galinae antigens described herein together with a liquid carrier or a finely divided solid carrier or both.

Compositions suitable for oral administration wherein the carrier is a solid are most preferably provided as unit dose formulations, such as boluses, capsules or tablets, each comprising a predetermined amount of one or more D. Galinae of the present invention. Contains antigen. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets contain free-flowing forms (eg powders or granules) of the active compound (eg one or more D. Galinae antigens), optionally binders, lubricants, inert diluents, lubricants, surfactants Alternatively, it can be prepared by mixing with a dispersant and pressing with a suitable machine. Molded tablets can be made by molding the active compound with an inert liquid diluent. Tablets can optionally be coated and, if not coated, can optionally be scored. Capsules can be prepared by filling the capsule shell with the active compound alone or mixed with one or more accessory ingredients, followed by sealing in the usual manner. A cachet is similar to a capsule and the active compound is sealed in rice paper along with any optional ingredients. The active compounds can also be formulated as dispersed granules, which can be suspended, for example, in water or sprinkled on food prior to administration. The granules can be packed into sachets, for example. Orally suitable formulations wherein the carrier is liquid can be prepared as aqueous or non-aqueous liquid solutions or suspensions, or as oil-in-water emulsions.
Compositions suitable for oral administration include controlled release dosage forms (eg, tablets), where the active compound (eg, one or more D. gallinae antigens) is formulated in a suitable controlled release matrix, Or coated with a suitable controlled release film. Such compositions can be particularly convenient for prophylactic use.
Compositions formulated for parenteral administration include sterile solutions or suspensions of the active compounds (eg, one or more D. galinae antigens) in aqueous or oily vehicles.

Injectable compositions (eg, including vaccines) can be adapted for bolus injection or continuous infusion. Such preparations are conveniently provided in unit dose or multi-dose containers, which are sealed until needed for use after introduction of the formulation. Alternatively, the active compound (eg, one or more D. galinae antigens) may be in powder form, which is configured prior to use with a suitable vehicle (eg, sterile, pyrogen-free water or PBS).
Compositions comprising one or more of the D. gallinae antigens of the invention can be formulated as long acting depot preparations, which can be administered by intramuscular injection or implantation (eg, subcutaneous or intramuscular implantation). The depot preparation can comprise, for example, a suitable polymer or hydrophobic material or an ion exchange resin. They can also include preparations or adjuvants known to enhance the affinity and / or longevity of the avian immune response (eg, an oil-in-water single or double emulsion). Such long acting compositions are particularly convenient for prophylactic use.
Compositions suitable (or formulated) for mucosal administration include compositions containing aerosol dispersing particles or compositions dispersed in drinking water. When dispersed, such compositions should desirably have a particle diameter in the range of 10 to 200 microns, for example, to allow retention in the nasal cavity. This can be achieved as appropriate by the use of powders of appropriate particle size or the selection of appropriate valves. Other suitable compositions include coarse powders with particle diameters ranging from 20 to 500 microns (for administration by rapid inhalation through a nasal passage from a container held close to the nose) and nasal drops (aqueous or oily solutions) Or containing 0.2 to 5% w / v of active compound in suspension).

In addition to the carrier components described above, the various compositions described herein include one or more suitable additional (pharmaceutically acceptable) carrier components such as diluents, buffers, fragrances, binders, It will be appreciated that surface active agents, swelling agents, lubricants, preservatives (including antioxidants), and the like, and substances added to make the target recipient's blood and formulation isotonic can be included. . Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1M and preferably 0.05M phosphate buffer or 0.8% saline. In addition to the foregoing, pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil (eg olive oil), mineral oil and injectable organic esters (eg 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, and sodium chloride, lactated Ringer's solution or non-volatile oil. Preservatives or other additives (eg, antimicrobial agents, antioxidants, chelating agents, inert gases, etc.) can also be added.
Compositions suitable for topical formulations can be provided as gels, creams or ointments, for example.
It will be appreciated by those skilled in the art that the generation of a local (skin or subcutaneous) immune response is particularly beneficial when treating or preventing infection / spread of ectoparasites.
The composition used by the veterinarian may conveniently be in powder or liquid concentrated form. In accordance with standard veterinary prescription practices, convenient water-soluble excipients (eg lactose or sucrose) can be incorporated into the powder to improve their physical properties. Thus, particularly suitable powders according to the invention are 50 to 100% w / w, preferably 60 to 80% w / w of active ingredient (eg one or more D. Galinae antigens), and 0 to 50% w / w. w, preferably 20 to 40% w / w of conventional veterinary excipients. These powders can be added, for example, to avian feed (possibly by an intermediate premix method) or diluted with animal drinking water.

The liquid concentrate of the present invention suitably contains one or more D. Galinae antigens, optionally further in an acceptable veterinary use water-miscible solvent (eg polyethylene glycol, propylene glycol, glycerol, glycerol formal or 30 % of such solvents mixed with up to% v / v ethanol). The liquid concentrate can be administered to the animal's drinking water.
In general, a suitable dose of one or more D. gallinae antigens provided by the present invention may range from about 10 to about 100 μg / bird. Furthermore, the one or more antigens described herein can be administered about 2 to about 5 times over a period of about 1 to about 10 weeks. In certain embodiments, each bird is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, one or more of the antigens described herein. 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 100 μg may be administered. Furthermore, each bird can be administered the antigen 2, 3, 4 or 5 times over a period of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. It will be appreciated that each bird can be administered the same or different doses of D. galinae antigen at each administration opportunity.
It should be noted that the various compositions, immunogenic compositions, and vaccines of the present invention may include one or more antigens described herein. For example, a variety of compositions can include two or more antigens described herein. The immunization protocol can include the use (administration) of one or more different compositions, wherein the compositions (eg, avian subjects) have already been administered one or more antigens of the present invention. Together. The antigens to be administered can be administered alone or together. Where more than one antigen is administered, the antigens can be administered separately and at different times together or simultaneously.

Transdermal administration can be accomplished by the use of impregnating shields, bandages, bandages, etc., or by using some form of transdermal delivery device.
The various antigens provided by the present invention can be provided as preparations derived directly from the D. gallinae antigen. For example, antigens used in the present invention can be obtained from total or fragmented parasites collected from the environment or habitat of donor animals and / or birds. The donor animal can be a naturally infected / spread animal or an animal that has been intentionally (experimentally) infected with D. galinae. In certain embodiments, D. Galinae capable of inducing antigens can be obtained from a number of different avian donor subjects (these avian subjects live in the same or different environments and / or habitats). If the bird species are farmed, D. Galinae may be obtained from and / or from one or more locations on the farm (eg, certain enclosures, poultry houses, cages or sheds on the farm) Can be obtained from one or more of the infected or prevalent avian hosts.
A vaccine or composition intended for use in a particular population of avian target animals that has been infected / spread (or at risk of being infected / spread) by D. Galinae is (i) administered a composition of the present invention. The environment of a particular population, one or more D. Galinae collected or collected from a habitat or field, and / or (ii) an environment that is linked to or closely related to the particular population to which the composition of the invention is administered. It should be understood that one or more D. galinae antigens derived from one or more D. galinae collected or collected from a habitat or site may be included. By way of example, if a farm includes intricate locations (perhaps spread over a particular geographic area), the present invention utilizes D. Galinae antigens obtained or collected from one or more of the intricate locations. These antigens could be used to provide compositions and / or vaccines for use in avian subjects that are farmed at each of the complex locations.

A person skilled in the art may refer to compositions and / or vaccines of the type described in the two paragraphs above as “self-composition” or “self-vaccine”. Accordingly, the present invention provides self-compositions and vaccines comprising one or more of the D. Galinae antigens described herein for use in eliciting an avian species immune response.
To obtain any D. galinae antigen used, the collected or collected D. galinae can be subjected to a homogenization protocol to produce a homogenized suspension of D. galinae components. The resulting homogenized D. Galinae suspension is subsequently subjected to one or more size / density separation techniques (eg, centrifugation) to remove unwanted D. from the fraction containing one or more antigens of the invention. Galina shrimp can be removed. Subsequently, the antigen-containing fractions can be subjected to a sterilization procedure, making them suitable for use in the present invention. The antigen-containing fractions thus prepared can be further processed to produce fractions containing lesser, more highly purified (or cleaner) / concentrated antigens or specific or selected antigens ( The ultimate goal is to generate one or more fractions that do not contain all but the antigens matching the antigens described herein or any fragment thereof). For example, using techniques such as anion exchange, gel filtration, and / or affinity chromatography for the preparation of the antigen fraction used and / or the extraction of one or more specific antigens. Can do. For example, an antibody extracted from an avian subject animal to which one or more of the antigens of the present invention has been administered is immobilized on an appropriate base, and the present invention is applied under conditions that allow binding between the antibody and the target antigen. Can be contacted with a solution containing one or more of the antigens. For example, the solution can contain a pool of various antigens. In this way, one or more specific antigens can be extracted or concentrated from the mixed antigen population.

The antigens of the present invention can be used to elicit egg yolk (IgY) immune responses. Egg yolk IgY can be fixed on a suitable foundation.
The D. gallinae antigen used in the present invention can be prepared for low temperature or cryopreservation by the addition of one or more cryopreservation / cryoprotectants.
In certain embodiments, the antigens and compositions for use, medicaments and methods provided herein comprise one or more D. Galinae antigens, said antigens comprising:
(A) SEQ ID NO: 1-22,
(B) At least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% with any one of (a) , 98% or 99% homologous or identical sequences, and (c) at least 30%, 40%, 50% with an immunogenic fragment of any one of (a), (b) or (c), 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% selected from the group consisting of homologous or identical sequences Contains or is encoded by a sequence.
In addition to providing D. Galinae antigen amino acid sequences (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22) as described, the present invention further encodes the above To nucleic acid sequences (SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21) and / or fragments thereof (preferably fragments encoding immunogenic peptides) . The nucleic acid sequence can be modified to replace one or more of the naturally occurring residues with matching or equivalent synthetic or chemically or enzymatically modified nucleotides. The nucleic acid sequences disclosed herein can be further modified to allow for degeneracy of the nucleic acid code. That is, any wild type sequence can be modified to modify one or more codon sequences without affecting any of the primary amino acid sequences.

Nucleic acid sequences that are substantially complementary to all or part of any of the sequences provided by SEQ ID NOs: 1-22 (especially nucleic acid sequences) are within the scope of the invention. Such complementary sequences can be useful as primers or probes. The complementary sequence may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 50 or more (eg, up to 100, 200 or 200 or more) breaks of any sequence disclosed herein. It may be complementary to a sequence representing a continuous or contiguous base.
The term “substantially complementary” refers to nucleic acids that exhibit some degree of identity / homology with respect to any nucleic acid sequence complementarity with any of the above SEQ ID NOs: 1-22 or fragments thereof (particularly immunogenic fragments) It should be understood to encompass molecules. A nucleic acid sequence having a level of identity or homology with respect to sequence complementarity with any of SEQ ID NOs: 1-22 (or immunogenic fragments thereof) is at least 30% with a full-length complementary sequence or portion thereof 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% showing identity or homology Can do.
The invention can further relate to natural or artificially generated variants or analogs of any of the protein / nucleic acid sequences described herein. Such variants may include, for example, one or more amino acid / nucleic acid deletions, additions, substitutions relative to a reference sequence (eg, a sequence disclosed herein, including those disclosed in SEQ ID NOs: 1-22). And / or insertion may be indicated. In certain embodiments, the substitution can be a conservative substitution. Conservative substitutions require replacement by alternative amino acids with similar properties of one or more amino acids of a protein or peptide sequence, conservative substitutions are physicochemical properties of native (or wild-type) proteins and / Or does not substantially change structure or function.

The nucleic acid molecules provided by the present invention can take the form of nucleic acid constructs or vectors, such as cloning or expression cassettes / vectors, including phage vectors for use as described, for example, in EP1370284. The term “nucleic acid construct” or “vector” can further include constructs intended for use as DNA vaccines.
The vectors provided by the present invention can direct the expression of a nucleic acid sequence encoding the D. Galinae antigen in, for example, bacteria, fungi, animals (including protozoa, avian species and mammals) and / or insect cells. .
Accordingly, the present invention provides a vector, preferably an expression vector, having a nucleic acid sequence encoding the D. Galinae antigen of the present invention, or having a sequence selected from the group consisting of: provide:
(A) SEQ ID NO: 1-22,
(B) At least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% with any one of (a) , 98% or 99% homologous or identical sequences, and (c) an immunogenic fragment of any one of (a) or (b) and at least 30%, 40%, 50%, 60%, 65 %, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous or identical sequences.
Suitable expression vectors used in this aspect of the invention may further direct expression in prokaryotic or eukaryotic cells (eg, protozoa, birds, mammals, fungi, bacteria, plants and / or insect cells). One or more promoter sequences can be included.

The vector provided by the present invention may be circular or linear, single-stranded or double-stranded, and may contain DNA, RNA, or a combination or modification thereof. Furthermore, the vectors of the invention can be, for example, plasmids, cosmids or viral vectors (eg retroviral or bacteriophage vectors). The vectors provided by the present invention may further comprise a selection or marker element, such as an antibiotic resistance gene and / or optionally a detectable tag. A number of suitable vectors are known and further information is available from the following literature: Pouwels et al. Cloning Vectors: a Laboratory Manual (1985 and addendum), Elsevier, NY; and Rodriquez, et al. (Eds. Vectors: a Survey of Molecular Cloning Vectors and their Uses, Buttersworth, Boston, Mass (1988), both of which are hereby incorporated by reference.
Therefore, in addition to the technique of directly extracting or purifying the D. Galinae antigen collected or collected from the D. Galinae antigen, the antigen used in the present invention can be obtained by using a recombinant technique. In certain embodiments, one or more recombinant D. Galinae is used with an expression vector comprising one or more nucleic acid sequences encoding the corresponding D. Galinae antigen (eg, any of the antigens described herein). An antigen can be generated.
Accordingly, in yet another aspect, the present invention provides a host cell transfected with or transformed with the vectors described herein. Eukaryotic cells or prokaryotic cells (eg, protozoa, birds, plants, insects, mammals, fungi and / or bacterial cells) can be transfected with one or more vectors described herein. Techniques used to introduce heterologous or foreign nucleic acid sequences (eg, expression vectors) into cells are well known to those of skill in the art, and include one or more to induce heat shock treatment, transformation / transfection, for example. Use of chemicals such as calcium phosphate, viral carriers, microinjection and / or the use of techniques such as electroporation. More information on transformation / transfection techniques can be found in the following literature: Current Protocols in Molecular Biology, Ausuble, FM, et al., John Wiley & Sons, NY (1989), which is hereby incorporated by reference. Included in the description).

In view of the above, the present invention generates a D. Galinae antigen comprising or encoded by the sequence of SEQ ID NO: 1-22 (or an immunogenic fragment thereof) and used in eliciting an avian immune response Provide a process. The method comprises the following steps: (a) transforming a host cell with a nucleic acid sequence of the invention or transfecting a host cell with a nucleic acid construct of the invention; (b) expression of the nucleic acid (more Suitably culturing the cells obtained in (a) under conditions that result in expression of the protein encoded by said); and (c) cell culture and / or culture of the expressed recombinant protein or peptide. Isolating from the supernatant.
The recombinant protein / peptide produced according to the above method can be partially purified from the host cell prior to use in a vaccine or vaccine composition. If the polypeptide is secreted from the host cell, the cell can be separated from the medium by centrifugation. In such situations, the supernatant (including the secreted polypeptide) can be used directly as a vaccine or in a vaccine composition. Alternatively, the polypeptide may be partially purified from this supernatant using, for example, affinity chromatography.
Any of the D. Galinae antigens described herein can be mixed with another component (eg, another polypeptide and / or adjuvant, diluent or excipient).
For example, the compositions, immunogenic compositions, and / or vaccine / vaccine compositions provided by the present invention may be used to control other avian diseases / infections or spreads, such as viral antigens, fungal antigens, Bacterial antigens or other parasite antigens can be included.
For example, the vaccine or vaccine composition can be incorporated into a multivalent vaccine containing antigens against other avian (eg chicken) diseases. These vaccines can include, for example, the vaccines and target organisms listed in Table 1 below.

Table 1: Exemplary avian vaccine

Adjuvants used in the present invention or added to the compositions described herein include, for example, aluminum salts (alum), oil emulsions, saponins, immune stimulating complexes (ISCOM), liposomes, microparticles, nonionic Sex block copolymers, derived polysaccharides, cytokines, and / or bacterial inducer forms can be included.
In yet another aspect, the invention has been treated with a vaccine or composition described herein and / or a vaccine or composition comprising one or more D. Galinae antigens described herein, A vaccinated or immunized bird population, such as a chicken farming population, is provided.
It will be appreciated by those skilled in the art that the vaccines described in the present invention may take the form of subunit-type vaccines in which one or more D. Galinae antigens are used for inoculation of animals. In addition or alternatively, the vaccine comprises a nucleic acid molecule encoding one or more antigens provided by the present invention or immunogenic fragments thereof and can be expressed by the cells of the animal to be vaccinated (DNA vaccine) Known as). In this way, in a vaccinated host (eg, a vaccinated chicken), constitutive expression of the D. gallinae antigen can elicit a constitutive protective immune response.
In addition to providing D. Galinae antigens used in eliciting an immune response in animals, the present invention also provides polyclonal antibodies that bind (or have affinity or specificity) to any of the described D. Galinae antigens and / or Alternatively, monoclonal antibodies (or antigen binding fragments thereof) can be provided. Generation 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, the following literature: “Basic methods in Antibody production and characterisation” Howard & Bethell, 2000 (Taylor & Francis Ltd.). Such antibodies can be used in diagnostic procedures to detect or diagnose passive immunity as well as, for example, avian species D. Galinae infection / spread. The term “antibody” includes egg yolk antibody (IgY).

The present invention further provides a vaccine for use in the prevention or control of D. galinae infection / spread and associated diseases in avian hosts. The vaccine can be a polypeptide vaccine or a polynucleotide vaccine.
The present invention further provides a method of immunizing birds against D. gallinae infection / spread and associated diseases such as secondary infections, said method comprising the step of administering the vaccine of the present invention to the birds .
The present invention further provides a method for diagnosing or detecting D. gallinae infection, spread and / or sample contamination. The method can utilize one or more D. Galinae antigens of the present invention and / or antibodies having specificity and / or affinity for any of the antigens of the present invention. The antibodies and / or antigens used can be immobilized to the foundation (possibly via some form of binding moiety (eg, antibody)).
Suitable foundations may include those containing nitrocellulose, glass and / or plastic, such as microtiter plates. The antibodies and / or antigens used can be immobilized as an array containing a number of separate spots. Any given spot can contain a single type of antibody / antigen or multiple antibodies with a single antigen specificity / affinity, each separately. In addition or alternatively, any given spot can contain different types of antibodies or antigens or multiple antibodies with specificity / affinity for different antigens.
The diagnostic or detection method can include contacting the sample with an antigen or antibody (optionally immobilized) of the present invention. Permits binding of any antibody and / or antigen of the sample and the present invention (optionally immobilized) to any antibody and / or antigen of the present invention and (optionally immobilized) of the present invention Can be contacted under the following conditions.
Thus, using the detection and / or diagnostic methods of the present invention, a sample for the presence of an antibody that reacts (or has specificity and / or affinity) with one D. Galinae antigen and / or multiple D. Galinae antigens Can be probed. Depending on the source and nature of the sample, the presence of an antibody that reacts (or has specificity and / or affinity) with one D. Galinae antigen and / or multiple D. Galinae antigens in the sample is confirmed. For example, it can be an indicator of infection, spread and / or contamination of D. gallinae.

The presence of an antibody that reacts (or has specificity and / or affinity) with one D. Galinae antigen and / or multiple D. Galinae antigens can be confirmed by detection of the antibody / antigen complex. For example, if the method is used to detect the presence of an anti-D. Galinae antibody in a sample, under appropriate conditions that allow binding of any antibody in the sample to the antigen with which the sample is contacted The sample can then be contacted with any of the (optionally immobilized) antigens of the present invention. A secondary antibody (or other, eg, labeled with fluorescence or chemiluminescence) that has an affinity for the antibody present in the sample, eg, optionally after washing to remove unbound material Can be used to detect antibody / antigen complexes. Similar methods can be performed to detect the presence of D. gallinae antigen in the sample. In this type of method, the antigen can be any of the antigens described herein, and the sample can be contacted with an antibody (specifically immobilized) that has specificity and / or affinity for the antigen. After washing to remove unbound material, detection of the antigen / antibody complex is accomplished by labeling (eg, fluorescent or chemiluminescent label) secondary antibody (or other) that binds to the complex or its component (eg, the antigen component). This can be achieved by using a connecting part). Samples can include biological samples such as blood samples (eg, avian / chicken blood, whole blood, plasma or serum), egg yolk, feces, urine, sweat, tissue, skin, exfoliates, secretions, and the like. . Samples can include environmental or field samples such as samples of nesting material, straw, soil, grass, feed, drinking water and the like.
The present invention further provides a kit, wherein the kit is optionally used in a method for detecting and / or diagnosing D. Galinae infection, spread and / or contamination, wherein one or more antigens and And / or an antibody having specificity and / or affinity for any of the antigens of the invention. Antigens and / or antibodies used in the kit can be immobilized on a suitable foundation. The kit can further provide instructions for use, containers for the reaction, and diluents, buffers and / or wash solutions used in the detection and / or diagnostic methods.
The invention will now be described in detail with reference to the following drawings.

In vitro blood-sucking device used to deliver a given blood feed to Dermanius gallinae The device is a modification of the device described by Wright et al. (2009) [9]. Specific modifications include: Replace the chicken skin blood-absorbing membrane with pre-stretched parafilm “M” (Bemis ™ Flexible packaging) and place a lamb castration rubber ring (NetTex) around the pastet valve to create a seal and prevent blood feed leakage prevent. Different immune response profiles of Delmanius Galinae soluble protein pool (IEX-pool) after ion-exchange chromatography 2 μg of IEX-pool 1 to 5 and 2 μg of soluble mite protein (SMP, pool 6) 12% Bis-Tris Isolate on Novex gel (Invitrogen), transfer to nitrocellulose and probe with 80 μg / mL IgY made for SMP (left blot) or 80 μg / mL IgY obtained from naive hens (right blot) did. Bound IgY was detected with rabbit anti-IgY-peroxidase (Sigma) and visualized with SIGMA FAST ^™ 3,3′-diaminobenzidine substrate (Sigma). Survival of Delmaniss gallinae after blood intake of vaccinated hens Heparin from hens vaccinated with IEX-pool (group (Gp) 1-5), soluble mite extract (group 6) or adjuvant alone (group 7) Mites were raised with added whole blood. Survival analysis was performed by Cox proportional hazard model using frailty function. Average proportions of surviving mites 24, 48, 72 and 96 hours after blood sucking are plotted along with corresponding 95% confidence intervals (shaded areas). 2-D gel analysis of IEX group 1 proteins and their corresponding immune reaction spot profiles IEX group 1 proteins were separated by replicate gels in the pI range of 7-11. One gel stained with SimplyBlue ^™ SafeStain (Life Technologies) (Panel C), immunoblot performed with two replicate gels, and 100 μg / mL yolk IgY (Panel A) of hens vaccinated with Group 1 or Control (Group 7) hens were screened with 100 μg / mL egg yolk IgY (panel B). Bound IgY was detected with rabbit anti-IgY-peroxidase IgG and visualized with a chemiluminescent substrate. After LC-ESI-MS / MS, spots that are cut out and identified are circled and numbered. Immunoaffinity enrichment of IEX group 4 protein Purified egg yolk IgY generated against IEX group 4 protein was cross-linked to a HiTrap NHS-activated column, and IEX group 4 protein was selectively enriched by immunoaffinity chromatography. Group 4 proteins before affinity purification (panel A, lanes 1 and 3) and eluted affinity enriched material (panel B, lanes 2 and 4) were separated on a 12% Bis-Tris Novex gel (Invitrogen) and nitrocellulose Moved to. Immunoblots lanes 1 and 2 (Panel A) were probed with pool sera from group 7 (adjuvant only control) hens (1: 200 dilution), lanes 3 and 4 after immunization of group 4 hens Probed with pooled serum (1: 200 dilution). Bound IgY was detected with rabbit anti-IgY-peroxidase (Sigma) and visualized with SIGMA FAST ^™ 3,3′-diaminobenzidine substrate (Sigma). The eluted immunoaffinity enriched material was further concentrated, separated by electrophoresis, and stained with SimplyBlue ™ SafeStain (panel B, lane 5). The stained concentrated protein profile appearing in lane 5 was cut into 24 equal sized horizontal slices and the extracted protein was subjected to LC-ESI-MS / MS. Effects of hen immunization with recombinant Delmanius Galinae protein on the survival of ticks reared in blood of immunized hens. A graphical summary of survival analysis of ticks reared in blood of hens immunized with recombinant vaccinia antigen. Shown (tick survival is plotted against time after blood sucking). The 95% confidence interval is represented by the shaded area. As abbreviations in this figure, Deg-SRP-1 is “Serp-02564”, Deg-HGP-1 is “HemeLGP-13207”, Deg-VIT-1 is “Vit-12013”, Deg-PUF-1 is “UK” -11549 ", Deg-ASP-1 is" Asp-00293 ", Deg-PUF-2 is" UK-13089 ", Deg-SRP-2 is" Serp-01514 ", Deg-CPR-1 is" PepC1A-13094 “, Deg-GPD-1 is“ PGDH-00877 ”, and Deg-PUF-3 is“ UK-00186 ”.

Materials and Methods Collection and conditioning of D. gallina ticks used for tick-sucking assays and protein extraction From a Scottish industrial egg production unit, a 75 cm ² ventilated tissue culture flask containing a mix of growth and gender (Corning). The mites were removed from the waste material and flash frozen in liquid nitrogen and stored at -80 ° C until needed. Mites that are snap frozen within 24 hours of collection constitute a “vampire” population. This is because most of these mites have not had time since blood sucking. “Starved ticks” were conditioned by initial incubation at room temperature (RT) for 7 days followed by storage at 4 ° C. for 14 days. Starved ticks were used in blood sucking assays or snap frozen in liquid nitrogen and stored at −80 ° C. until needed.

Extraction of soluble mite protein (SMP) Soluble mite protein (SMP) was prepared from flash frozen mites as follows: 1 g of frozen mites were suspended in 10 mL of ice-cold phosphate buffer (PBS) and 2 × 30 on ice. Homogenized with a second pulse (Ultra Turrex ™ T 25 D-S2, IKA with S25N-8G dispersive element). Insoluble material and debris were removed by centrifugation (25,000 × g, 4 ° C., 20 minutes). Soluble material was decanted and the SMP further clarified by a second centrifugation. The SMP was immediately frozen in liquid nitrogen and stored at -80 ° C. The concentration of SMP was estimated using the bicinchoninic acid (BCA) assay (Peirce) according to the manufacturer's protocol.

Fractionation of SMP by ion exchange chromatography (IEX) Prior to ion exchange chromatography (IEX), 20 mg of SMP was desalted with a PD-10 desalting column (GE healthcare). IEX was performed at room temperature using a 1 mL HiTRAP Q HP anion column (GE healthcare) combined with an AKTA high performance liquid protein chromatography (FPLC) system (Amersham Biosciences) and 20 mM Tris-HCL (pH 7.4). ) And a subsequent isocratic step (20 mM Tris-HCL, 1 M NaCl (pH 7.4)). The eluted protein was collected in 1 mL fractions, immediately snap frozen in liquid nitrogen and stored at −80 ° C. Immunoblots of IEX-SMP fractions were immunized with total SMP in adjuvant or adjuvant / PBS alone (control group) (described below) and 5 separate pools (groups 1-5 according to their immunogenicity profile) Probed with IgY of pooled hens. Each of the IEX-SMP pool and unfractionated SMP was concentrated with an Ultracel ™ -10K Amicon ™ Ultra-15 centrifugal filter unit (Merck Millipore Ltd) and 0.22 μM Millex-GV 13 mm PVDF unit (Merck Millipore Ltd.) and the concentration was determined before immunizing hens (BCA kit, Pierce).

Polyacrylamide gel electrophoresis (PAGE) and immunoblot development For one-dimensional gel electrophoresis (1D-PAGE), a predetermined concentration of tick protein is 12% in NuPAGE ™ MES SDS running buffer (GE healthcare). Separated on Bis-Tris Novex gel. Proteins were visualized in situ by silver staining (SilverQuest, Invitrogen) or transferred to a nitrocellulose membrane according to the manufacturer's instructions with an Xcell II blot module (GE healthcare) for immunoblotting. For immunoblot development, the membrane holding the electroblotted protein is incubated in 5% (w / v) milk powder in PBST (PBS, 0.05% (v / v) Tween 20 (Sigma)) did. After washing 3 times with PBST, the immobilized protein was probed with 80-100 μg / mL egg yolk-derived IgY in PBS (egg yolk-IgY) or with serum diluted 1:50 to 1: 200 in PBS. IgY and serum were derived from hens immunized with SMP in Quil A adjuvant or adjuvant / PBS alone as described in [9]. The blot was washed and bound IgY was detected by incubation in rabbit anti-IgY-peroxidase conjugate (diluted 1: 20,000 with PBS (Sigma)). Blots were washed 5 times in PBST and then developed in a colorimetric substrate: SIGMA FAST ^™ 3,3′-diaminobenzidine (Sigma) or SuperSignal West Pico chemiluminescent substrate (Thermo Scientific). Blots were captured using an ImageQuant LAS4000 luminescence image analyzer (GE healthcare).

Evaluation of immunity and miticide effect of hens by IEX-SMP fractionation
14 ISA-Warrens hens (24 weeks old) were randomly assigned to 7 groups as follows (5 IEX-pools (Group 1-5), all SMPs (Group 6), and QuilA adjuvant alone (Control, group 7)), immunized with the appropriate immunogen three times at biweekly intervals. Each 200 μL vaccine dose was formulated and contained 200 μg of the appropriate protein and 200 μg of QuilA adjuvant (Brenntag Bioserve) (Groups 1 to 6), or 200 μg of QuilA alone (Group 7). The vaccine was administered intramuscularly to alternating chest muscles. Eggs were collected before vaccination and throughout this experiment as a source of egg yolk-IgY. IgY was purified from egg yolk using an IgY purification kit according to the manufacturer's instructions (Pierce). Whole blood was collected from the wing brachial vein into heparinized (final concentration 36 USP / mL blood) tubes before each vaccination and 3, 5, and 6 weeks after the final vaccination. To assess the effects of blood intake of vaccinated hens on tick survival, two groups of heparinized blood were pooled in each group, and a modified version of the previously described in vitro blood sucking device [9] (Figure 1) was used to condition D. Galinae. Ten blood collection chambers were set up for each group, and ticks were sucked at 24% intervals at 75% relative humidity and 39 ° C. Blood sucking ticks were removed from the blood sucking chamber and isolated in 96 well microtiter plates and immediately scored for viability at 48, 72 and 96 hours after blood sucking. Blood sucking assays were performed at 3, 5 and 6 weeks after the final immunization.

Two-dimensional (2-D) gel electrophoresis and identification of immune reaction spots
Group 1 and 4 proteins from IEX-enrichment were separated by two-dimensional (2-D) electrophoresis and constitutive immune response proteins were identified using Western blot analysis combined with proteomic analysis. All kits, materials and equipment used in the 2-D procedure are obtained from the manufacturer (GE healthcare), and unless otherwise noted, all procedures are described in the manufacturer's handbook (GE healthcare, 2004). Followed the detailed methodology posted.
After removing impurities from 100 μg aliquots of group 1 and 4 proteins, the above was resuspended in the following (7M urea, 2% (w / v) CHAPS, 2M thiourea, 0.3% (w / v) DTT, 0.002% (W / v) bromophenol blue, 2% (v / v) IPG buffer (IPG buffer 7-11 for pool 1; 3-10 for pool 4)), 16 hours on appropriate Immobiline DryStrips, Rehydrated at room temperature and subjected to isoelectric focusing as follows: 30 minutes at 300V, increased to 1000V over 30 minutes, then increased to 5000V over 90 minutes, followed by 5000V for 25 minutes. Denature the focused protein with the following solution for 15 minutes (75 mM 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)), followed by incubation for 15 minutes in equilibration buffer containing 25 mg / mL iodoacetamide, followed by Excel SDS two-dimensional homogeneous gel 12.5 on Multiphor II electrophoresis system % (25 cm × 11 cm) was used for separation in two dimensions.
One replicate of the two-dimensional gel was cut and the protein was stained with SimplyBlue ^™ SafeStain (Invitrogen). The remaining two replicate gels were electroblotted onto Hybond-C nitrocellulose membranes and the following 100 μg (yolk-IgY (positive) in PBS made in hens for group 1 or 4 proteins or control group with adjuvant alone Immunoscreening was performed as described above using 7 yolk-IgY (control). Co-localized staining and further immunolabeled protein spots were excised from SimplyBlue ^™ stained gels, and proteins were subjected to LC-ESI-MS / MS to generate peptide data. Search the NCBInr database (09-Jan-2015) and custom D. Galina ettranscriptome database (Bartley et al., 2015 (in preparation)) for stringent identification criteria (described in [13]) Was used to identify the DNA sequence encoding the protein of interest.

Immunoaffinity enrichment of SMP
To enable enrichment of IEX group 4 protein, 1 mL HiTrap NHS-activated HP column (GE healthcare) was cross-linked with egg yolk-IgY from hens immunized with IEX group 4 protein (IEX group 4 protein is Conjugated with IgY under native conditions as opposed to reducing, denaturing conditions). Briefly, purified egg yolk-IgY using a PD-10 column (GE healthcare) equilibrated with coupling buffer (0.2 M NaHCO ₃ , 0.5 M NaCl, pH 8.3) according to the manufacturer's spin procedure. Buffer exchange was performed. A HiTrap NHS-activated HP column was primed with 6 mL ice-cold HCL (1 mM) and egg yolk-IgY (3.75 mg) in coupling buffer was recirculated at 4 ° C. for 3 hours. Egg yolk-IgY is cross-linked to a Sepharose matrix and 2 mL of buffer A (0.5 M ethanolamine, 0.5 M NaCl (pH 8.3)) and 2 mL of buffer B (0.1 M sodium acetate, 0.5 M NaCl (pH 4)) The column was deactivated by three rounds of sequential washing with followed by equilibration with 12 mL PBS. IEX group 4 protein (1.4 mg) was recycled to the column at 4 ° C. for 16 hours. Unbound material was washed from the column with 10 mL PBS and bound protein was eluted with 4 mL elution buffer (0.1 M glycine, 6 M urea (pH 2.5)). Concentrated immune reaction components were eluted from the column and separated on an SDS-PAGE gel (4-12% Bis-Tris Novex gel (GE healthcare) in NuPAGE ™ MES SDS running buffer). The gel was subsequently divided into 24 pieces of 2.5 mm sections and each section was subjected to LC-ESI-MS / MS as previously described [12]. A mascot search of the custom D. Galinae transcriptome database (Bartley et al., 2015 (in preparation)) and NCBInr database (09-Jan-2015) was used to identify the components within each gel section.

Production of recombinant protein and assessment of vaccine potential Ten proteins identified from IEX groups 1 and 4 by 2-D immunoblotting or immunoaffinity purification as above were selected as vaccine candidates for further analysis ( Table 3). The coding sequence (CDS) of the open reading frame (ORF) of the transcript encoding each of these proteins can be inferred from bioinformatics analysis of their corresponding contigs or, in the case of Deg-SRP-2, at the end of the cDNA. Induced by rapid amplification (RACE). The protein named “Deg-PUF-2” had two variant contig sequences. Both CDS of these Deg-PUF-2 variants were synthesized (MWG operon), while the CDS of all remaining vaccine candidates was amplified by RT-PCR from total RNA as previously described [14]. Both Deg-PFU-2 variants 1 and 2 and Deg-CPR-1 CDS were subcloned with the expression vector pET22b (Novagen), and all other vaccine candidate CDS were subcloned with pET SUMO (expected signal peptide The coding sequence was omitted where appropriate). After transformation of E. coli BL21 competent cells with each of the plasmids, recombinant forms of each protein were generated and affinity purified as previously described [8,14]. Due to their size (and thus the difficulty of expression), Deg-HGP-1 is expressed as two halves and only the N-terminal half of Deg-VIT-1 is expressed and used as an immunogen It was. The vaccine potential of each recombinant protein was assessed as follows: 200 μg of each of the proteins listed in Table 3 as adjuvants in the laying hen's muscle (chest) 3 times at 2 week intervals. Injection with QuilA. An adjuvant-only control group was also added. Blood was collected from the brachial vein of each hen's wings for 1 week interval starting from 2 weeks after each vaccination and after the third vaccination for monitoring of serum antibody levels and in vitro blood absorption assays. Using the modified in vitro blood-sucking device shown above and shown in Figure 1, blood of the immunized hen was given to the mites (blood was collected 2, 3 and 4 weeks after the third immunization), and the mortality rate over 5 days Measurements were taken at 24 hour intervals. The blood absorption assay was performed in triplicate. An increase in mean mortality was obtained compared to the corresponding adjuvant-only control group.

Statistical analysis Tick mortality data were analyzed using two different statistical approaches. Survival analysis (incorporating treatment groups as explanatory variables) was performed based on the Cox proportional hazards model using a frailty function. Mites that survived after 96 hours were processed as censored data. The frailty function incorporated a replica within each experiment as a random effect, and an estimate of the random effect obtained using the restricted maximum likelihood method assuming a Gaussian distribution. In the second approach, cumulative mite mortality at 24, 48 and 96 hours for each treatment group was analyzed with a separate generalized linear mixed model (GLMM), and differences in mean mortality between groups at these time points were analyzed. Examined. The model included treatment groups as a parameter effect and replicates within each experiment as a random effect. Data overdispersion was tricked by including individual level random effects. A generalized linear mixed model was fitted using a binomial distribution and a logit link function.

result
Fractionation of SMP by ion exchange chromatography (IEX)
IEX is a complex protein mixture contained in SMP, “flow-through” (FT) fractions containing unbound protein (Group 1 pool), and four groups with minimal protein overlap and immunoreactivity profiles (Group 2-5) could be separated into continuously eluting fractions that could be pooled (Figure 2). Minimal immunoreactivity was detected when IEX-fractions or SMPs were incubated with IgY from non-immunized hens, except that two proteins with molecular weights of about 25 and 65 KDa (previously referred to as IgY light chain and heavy weight). Are matched). IgY present in IEX-fractions and SMPs is derived from blood feed present in the intestine of ticks, which is detected by anti-IgY-peroxidase conjugates.

Evaluation of immunity and miticide effect of hens by IEX-SMP fractionation
Five IEX-pools (Group 1-5), total SMP (Group 6) and QuilA adjuvant alone (Control, Group 7) were used to immunize hens and blood of the immunized hens was given to ticks. Mites ingesting the blood feed were scored for mortality 24 hours after blood sucking and daily thereafter. The average mite mortality rate was lower in all immunization groups (except group 2) than in the control group (p <0.001) (Table 2, Figure 3). Groups 1 and 4 ticks had the highest mortality risk at any time during the experiment, with a probability of death of 3.06 to 3.72 times greater than the control group (p <0.001). Similarly, a GLMM analysis of the cumulative mortality of mites in each treatment group 96 hours after blood sucking also identified group 1 and group 4 as having the highest mortality (0.37 and 0.40, respectively) (p <0.05). At earlier time points after 24, 48 and 72 hours of blood sucking, only Groups 1 and 4 showed a consistent and substantially significant increase in mortality compared to the control group. Both analyzes suggest that vaccine immunization with IEX-Pools 1 and 4 has the strongest effect on tick survival, so IEX-Pools 1 and 4 are immunoreactive and potential for these IEX-Pools. Selected for thorough analysis to identify the different protective protein components.

Identification of vaccine candidates using 2-D gel electrophoresis and immunoaffinity purification 2-D gel electrophoresis combined with immunoblotting was performed using IEX-Group 1 pool (Figure 4) and Group 4 pools (data not shown) High resolution separation and a clear presence of immunoreactive spots. However, protein resolution on a 2-D gel platform depends on efficient denaturation of the protein under reducing conditions (reduction can result in loss of steric epitopes). Therefore, to complement the data obtained from 2-D PAGE analysis, proteins were identified using a second non-denaturing immunoaffinity enrichment approach (FIG. 5). The entities of all immunoreactive proteins in groups 1 and 4 obtained from immunoblotting and immunoaffinity purification are summarized in Table 5 (Group 1 protein) and Table 6 (Group 4 protein). A total of 14 and 16 spots, respectively, were identified from group 1 and group 4 by 2-D PAGE immunoblotting.
Immunoaffinity enrichment of group 4 proteins (not group 1) was successful (Figure 5). Comparison of immunoblots between non-enriched (Figure 5A, lane 3) and affinity enriched (Figure 5A, lane 4) shows that there is specific enrichment of some proteins following the immunoaffinity enrichment step Is clear. Most of the concentrated protein has a molecular weight above 55 KDa, and some intensities of these bands in the immunoblot suggest strong immunoreactivity and / or abundance.

Production of recombinant proteins and assessment of vaccine potential Recombinant proteins were produced for 10 selected candidate antigens (Table 3) that were certified with IEX-group 1 and group 4 proteins by their immunoreactivity and hypothetical function. These recombinant antigens were successfully expressed in E. coli, affinity purified using nickel affinity chromatography, and could be refolded prior to hen immunization. Tick was raised in vitro in the blood of immunized hens and all data obtained from the blood sucking assay was analyzed using the Cox proportional hazard model applied over 4 days after the blood sucking period (Table 4, Figure 6). In further analysis, a generalized linear mixed model (GLMM) was applied to mortality data at 24 and 120 hours after blood sucking (Table 4). Strong statistical evidence showing that immunization with Deg-SRP-1, Deg-PUF-1, Deg-HGP-1 and Deg-VIT-1 results in the greatest increase in tick mortality was used in both studies Occasionally present (p <0.05).

Conclusion
1) Ion-exchange chromatographic fractionation of unpurified soluble D. gallinae extract resulted in the identification of a semi-purified protein pool with significant vaccine potential when tested in vitro.
2) Immunoaffinity analysis and immunoblotting of 2-D separation of these proteins, together with proteome analysis, revealed the substance of the most powerful immunoreactive protein.
3) Recombinant forms of four of these proteins, serpin, vitellogenin, heme lipoglycoprotein and proteins of unknown function, are significant in in vitro blood-sucking analysis when fed hen blood immunized with these proteins to mites The level of mite mortality was presented.

Table 2: Effects on tick mortality after blood intake in immunized and control hens
mite killing risk ratio in the vaccinated group (1-6) during the experiment period compared to ^a control group 7.
LCI and UCI: lower and upper 95% confidence intervals

Table 3: Summary selection of 10 proteins selected as vaccine antigen candidates is based on proteome discrimination using 2-D electrophoresis with immunoblotting and immunoaffinity protein enrichment. 7 antigens have functions that are estimated based on homology with known proteins of other species, and 3 antigens have unknown functions.

Table 4: Survival analysis of ticks given blood of hens vaccinated with individual antigens The treatments leading to a significant reduction in the survival of ticks are highlighted in pink.

Table 5: Proteomic analysis of IEX group, 2-D gel immune reaction spots

Table 6: Proteomic analysis of IEX group 4, 2-D gel immune reaction spot and immunoaffinity enrichment group 4 protein

References
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2. Van Emous R., 2005. Wage war against the red mite! Poultry Int. 44, 26-33.

3. Valiente Moro C, De Luna CJ, Tod A, Guy JH, Sparagano OA, Zenner L., 2009. The poultry red mite (Dermanyssus gallinae): a potential vector of pathogenic agents.Exp Appl Acarol. 48, 93-104 .

4. Marangi M, Cafiero MA, Capelli G, Camarda A, Sparagano OA, Giangaspero A., 2009. Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility to some acaricides in field populations from Italy.Exp Appl Acarol 48, 11-18.

5.Guy JH, Khajavi M, Hlalel MM, Sparagano O., 2004, Red mite (Dermanyssus gallinae) prevalence in laying units in Northern England, Br.Poult.Sci., 45 Suppl 1, S15-S16.

6. Willadsen P., 2004. Anti-tick vaccines. Parasitology 129 (Supplement); S367-S387.

7. de la Fuente J. and Kocan KM., 2003. Advances in the identification and characterization of protective antigens for recombinant vaccines against tick infestations. Expert Rev Vaccines 2, 583-593.

8. Bartley, K., Nisbet, AJ, Offer, J., Sparks, N., Wright, H, Huntley, JF, 2009. Histamine Release Factor from Dermanyssus gallinae (De Geer): Characterisation and in vitro assessment as a protective Int. J. Parasitol. 39, 447-456 doi: 10.1016 / j.ijpara.2008.09.006

9. Wright, HW, Bartley, K., Nisbet, AJ, McDevitt, R., Sparks, N., Brocklehurst, S., Huntley, JF, 2009. The testing of antibodies raised against poultry red mite antigens in an in vitro feeding assay; preliminary screen for vaccine candidates. Exp Appl Acarol 48, 81-91.

10. Harrington D, Canales M, de la Fuente J, de Luna C, Robinson K, Guy J, Sparagano O., 2009. Immunisation with recombinant proteins subolesin and Bm86 for the control of Dermanyssus gallinae in poultry.Vaccine. 27; 4056 -4063.

11. Harrington D, Din HM, Guy J, Robinson K, Sparagano O., 2009. Characterization of the immune response of domestic fowl following immunization with proteins extracted from Dermanyssus gallinae. Vet Parasitol 160: 285-294.

12. Ellis S, Matthews JB, Shaw DJ, Paterson S, McWilliam HE, Inglis NF, Nisbet AJ. (2014). Ovine IgA-reactive proteins from Teladorsagia circumcincta infective larvae. Int J Parasitol. 44, 743-50.

13. Bartley K, Deane D, Percival A, Dry IR, Grant DM, Inglis NF, Mclean K, Manson ED, Imrie LH, Haig DM, Lankester F, Russell GC. 2014. Identification of immuno-reactive capsid proteins of malignant catarrhal fever viruses. Vet Microbiol. 17; 173 (1-2): 17-26.

14. Bartley K, Huntley JF, Wright HW, Nath M, Nisbet AJ. 2012. Assessment of cathepsin D and L-like proteinases of poultry red mite, Dermanyssus gallinae (De Geer), as potential vaccine antigens. Parasitology. 139, 755 -65.

Claims (18)

One or more Dermanyssus gallinae (D. Galinae) antigens used to elicit an immune response in an avian species, wherein the one or more D. Galinae antigens are:
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
The antigen selected from the group consisting of (x) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x) .   2. One or more D. gallinae antigens according to claim 1, for use according to claim 1, wherein the immune response is a protective response.   The use according to claim 1, for use according to claim 1, wherein the avian species is selected from the group consisting of domesticated or hunting bird species, chicken, pheasant, grouse, turkey, guinea fowl and duck species. One or more D. Galinae antigens.   One or more D. Galinae antigens according to claim 1, for use according to claim 1, wherein the avian species is Gallus gallus domesticus. A composition, immunogenic composition or vaccine comprising one or more Dermanius Galinae (D. Galinae) antigens selected from the group consisting of:
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
(X) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x).   A composition, immunogenic composition or vaccine comprising Serpin (Deg-SRP-1) or an immunogenic fragment thereof of Dermanius Galinae (D. Galinae) for use in raising an immune response in an animal. (I) Serpin (Deg-SRP-1) or an immunogenic fragment thereof of (D) Galinae (D. Galinae); (ii) Hemlipoglycoprotein (Deg-HGP-1) or an immunogenic fragment thereof; (iii) Used to elicit an immune response in avian species, including vitellogenin (Deg-VIT-1) or an immunogenic fragment thereof; and (iv) a protein of unknown function 1 (Deg-PUF-1) or an immunogenic fragment thereof A composition, an immunogenic composition or a vaccine.   The composition, immunogenic composition or vaccine further comprises one or more additional selected from the group consisting of (i) an adjuvant, (ii) another polypeptide, and (iii) a diluent and / or excipient. A composition, immunogenic composition or vaccine according to claim 5, 6 or 7 for use according to claim 5, 6 or 7, comprising (I) the serpin (Deg-SRP-1) is at least partially encoded by SEQ ID NO: 1 and / or comprises SEQ ID NO: 2;
(Ii) the heme lipoglycoprotein (Deg-HGP-1) is at least partially encoded by SEQ ID NO: 3 and / or comprises SEQ ID NO: 4;
(Iii) vitellogenin (Deg-VIT-1) is at least partially encoded by SEQ ID NO: 5 and / or comprises SEQ ID NO: 6;
(Iv) the protein of unknown function 1 (Deg-PUF-1) is at least partially encoded by SEQ ID NO: 7 and / or comprises SEQ ID NO: 8;
(V) aspartyl proteinase / catespin D (Deg-ASP-1) is at least partially encoded by SEQ ID NO: 9 and / or comprises SEQ ID NO: 10;
(Vi) protein of unknown function 2 (Deg-PUF-2): variant 1 and / or 2 is at least partly encoded by SEQ ID NO: 11/12 and / or SEQ ID NO: 13/14 Including;
(Vii) the serpin (Deg-SRP-2) is at least partially encoded by SEQ ID NO: 15 and / or comprises SEQ ID NO: 16;
(Viii) the peptidase C1A cysteine proteinase (Deg-CPR-1) is at least partially encoded by SEQ ID NO: 17 and / or comprises SEQ ID NO: 18;
(Ix) the phosphoglycerate dehydrogenase (Deg-GPD-1) is at least partially encoded by SEQ ID NO: 19 and / or comprises SEQ ID NO: 20;
(X) A protein of unknown function 3 (Deg-PUF-3) is at least partially encoded by SEQ ID NO: 21 and / or comprises SEQ ID NO: 22 Antigens, compositions, immunogenic compositions or vaccines for the described uses.   10. The composition according to any of claims 1 to 9, wherein the composition, immunogenic composition or vaccine is intended to be administered prophylactically in order to prevent colonization of D. Galinae infection / spread in avian species. 10. A composition, immunogenic composition or vaccine according to any of claims 1 to 9 for use. A method of probing a sample for the presence of one or more D. Galinae antigens or an antibody capable of binding to said method, said method comprising:
A sample and (a) one or more D. Galinae antigens selected from the group consisting of (i)-(xi) below:
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
(X) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x), or (b) above (a) ( i)-(a) contacting with one or more antibodies capable of binding to one or more of the D. Galinae antigens of (xi),
The method wherein the sample and antibody / antigen are contacted under conditions that allow binding between any antibody and / or antigen.   12. The method of claim 11, wherein the sample is contacted with an immobilized antibody / plural antibodies and / or a plurality of antigens.   13. A method according to claim 11 or 12, wherein the presence of one or more D. gallinae antigens or one antibody capable of binding to said is confirmed by detection of an antibody-antigen complex. An isolated or recombinant nucleic acid encoding a sequence selected from the group consisting of:
(A) any one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or 21;
(B) any sequence of (a) and at least 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98% or 99% homologous or identical sequences; and (c) at least 30%, 40%, 50%, 60%, 65 with a fragment of any sequence of (a) encoding an immunogenic antigen Sequences that are%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous or identical.   15. The isolated or recombinant nucleic acid according to claim 14, which is used for raising an immune response in birds. A method of eliciting an immune response in an avian species, said method comprising one or more Delmanius Galinae (D. Galinae) antigens selected from the group consisting of Said method comprising the step of administering to the species:
(I) Serpin (Deg-SRP-1);
(Ii) heme lipoglycoprotein (Deg-HGP-1);
(Iii) vitellogenin (Deg-VIT-1);
(Iv) protein of unknown function 1 (Deg-PUF-1);
(V) aspartyl proteinase / catespin D (Deg-ASP-1);
(Vi) Protein of unknown function 2 (Deg-PUF-2): Variant 1 and / or 2;
(Vii) Serpin (Deg-SRP-2);
(Viii) peptidase C1A cysteine proteinase (Deg-CPR-1);
(Ix) phosphoglycerate dehydrogenase (Deg-GPD-1);
(X) a protein of unknown function 3 (Deg-PUF-3); and (xi) an immunogenic fragment, variant or derivative of any one of (i) to (x).   11. The use according to any one of claims 1 to 10 for use according to any one of claims 1 to 10, wherein the composition, immunogenic composition or vaccine is contained within a multivalent vaccine. Composition, immunogenic composition or vaccine.   18. A composition, immunogenic composition or vaccine according to claim 17, for use according to claim 17, wherein the multivalent vaccine comprises an antigen against other avian diseases and / or a vaccine listed in Table 1. .