EP3270956A1

EP3270956A1 - Ovine vaccine

Info

Publication number: EP3270956A1
Application number: EP16713019.4A
Authority: EP
Inventors: Alasdair Justice NISBET; Stewart Thomas George BURGESS; John Frederick HUNTLEY
Original assignee: Moredun Research Institute
Current assignee: Moredun Research Institute
Priority date: 2015-03-18
Filing date: 2016-03-18
Publication date: 2018-01-24
Also published as: WO2016147008A1; GB201504579D0

Abstract

The present disclosure is based upon the identification of a number of antigens derived from species of the genus Psoroptes, which can be used to raise immune responses in animals – particularly those animals susceptible or predisposed to infection/infestation by (or with) one or more Psoroptes species. The antigens provided may be exploited to provide compositions for raising protective immune responses in animals and as vaccines.

Description

OVINE VACCINE

FIELD OF THE INVENTION

The present invention provides a cohort of P. ovis antigens and compositions, vaccines, methods and uses comprising/exploiting the same. The antigens, compositions, vaccines, methods and uses described herein may be exploited as a means to raise immune responses, for example protective immune responses, in animals, for example ovine animals.

BACKGROUND OF THE INVENTION

Infestation of the skin with Astigmatid mites results in morbidity and mortality in humans and production animals worldwide. Psoroptic mange, or sheep scab, caused by infestation of sheep skin with the mite Psoroptes ovis, is highly contagious, causes intense pruritus and is a major welfare and economic concern [1] [2]. Currently, disease control relies on chemotherapy; however issues such as chemical residues, eco-toxicity and acaricide resistance have raised concerns regarding the sustainability of this strategy and novel means of control are therefore desperately needed [3]. The concept of controlling sheep scab by vaccination is supported by the demonstration of a degree of protective immunity in sheep following previous infestation with P. ovis [4-6]: During primary infestation an initial 'lag phase', with small numbers of mites and tight, focal lesions, is followed by a more rapid 'growth phase', with increasing mite numbers, expanding lesions and increasing serum anti- . ovis IgG levels [from ~2 weeks post-infestation (wpi)]. During subsequent infestations an extended 'lag phase', with lower mite numbers, reduced lesion size and a more rapid induction of mite-specific IgE antibodies (peaking within 1 wpi,) are characteristic [4, 5].

Partial protection of rabbits against lesions induced by P. cuniculi (believed to be conspecific with P. ovis) [7] and partial protection of cattle against P. ovis [8] have been induced by immunisation with extracts of these mites . Previously, vaccination of sheep with fractionated extracts of P. ovis resulted in a 13 -fold reduction in mite numbers with lesion growth limited to less than a third of that seen in control sheep following parasite challenge [9]. However, sub-fractionation of these extracts failed to identify the major immunogenic components involved and the practicality of a vaccine based on native P. ovis fractions is limited due to a failure to successfully culture P. ovis in vitro, meaning that native antigen extracts could be prohibitively expensive to produce.

SUMMARY OF THE INVENTION The present invention is based upon the identification of a number of antigens derived from species of the genus Psoroptes, which can be used to raise immune responses in animals - particularly those animals susceptible or predisposed to infection/infestation by (or with) one or more Psoroptes species.

The antigens provided by this invention may be exploited to provide compositions for raising protective immune responses in animals and as vaccines (which may also raise protective immune responses).

In a first aspect, the present invention provides one or more Psoroptes antigen(s) or a fragment(s) thereof, for use in raising an immune response in an animal.

As stated, the inventors have discovered that animals administered the Psoroptes antigens of this invention and exposed to the Psoroptes pathogen exhibit reduced lesion size and pathogen burden (mite numbers). Without wishing to be bound by theory, it is suggested that the antigens described herein elicit immune responses which offer at least some protection against the symptoms of psoroptic mange (caused by Psoroptes species and characterised by the development of lesions which, in untreated animals, may extend to cover almost the entire body). The raised immune response(s) may also be at least partly effective in reducing the pathogen burden - that is, the overall number of mites in any given infection/infestation.

An immune response which contributes to an animal's ability to resolve an infection/infestation and/or which helps reduce the symptoms associated with an infection/infestation may be a referred to as a "protective response". In the context of this invention, the immune responses raised through exploitation of the antigens described herein may be referred to as "protective" immune responses. The term "protective" immune response may embrace any immune response which: (i) facilitates or effects a reduction in host pathogen burden; (ii) reduces one or more of the effects or symptoms of an infection/infestation; and/or (iii) prevents, reduces or limits the occurrence of further (subsequent/secondary) infections.

Thus a protective immune response may prevent an animal from becoming infected/infested with a particular pathogen and/or from developing a particular disease or condition. In the context of this invention, a protective immune response may prevent an animal from becoming infected/infested with Psoroptes ovis (a Psoroptes species responsible for psoroptic mange); limit the extent of a Psoroptes ovis infection/infestation; and/or may prevent an animal from developing psoroptic mange and/or one or more symptoms associated therewith.

In the case of animals infected with Psoroptes parasites, a protective immune response elicited through use of the antigen(s) described herein may result in a reduction in the pathogen burden and/or lesion size.

One of skill would appreciate that any reduction in pathogen burden and/or lesion size achieved through use of the antigen(s) described herein, may be assessed by comparison to the pathogen burden/lesion size observed in an infected animal not exposed to (or administered) the antigen(s) provided by this invention.

A second aspect of this invention provides a composition, an immunogenic composition and/or a vaccine composition comprising one or more of the Psoroptes antigens described herein. The composition, immunogenic composition and/or vaccine composition may be for use in raising an immune response in an animal. As stated, the raised immune response may be a protective immune response.

In a third aspect, the invention provides the use of one or more Psoroptes antigens or a fragment(s) thereof, for the manufacture of a medicament for use in the treatment and/or prevention of psoroptic mange and/or an infection/infestation/colonisation by/with a Psoroptes pathogen.

In a fourth aspect, the invention provides a method of raising an immune response, for example an anti- Psoroptes immune response in an animal, said method comprising the step of administering to an animal, an amount of one or more Psoroptes antigen(s) or fragment(s) thereof, sufficient to induce an immune response or anti- Psoroptes immune response.

Advantageously, the one or more Psoroptes antigens (or fragments thereof) are derived from Psoroptes ovis. Additionally, references to a Psoroptes "species" or "pathogen" may relate to Psoroptes ovis

The term "animal" encompasses animals collectively known as ovine animals. As such, the invention provides antigens and compositions for use in raising immune responses in ovine subjects such as, for example sheep and goats. The various antigens, compositions, methods and uses of this invention may also find application in other animals including any that are recognised as hosts for Psoroptes mites, including the Psoroptes ovis mite. Such animals might include, for example, cattle, horses, rabbits and camelids.

This invention provides: (i) one or more antigens derived from Psoroptes ovis;

(ϋ) compositions (including immunogenic and vaccine compositions) and medicaments comprising one or more antigens derived from Psoroptes ovis; and

(iii) methods exploiting one or more antigens derived from Psoroptes ovis; for use in raising immune responses in ovine animals (including sheep and/or goats).

e present invention may relate to one or more of the following P. ovis Cathepsin L;

(ϋ) mu class glutathione S transferase (muGST);

(iii) Pso o 1

(iv) Pso o 2:

(v) Pso o 3

(vi) Pso o 10; and

(vii) Cyclophilin.

An exemplary P. ovis cathepsin L encoding sequence is deposited under the accession number BQ834906.1. A useful P. ovis cathepsin L antigen is at least partly encoded by the nucleic acid sequence given below as SEQ ID NO: 1.

SEP ID NO: 1

ATGGCCGGGAATAAAAAGCTGCTCGTGGCCAAAAAAATTTTACATTATCAGTAAATCAAT TTGCTGATCTTATGCATCATGAATTTGTCAATATTATGAATGGATATAAATATGATCCGA ATAGACGTTTGAAAAATGGTGCCAGTTTATTTTTAAGTCCTCATAATATTAATGTACCTG AAGAAGTTGACTGGCGTAAACATGATCTTGTTACACCAGTCAAAAATCAAGGACGTTGTG GTTCATGTTGGTCATTTAGTGCTACTGGTGCATTAGAAGGACAACATGCTCGTAAAACAG GCAAATTAGTTTCATTGAGTGAACAAAATCTTGTTGATTGTTCAGCAAAATATGGTAATA ATGGTTGTAATGGTGGCCTCATGGATTATGCATTCCAATATATTAAATCCAATCATGGTA TTGATACTGAACAAAGTTATCCATATGAAGGAAAAGTTGGTAAATGTCGTTACACACAAC GTGCAGTCGGTGCTACTGATACTGGTTTTGTTGATATACCTGAAGGTAATGAAGAAAAAT TGAAAGAAGCTATCGCTACTGTTGGTCCAGTATCGATTGCAATCGATGCATCACAACCAA CATTTCAATTCTATAATGATGGTGTTTATGATGAACCTGATTGTTCATCAACTGAATTAG ATCATGGTGTATTGGCTGTTGGTTATGGTACTGATGAAGAAACTGGTCAAGATTATTGGT TA An exemplary P. ovis muGST sequence is deposited under the accession number AM991140.1. A useful P. ovis muGST antigen is at least partly encoded by the nucleic acid sequence given below as SEQ ID NO: 2.

SEP ID NO: 2

atggacacta aaccggtgct tggttactgg gatgctcgag gtcttggtca agccatacga ctactgttaa catatgctga tgtcgatttc attgataaac gttataaagt gggaccagcc ccaaattatg atcgttcaga atggttaaac gataaatata atcttggttt ggattttcca aattgcccat attatttaga tggcaatgtg cgattgaccc aatcgatagc aattttacgt tatattgccc gcaaatataa tttaaatggt aacaatgaaa tggaaaaaat acgcatttca ttagctgaac aacaaatagt cgacttgaat caagcattgt ctcgtgtggc atataatcct aattgcgaaa aattgaagcc agaatatctg gaaaaattgc cagaacaatt ggaattattg tcgaaatttc taaacaatca accattcata tcgggtacaa acatatcata tgctgatttt ttattgtatg aatatctgac taaaatcaaa gtgttgatgc cagaattgtt tgttaaaaat gataatctta aaaaatttca cgaacgtatc gaagcattac caagaatcaa tgaatatatc aagaaacagc aaccgaaatc gtttcatggc ccaacagcat tgtggaatgg tcaatacgct taa

The P. ovis muGST antigen comprises the amino acid sequence provided below as SEQ ID NO: 3.

SEP ID NO: 3:

MDTKPVLGYWDARGLGQAIRLLLTYADVDFIDKRYKVGPAPNYDRSEWLNDKYNLGLDFPNCPYYL DGNVRLTQSIAILRYIARKYNLNGNNEMEKIRISLAEQQIVDLNQALSRVAYNPNCEKLKPEYLEK LPEQLELLSKFLNNQPFI SGTNISYADFLLYEYLTKIKVLMPELFVKNDNLKKFHERIEALPRINEY IKKQQPKSFHGPTALWNGQYA

An exemplary P. ovis Pso o 1 sequence is deposited under the accession number AM269885.1. A useful Pso o 1 antigen is at least partly encoded by the nucleic acid sequence given below as SEQ ID NO: 4.

SEP ID NO: 4:

atgaaatttg ttttggccat cgcctcattg ttggtgttga gcgttgttta cgcttaccca tcagaaatca gaacattcga agaattcaaa aaagcattca acaaacacta tgtaacacct gaagcagaac aagaagctcg tcaaaatttc ttagcttcat tggaacacat tgaaaaagct ggaaaaggtc gcatcaatca attctcagat atgtcattgg aagaattcaa aaaccaatat ttgatgagtg atcaagcata cgaagcttta aaaaaagaat ttgatttaga tgctggagct caagcttgcc aaatcggcgc cgtaaacatt ccaaatgaaa ttgatttacg tgctttgggt tatgtaacaa aaatcaagaa tcaagttgcc tgtggttcat gctgggcttt ctctggtgtt gctacagtcg aatcaaatta tttatcatac gataatgtat cattagatct ttctgaacaa gaattggttg actgtgcttc acaacacggt tgcggtggtg atacagttct aaacggttta cgttatattc aaaagaatgg tgtggttgaa gagcaaagct atccatacaa agctcgcgaa ggacgatgcc aaagaccaaa tgctaaacga tacggtatca aagatttatg tcaaatttat ccaccaaacg gtgataaaat ccgtacctat cttgcaacaa aacaagcagc cctttcagtc atcattggta tccgagattt agattctttc cgtcattatg atggccgaac aattttgcaa agtgataatg gtggcaaacg aaatttccat gcaatcaata ttgttggtta cggatcaaaa caaggcgttc gatattggat tatacgaaac agttgggaca caacttgggg tgataaaggc tatggctatt ttgttgctga taaaaacttg atgggtatcg aaaaattccc attggctgca atgctttga

The P. ovis Pso o 1 antigen comprises the amino acid sequence provided below as SEQ ID NO: 5.

SEP ID NO: 5:

MKFVLAIASLLVLSWYAYPSEIRTFEEFKKAFNKHYVTPEAEQEARQNFLASLEHIEKAGKGRINQFS DMSLEEFKNQYLMSDQAYEALKKEFDLDAGAQACQIGAVNIPNEIDLRALGYVTKIKNQVACGSCWAFS GVATVESNYLSYDNVSLDLSEQELVDCASQHGCGGDTVLNGLRYIQKNGVVEEQSYPYKAREGRCQRPN AKRYGIKDLCQIYPPNGDKIRTYLATKQAALSVIIGIRDLDSFRHYDGRTILQSDNGGKRNFHAINIVG YGSKQGVRYWIIRNSWDTTWGDKGYGYFVADKNLMGIEKFPLAAML

An exemplary P. ovis Pso o 2 sequence is deposited under the accession number AF187083.1. A useful Pso o 2 antigen is at least partly encoded by the nucleic acid sequence given below as SEQ ID NO: 6.

SEP ID NO: 6

cctcgcggcc tcgtcgaccc caattaaaac taaaaaataa ttttaaaaaa atcaaaatga tgaaaacttt ggtagttctc gccatcactt tggctgttgt atcagctggs aargtcaart tycaagactg tggaaaaggr gaagttgaat ctcttgaagt tgaaggctgt tcaggtgatt actgcgtcat tcacaaaggt aaaaaacttg atttagccat cagtgtaaca tcgaaccaag attcagccaa tttgaaactc gatattgttg ccgatatcaa cggtgtacaa attgaagttc ctggcgttga tcatgatggt tgccattacg tcaaatgtcc aatcaagaaa ggccaacact ttgacgtcaa atacacatac agcattccag caatcttgcc aactaccaaa gctaaaatca ttgctaaaat tattggtgat aaaggtcttg gtggttgtat cgtaatcaat ggtgaaattc aagactaaat caataaaaac ctaaaaatat tttgatgaaa ttagatttgt tatttttatt tctcatttta ttcaaaatta aaaagtattc agtcgacgag gccgcgag

The P. ovis Pso o 2 antigen comprises the amino acid sequence provided below as SEQ ID NO: 7.

SEP ID NO: 7 MMKTLVVLAITLAVVSAGKVKFQDCGKGEVESLEVEGCSGDYCVIHKGKKLDLAISVTSNQDSANLKLD IVADINGVQIEVPGVDHDGCHYVKCPIKKGQHFDVKYTYSIPAILPTTKAKI IAKI IGDKGLGGCIVIN GEIQD

An exemplary P. ovis Pso o 3 antigen comprises the amino acid sequence provided below as SEQ ID NO: 8 (please note, the signal peptide portion is highlighted as bold/underline characters).

SEQ ID NO: 8

MTSTQLAIIITTLLAINNTNAYRMFPLPPLPPDADIVGGI IAPNGSCPYQISLQSGDEHFCGGTILDEY WILTAAHCVYRRSLDGLSIRYDSLTHASGGEKIKVSKIFIHENFNLWMADNDIALLRLQKPMKLGQKNA NSIPLPKSNSDVQSNQKLRVSGWGYLGENSLKLPEKLRLVDIDVVSRNECADLYRNVNAI ITDNMICGG NVTDGGVDSCGGDSGGPVIDLKSKTIVGIVSWGLGCARKHYPGVYTRVGNYIDWIESKRLQ

An exemplary P. ovis Pso o 10 antigen sequence is deposited under the accession number AMI 14276.1. A useful Pso o 10 antigen is at least partly encoded by the nucleic acid sequence given below as SEQ ID NO: 9.

SEP ID NO: 9

atggaggcca tcaagaagaa aatgcaggca atgaagctcg aaaaagataa tgctatcgat cgagctgaaa ttgccgaaca aaaagcccgt gatgcaaatt tacgtgctga aaaatccgaa gaagaggtcc gcggattaca gaaaaagatt caacaaattg aaaatgaatt ggatcaagta caagaacaat tatcagcagc aaacaccaaa ttggaagaaa aaaaaaaagc tttacagacc gccgaaggtg atgtagccgc attgaatcgt cgtattcaat tgattgaaga agatttggaa cgttccgaag aacgactcaa gattgctacg gctaaattgg aagaagcatc gcaatcggcc gatgaatctg aacgtatgcg taaaatgctt gaacatcgtt caatcaccga tgaagaacgt atggatggct tagaaaatca actcaaagaa gcccgtatga tggctgaaga tgctgaccga aaatatgatg aagttgcacg taaattggca atggttgaag ctgatttaga gcgtgctgaa gaacgtgccg aaaccggtga atccaaaatc gtcgaactcg aagaagaatt acgtgttgtt ggcaataatc ttaaatcatt ggaagtgagc gaagaaaaag cacaacaacg tgaagaagca catgaacaac aaattcgtat tatgacagct aaacttaaag aagctgaagc acgtgccgaa tttgccgaac gttcagtaca aaaacttcaa aaagaagtcg atcgattgga agatgaattg gtacatgaaa aagaaaaata taaatcaatt tcagatgaat tggaccaaac atttgccgaa cttactggat attaa

The P. ovis Pso o 10 antigen comprises the amino acid sequence provided below as SEQ ID NO: 10.

SEP ID NO: 10

MEAIKKKMQAMKLEKDNAIDRAEIAEQKARDANLRAEKSEEEVRGLQKKIQQIENELDQVQEQLSAANT KLEEKKKALQTAEGDVAALNRRIQLIEEDLERSEERLKIATAKLEEASQSADESERMRKMLEHRSITDE ERMDGLENQLKEARMMAE DADRKYDEVARKLAMVEADLERAEERAE TGE SKIVE LEEE LRWGNNLKS L EVSEEKAQQREEAHEQQI RIMTAKLKEAEARAEFAERSVQKLQKEVDRLE DE LVHEKEKYKS I S DE LDQ TFAE LTGY

An exemplary P. ovis cyclophilin antigen sequence is deposited under the accession number AAP03083.1. A useful P. ovis cyclophilin antigen comprises the amino acid sequence provided as SEQ ID NO: 11 below.

SEP ID NO: 11

AL STGEKGFGYKGSCFHRI I PGFMCQGGDF TRHNGTGGKS IYGEKF DDENF I LKHTGPGI L SMANAGPN TNGSQFF I CTAKTEWL DGKHWFGKVKEGMNIVEAMERFGSRN

SEQ ID NOS: 1-11 above (and any fragments, variants or derivatives thereof as described herein), may be regarded as reference sequences - against which sequences of the various immunogenic fragments, variants and derivatives described herein may be compared. Additionally or alternatively, a "reference sequence" may be any of the wild-type sequences of any of the P. ovis antigens described herein.

It should be understood that all references to "antigen" or "Psoroptes antigen" encompass the whole or native antigens described herein; antigens comprising or encoded by any of SEQ ID NOS: 1-11 and immunogenic/antigenic fragments, variants, recombinant forms and/or derivatives of any of these.

An immunogenic/antigenic fragment may be any P. ovis antigen fragment (for example a fragment of any of the P. ovis antigens described herein) capable of eliciting an immune response when administered to an animal - in particular an ovine animal. An "immune response" may be regarded as any response which elicits antibody (for example IgA, IgM and/or IgG or any other relevant isotype) responses and/or cytokine or cell mediated immune responses. For example, the various P. ovis antigen fragments provided by this invention may be capable of eliciting an immune response which is substantially identical or similar to, an immune response elicited by the complete antigen from which the fragment is derived. In one embodiment, the antigen fragments provided by this invention are capable of providing protective immune responses in ovine animals.

One of skill will appreciate that the P. ovis antigens of this invention define one or more epitopes and as such, the term "antigen" may also embrace proteins or peptides which comprise one or more Psoroptes antigen epitopes. Further, the term "antigen" embraces recombinant forms of any of the antigens described herein. The term "antigen" also includes recombinantly prepared immunogenic fragments of any of the Psoroptes antigens of this invention.

In other embodiments, the term "antigen" or "antigen fragment" may encompass variants or derivatives of any of the antigen(s) described herein - such antigens being referred to as "variant" or "derivative" antigens. Again, it should be understood that these terms include variants/derivatives of any of the P. ovis antigens of this invention or comprising/encoded by, any of SEQ ID NOS: 1-11. The skilled man would understand that any variant or derivative antigen may also be immunogenic/antigenic in that it elicits an immune response which is similar or substantially identical to an immune response elicited by the corresponding complete or native antigen in the same host - such variants/derivatives may be referred to as "immunogenic variants/derivatives". An immunogenic variant/derivative may comprise or be encoded by, a protein/peptide sequence or nucleic acid or amino acid sequence which comprises one or more nucleobase and/or amino acid substitutions, inversions, additions and/or deletions relative to a reference sequence (for example sequences of or encoding the P. ovis antigens of this invention).

One of skill will appreciate that the term "substitution" may encompass one or more conservative substitution(s). One of skill in this field will understand that the term "conservative substitution" is intended to embrace the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physico-chemical properties and/or structure or function of the native (or wild type) protein.

In the context of this invention, a variant/derivative antigen may comprise or be encoded by a mutant sequence which when compared to a reference sequence (such as for example a wild type sequence (including sequences encoding any of the specific P. ovis antigens given as (i)-(viii) above) or antigens comprising or encoded by any of SEQ ID NOS: 1-11 (or fragments thereof) above), is found to contain one or more amino acid/nucleotide substitutions, additions, deletions and/or inversions.

An antigen which may be regarded as a derivative may further comprise one or more features of a fragment or variant described herein optionally in combination with one or more modifications to the structure of the antigen or one or more of the amino acid residues thereof. In addition to being immunogenic (as described above), useful fragments, variants, mutants and/or derivatives may comprise anything from about 5 to about 10 residues (amino acids and/or nucleic acids) of the complete amino acid or nucleic acid sequence (n) of (or encoding) the relevant complete wild-type or native Psoroptes ovis antigen, to about n-1 residues. In certain embodiments, the fragments, variants and/or derivatives provided by this invention comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300 residues - the upper limit (n-1) depending upon the size (n) of the nucleic acid encoding the complete antigen or the number (n) of amino acid residues comprising the primary sequence of the antigen.

Additionally, or alternatively, the antigenic fragments, variants and/or derivatives provided by this invention comprise sequences or are at least partially encoded by sequences, which are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous or identical to the various reference (exemplary) sequences provided herein.

The degree of (or percentage) "homology" between two or more (amino acid or nucleic acid) sequences may be determined by aligning two or more sequences and determining the number of aligned residues which are identical or which are not identical but which differ by redundant nucleotide substitutions (the redundant nucleotide substitution having no effect upon the amino acid encoded by a particular codon, or conservative amino acid substitutions.

A degree (or percentage) "identity" between two or more (amino acid or nucleic acid) sequences may also be determined by aligning the sequences and ascertaining the number of exact residue matches between the aligned sequences and dividing this number by the number of total residues compared - multiplying the resultant figure by 100 would yield the percentage identity between the sequences.

Thus, this invention relates to:

(i) one or more of the P. ovis antigens selected from the group consisting of (i)-(vii) above;

(ii) antigens comprising or at least partially encoded by one or more of the sequences provided as SEQ ID NOS : 1 - 11 ;

(iii) immunogenic fragments, variants or derivatives of any antigen provided by (i) or (ii) above; and/or

(iv) recombinant forms of any of the antigens provided by (i)-(iii) above; for raising immune responses in animals - in particular ovine animals such as sheep and goats.

The invention provides compositions, immunogenic compositions and vaccines comprising:

(i) one or more of the P. ovis antigens provided as (i)-(vii) above;

(ii) antigens comprising or (at least partly) encoded by any of the sequences provided as SEQ ID NOS: 1-11;

(iii) immunogenic fragments, variants or derivatives of any antigen provided by (i) or (ii); and/or

(iv) recombinant forms of any of the antigens provided by (i)-(iii);

for use in raising immune responses (for example protective immune responses).

The various methods and uses described herein may exploit one or more of the P. ovis antigens. For example, the invention may exploit combinations of the antigens described herein. The compositions, immunogenic compositions and vaccines of this invention may, for example, comprise two, three, four, five, six or all seven of the P. ovis antigens described herein. Additionally, or alternatively, the various uses and methods of this invention may exploit one or more of the P. ovis antigens; for example, the methods/uses may exploit combinations (for example two, three, four, five, six or all seven) of the antigens described herein.

Where a composition comprises more than one of the P. ovis antigens of this invention, such compositions may be referred to as multi-component compositions.

Each antigen of a multi-component composition of this invention may be provided at a defined, predetermined and/or specific concentration/amount. Each antigen may be provided at the same or a different amount. For example, anywhere between about 1 μg and 100 g of any given antigen may be used in the various compositions, vaccines, methods and uses of this invention. For example, about 5 μg, about 10 μg, about 15 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg or about 90 μg of any given antigen may be used. For example, a composition comprising all seven of the P. ovis antigens of this invention may comprise about 50 μg of each of the relevant antigens. Thus, a composition or vaccine comprising seven P. ovis antigens, may comprise about 350 μg of antigen. It should be noted that the term "about" refers to the stated amount +/- 1, 2, 3, 4, 5, 6, 7, 8 θΓ 9 μ_β. A composition or vaccine of this invention may be formulated as a sterile composition and may comprise one or more excipients or diluents -for example one or more pharmaceutically acceptable excipients or diluents.

The compositions and vaccines of this invention may be formulated for oral, topical (including dermal and sublingual), intramammary, parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and/or mucosal administration.

A method of raising an immune response in an animal according to this invention may exploit one or more multi-component compositions. For example, one composition may comprise one set of antigens to be administered and one or more other compositions may comprise other P. ovis antigens for administration.

Thus, the invention provides multi-component compositions, multi-component immunogenic compositions and multi-component vaccines for use in raising an immune response in an animal, the various compositions and/or vaccines comprising, consisting or consisting essentially of, two, three, four, five, six or each (i.e. all seven) of the following P. ovis antigens:

(i) Cathepsin L;

(ϋ) muGST;

(iii) Pso o 1 ;

(iv) Pso o 2;

(v) Pso o 3;

(vi) Pso o 10; and

(vii) Cyclophilin.

Compositions, immunogenic compositions and vaccines comprising, consisting essentially of or consisting of each of the seven P. ovis antigens listed above (and at least partially encoded by or comprising any of the sequences provided by SEQ ID NOS: 1-11) may find particular application in methods of raising immune responses in animals, in particular ovine animals and in methods of raising immune responses protective (or effective) against psoroptic mange and the causative pathogen, P. ovis.

Where multiple different compositions, immunogenic compositions and vaccines are to be administered to an animal as part of a protocol to raise (protective) immune responses, those various compositions may have the same or different formulations. Together, the various compositions may comprise, consist essentially of or consist of all seven of the P. ovis antigens of this invention such that once all of the compositions have been administered, the animal has been exposed to all of the P. ovis antigens.

The various compositions, immunogenic compositions, vaccines, uses and methods of this invention may exploit one or more adjuvant components. For example, the various compositions and vaccines of this invention may further comprise an adjuvant, for example, QuilA.

The inventors have discovered that animals (in particular sheep) administered a number (for example all seven of the P. ovis antigens of this invention) develop an immune response which confers a level of protection which reduces (a) the number of lesions and/or the overall rate of lesion development (by about 50-60%) and (b) a significant (about 50-60%) reduction in the number of mites at the leading edge of the sheep scab lesion; both (a) and (b) being determined by comparison with animals infected with P. ovis but not exposed to any of the antigens of this invention.

The antigens, composition and vaccines of this invention may reduce the mean lesion size in infected animals by about 10%-90%, 15%-85%, 20%-80%, 25%-75% or 30%-70%. For example, the mean lesion size in an animal administered an antigen(s), composition or vaccine of this invention may be 50-60% less than the mean lesion size in untreated animals. Similarly, the mean mite count may be 10%-90%, 15%- 85%, 20%-80%, 25%-75%, 30%-70% or 50%-60% lower in animals administered an antigen(s), composition or vaccine of this invention.

As stated, P. ovis antigens (or indeed any immunogenic fragments thereof) to be exploited in this invention may be obtained using recombinant technology. In one embodiment, an expression vector comprising one or more nucleic acid sequences encoding a suitable P. ovis antigen (such as any of those described herein) may be used to produce one or more recombinant P. ovis antigens for use in raising immune responses in animals - particularly ovine animals.

In view of the above, the invention further provides vectors, for example expression vectors, comprising nucleic acid sequence(s) encoding one or more of the P. ovis antigens described herein (or fragments thereof). By way of example, the vectors provided by this invention may comprise plasmid expression systems such as those known as pET, pPICZ, pSUMO and/or pGST. Vectors according to this invention may otherwise be referred to as "nucleic acid constructs".

In a further aspect, the present invention provides host cells transfected or transformed with a vector as described herein. Eukaryotic or prokaryotic cells, such as, for example plant, insect, mammalian, fungal and/or bacterial cells, may be transfected with one or more of the vectors described herein. One of skill in this field will be familiar with the techniques used to introduce heterologous or foreign nucleic acid sequences, such as expression vectors, into cells and these may include, for example, heat-shock treatment, use of one or more chemicals (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information regarding transformation/transfection techniques may be found in Current Protocols in Molecular Biology, Ausuble, F.M., ea., John Wiley & Sons, N.Y. (1989) which is incorporated herein by reference.

A suitable host cell may be a bacterial cell such as, for example, an Escherichia coli cell.

The present invention further provides a process for the production of a recombinant P. ovis antigen encoded by any of the sequences described herein (or an immunogenic fragment thereof), which recombinant antigen (or immunogenic fragment thereof) is for use in raising an immune response in an animal (for example an ovine), said method comprising the step of (a) transforming a host cell with a nucleic acid sequence according to this invention (e.g. a nucleic acid encoding all or part of a P. ovis antigen) or transfecting a host cell with a nucleic acid construct of the invention; (b) culturing the cells obtained in (a) under conditions in which expression of the nucleic acid (or rather a protein encoded thereby) takes place; and (c) isolating the expressed recombinant protein or peptide from the cell culture and/or the culture supernatant.

Recombinant proteins/peptides produced according to the method described above may be partially purified from the host cell before being used in an immunogenic composition or vaccine. Where the polypeptide is secreted from the host cell, the cells may be separated from the media by centrifugation. In such a situation, the supernatant, which contains the secreted polypeptide, may be used directly as a vaccine, or in a vaccine composition. Alternatively, the polypeptide may be partially purified from this supernatant, for example using affinity chromatography.

Thus, the invention provides uses, methods, compositions and vaccines exploiting or comprising, consisting or consisting essentially of, each of the following recombinantly prepared P. ovis antigens: (i) Cathepsin L;

(ϋ) muGST;

(iii) Pso o 1 ;

(iv) Pso o 2;

(v) Pso o 3;

(vi) Pso o 10; and

(vii) Cyclophilin.

for use in raising an immune response in an animal (for example an ovine species - including sheep and goats).

In one embodiment, any of the P. ovis antigens described herein may be admixed with another component, such as another polypeptide and/or an adjuvant, diluent or excipient. In one embodiment, the vaccine compositions provided by this invention may comprise a QuilA adjuvant. Additionally, or alternatively, compositions or vaccines provided by this invention may, for example, contain viral, fungal, bacterial or other parasite antigens used to control other diseases/infections or infestations. For example, a composition or vaccine composition of this invention may be included within a multivalent/component vaccine, which includes antigens against other ovine (for example, sheep) diseases.

In a still further aspect, the present invention provides an ovine population, for example a farmed population of sheep and/or goats, administered, treated, vaccinated or immunised with one or more of the antigens described herein and/or a composition or vaccine composition of this invention.

The vaccines described in this invention may take the form of subunit-type vaccines whereby one or more of the P. ovis antigens disclosed herein (for example all seven) are used to inoculate an animal. Additionally or alternatively, a composition, immunogenic composition or vaccine of this invention may comprise a nucleic acid molecule (known as a DNA vaccine), which nucleic acid encodes one or more of the described P. ovis antigens or immunogenic fragments thereof, to be expressed by the cells of an animal to be vaccinated. In this way, constitutive expression of one or more of the P. ovis antigens or any immunogenic fragment thereof, in a host (such as, for example a vaccinated ovine subject (sheep or goat)) may elicit a constitutive protective immune response.

The (vaccine) compositions described herein, may comprise a discrete dosage unit and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association one or more of the P. ovis antigens described herein with liquid carriers or finely divided solid carriers or both.

Compositions (the term "composition" including vaccine compositions), suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of one or more of the P. ovis antigens of this invention. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound (for example one or more P. ovis antigen(s)) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water liquid emulsion.

Compositions suitable for oral administration include controlled release dosage forms, e.g., tablets wherein an active compound (for example one or more P. ovis antigens) is formulated in an appropriate release-controlling matrix, or is coated with a suitable release-controlling film. Such compositions may be particularly convenient for prophylactic use.

Composition and vaccine compositions formulated for parenteral administration include sterile solutions or suspensions of an active compound (for example one or more P. ovis antigens) in aqueous or oleaginous vehicles.

Injectable compositions and vaccines may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use. Alternatively, an active compound (for example one or more P. ovis antigens) may be in powder form that is constituted with a suitable vehicle, such as sterile, pyrogen-free water or PBS before use.

Compositions comprising one or more P. ovis antigens may also be formulated as long-acting depot preparations, which may be administered by intramuscular injection or by implantation, e.g., subcutaneously or intramuscularly. Depot preparations may include, for example, suitable polymeric or hydrophobic materials, or ion-exchange resins. They may also include preparations or adjuvants known to enhance the affinity and/or longevity of an animal (for example ovine) immune response, such as single or double emulsions of oil in water. Such long-acting compositions are particularly convenient for prophylactic use.

Compositions suitable (or formulated) for mucosal administration include compositions comprising particles for aerosol dispersion, or dispensed in drinking water. When dispensed such compositions should desirably have a particle diameter in the range 10 to 200 microns to enable retention in, for example, the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable compositions include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension.

It should be understood that in addition to the carrier ingredients mentioned above, the various compositions described herein may include, an appropriate one or more additional (pharmaceutically acceptable) carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient.

Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.

Compositions suitable for topical formulation may be provided for example as gels, creams or ointments.

Compositions for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water-soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus, particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) (for example one or more P. ovis antigens) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to, for example, animal feed - perhaps by way of an intermediate premix, or diluted in animal drinking water.

Liquid concentrates of this invention suitably contain one or more P. ovis antigens and may optionally further include an acceptable water-miscible solvent for veterinary use, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals.

In general, a suitable dose of each the P. ovis antigens provided by this invention may be in the range of about 10 to about 100 μg protein per animal. Furthermore, the one or more antigens described herein may be administered on about 1 to about 5, for example 2, 3 or 4 occasions over a period of about 1 to about 10 weeks (for example, 2, 3, 4, 5, 6, 7, 8 or 9 weeks) or on an annual boost basis. In one embodiment, each animal may be administered about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg of each (or a predetermined selection of) the one or more antigens described herein. As such, where the vaccine comprises seven antigens, the total protein content may range from about 70 μg to about 700 μg. Typically a useful composition or vaccine may contain a total protein (antigen) content of about 350 μg (i.e. 50 μg per antigen).

Furthermore, each animal may be administered the antigen(s) on 2, 3, 4 or 5 occasions over a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 week period. It should be understood that each animal may receive the same or a different dose of the P. ovis antigen(s) on each administration occasion.

In one embodiment, a vaccine formulated for administration to sheep may comprise approximately 5(^g of each P. ovis antigen. As such, where the vaccine comprises, for example, seven P. ovis antigens, the total protein (antigen) content may be in the region of 35(^g. Further, the vaccine may comprise an adjuvant and may be administered on three occasions, with a two week gap between each administration.

In addition to providing P. ovis antigens for use in raising immune responses in animals, the present invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have affinity or specificity for) any of the P. ovis antigens provided by this invention. Production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example "Basic methods in Antibody production and characterisation" Howard & Bethell, 2000, Taylor & Francis Ltd. Such antibodies may be used in diagnostic procedures, to for example, detect or diagnose P. ovis infection/infestations in animal (for example ovine) species, as well as for passive immunisation.

DETAILED DESCRIPTION

The present invention will now be described in detail with reference to the following Figures which show:

Figure 1: Lesion development over a 6 week period post-infestation with P. ovis across repeated vaccine trials. Lambs were infested with -50 mites following immunisation with a seven recombinant protein cocktail vaccine with QuilA adjuvant (vaccine) or adjuvant only (control). Data on lesion size are presented on transformed scale (square root of lesion size, cm²). Plot shows observed lesion size of each lamb of vaccine (triangles) and control (circles) groups, estimated mean lesion size of vaccine (solid line) and control (dashed line) groups and corresponding 95% CIs envelop (shaded region).

Figure 2: Mite numbers at the leading edge of the sheep scab lesion over a 6 week period post-infestation with P. ovis across repeated vaccine trials. Data on mite number are presented as log of mite number per log strip length (cm). Plot shows observed mite number on lambs of vaccine (triangles) and control (circles) groups accompanied with boxplots presenting summary statistics of the observed data, and estimated mean mite number on the log scale (square) and corresponding 95% CIs (error bar) for vaccine and control groups during both trials.

Figure 3: Antigen-specific antibody (IgG) levels in serum over the entire experimental period (pre- and post-infestation with P. ovis). Serum IgG responses specific for cathepsin L; Pso o 10; muGST; Pso o 1; Pso o 2, Pso o 3 and cyclophilin, respectively over the entire experimental period for Trial 1 (A) and Trial 2 (B). Data on IgG levels are presented on observed scale (OD₄₅o_nm)- Plot shows observed IgG levels of each lamb of the vaccine (triangles) and control (circles) groups, estimated mean IgG level of vaccine (solid line) and control (dashed line) groups and corresponding 95% CIs envelope (shaded region).

Summary

A 'rational approach' to recombinant sub-unit vaccine design has been adopted, [10- 14] elucidating host signalling pathways involved in the initial cutaneous proinflammatory response to P. ovis, upon which the mite relies to initiate its feeding and survival and to then use this information to identify the mite factors that may trigger these pathways. By targeting these mite-derived factors via immunisation, it is hypothesised that mite survival may be inhibited. Due to the complex nature of the host:parasite interaction in sheep scab, a successful vaccine may need to target multiple mite antigens, simultaneously influencing a number of host pathways. Similar approaches have been used in the development of a vaccine against Plasmodium falciparum, which targets multiple alleles of the apical membrane antigen 1 (AMAl) and also a multi-antigen vaccine for the human hookworm Necator americanus [15, 16].

Here we employed a recombinant sub-unit cocktail vaccine, employing seven P. ovis proteins, four of which were identified using this 'rational approach' (Pso o 1; Pso o 2; Pso o 3 and cyclophilin) with three additional antigens identified as homologues of known allergens (Pso o 10); proteins upregulated during feeding (cathepsin L) or by immuno- screening of P. ovis cDNA libraries (mu class GST) [10- 14, 17-22]. These antigens were combined as a sub-unit cocktail vaccine and efficacy tested in sheep using a previously developed P. ovis challenge model.

Materials and Methods Recombinant protein production

The sub-unit cocktail vaccine was composed of seven recombinant proteins as described in Table 1 [14, 18-24]. Pso o 1, Pso o 10, cyclophilin and the mu class glutathione-S-transferase (muGST) were soluble in phosphate buffered saline (PBS), whilst Pso o 2, Pso o 3 and Cathepsin L were insoluble in PBS and were instead formulated in Dialysis Buffer (DB) [20mM sodium phosphate, 0.5M NaCl, 2M urea pH7.4]. Insoluble proteins were expressed and purified as described previously [24] and then dialysed in DB for 24h at room temperature. Protein concentration was measured using a modified BCA protein assay (Pierce, UK) with BSA standards made up in DB. Pso o 10, cyclophilin and the muGST were induced and purified as above in the absence of urea and concentrations determined by BCA assay following dialysis against PBS. Pso o 1 was expressed in Pichia pastoris, strain X-33 by induction with methanol as described previously [21]. Following dialysis against PBS, Pso o 1 concentration was determined by BCA assay. After purification, all antigens were stored at A ° , except for Pso o 1, which was stored at -20 ° .

Immunisation and challenge protocols

Trial 1

Thirty sheep scab-naive Texel crossbred lambs (~6 months old) were randomly allocated into two equal-sized groups (vaccine and control) housed in separate pens. Lambs in the vaccine group were immunized on three occasions two weeks apart with 350μg of recombinant protein cocktail (50μg each of the 7 P. ovis antigens) plus QuilA adjuvant (Brenntag Biosector). PBS-soluble proteins were administered in PBS together with 5mg QuilA as a single sub-cutaneous (s.c.) injection into the lateral neck region, whilst insoluble proteins were administered via s.c. injection into the opposite side of the neck in DB with 5mg QuilA. Lambs in the adjuvant-only control group were immunized at the same time and via the same route as the vaccine group with equivalent volumes of PBS, DB and QuilA. Two weeks after the final immunisation all lambs were infested, between the withers, with -50 mixed-stage P. ovis mites. Lesion development was assessed weekly. Blood samples were collected from all lambs immediately prior to each injection, 1 day pre-infestation and then weekly throughout a six-week infestation period. At post mortem (pm) three skin strips (5cm x 1cm) at the leading edge of the lesion were removed from each lamb for enumeration of mites. Trial 2

Trial 2 was identical to Trial 1, with two exceptions: 10 lambs per group were used and 5 skin strips were taken at pm. Both trials were performed under the regulations of the UK Animal Procedures Act (1986) and a UK Home Office Project License. Experimental design and statistical power calculations were performed by Biomathematics and Statistics Scotland (BioSS) and were approved by the Moredun Research Institute Experiments and Ethics Committee.

Lesion size assessment

Single lesions were observed in all lambs, radiating outwards from the initial site of infestation. The lesion area was measured weekly following infestation by multiplying the length and width of the lesion at the broadest point.

Assessment of mite numbers

Mite numbers were estimated by counting parasites on skin strips from the leading edge of each lesion at pm and expressed as mites per cm of skin strip. In addition, an estimate of the total number of mites at the leading edge of the lesion was determined by multiplying the mite count value by the total lesion perimeter for each animal [estimated as 2xlesion length (cm) + 2xlesion width (cm)].

Serological analysis of antibody responses to individual vaccine antigens

Recombinant antigen-specific IgG levels in serum were assessed for all antigens by ELISA as described previously [24] with the following exceptions: ELISA for Pso o 3 used a horse-radish peroxidase (HRP) -conjugate of polyclonal antibodies raised in pig against sheep IgG (Dako, UK). ELISA for Pso o 2 was as described in [24] but the antigen was diluted in ddt^O rather than sodium carbonate buffer (pH 9.6). The responses for each antigen were assessed for each sample in triplicate. OD450nm values were corrected against a reagent blank (no sample control) and all plates incorporated positive (pooled 6 wpi sera) and negative (pre-bleed from sheep scab naive lambs) serum controls to account for inter- and intra-plate variation.

Statistical Analysis

Lesion size (cm²) measurements were square root transformed and mean difference between treatment groups across different time points assessed using a linear random coefficients model incorporating fixed effects of trial, treatment group, time and the interaction of treatment/time. The model considered random intercept and time- specific slope for each lamb, allowing the intercept and slope to vary for each lamb. Mite count data was assumed to follow a Poisson distribution and modelled using a generalised linear mixed model (GLMM). Fixed effects included treatment group, trial and the interaction of treatment/trial, the random effect of lamb was also included and a dispersion parameter was estimated to account for overdispersion in mite count data. The data for each antibody titre during the 12-week experimental period (including pre and post-infestation) was square root transformed, the transformed data was then analysed by an additive linear mixed model. The model included treatment group (vaccine and control) trial (2012 and 2013) and interaction effect of treatment group and trial as fixed effects. The model incorporated separate smoothing curves for the non-linear relationship of the antibody response with time by the treatment group. The model also considered a first-order autoregressive correlation structure between observations at 13 time points within the same animal. Heterogeneity in variance for each year was allowed in the model. Model selection was based on the Akaike's information criterion (AIC); optimal mixed models were fitted by residual maximum likelihood (REML). All models incorporated continuous variables as a deviation from the overall population mean. The overall statistical significance of a fixed effect was assessed from the corresponding p-value estimated from the -statistic. If the F test for an effect exceeded the critical value at the upper 5% level, it was considered as statistically significant (p < 0.05). Pertinent estimates from the models are given in the text together with standard errors. All statistical analyses were carried out using R software version 3.0.1 [26].

Results

Lesion size

There was no evidence that mean lesion size differed between trials, for both control and vaccinated lambs (p=0.641) therefore, the final model excluded the trial effect, with data from both trials combined in the subsequent analysis. Figure 1 presents the estimated mean lesion size (square root transformation, cm and corresponding 95% confidence intervals (CIs)) for vaccine and control groups along with observed lesion size for each animal at each wpi (across both trials). The mean lesion size increased over time for both groups, but the lesion size (rate of increase in the slope) in the control group (8.08+0.36 (sqrt (lesion size (cm)/time (wpi))) was statistically significantly (/?<0.001) higher than that of the vaccine group (5.20+0.36) and mean lesion size in the vaccine group was smaller compared with the control group during the entire experimental period (Figure 1). Considering the untransformed data, mean lesion size (95% CIs) in the vaccine and control groups were 52.68 (39.22, 68.13) and 106.46 (86.90, 128.00) cm², respectively at 1 wpi, increasing to 1105.72 (900.77, 1331.64) and 2574.47 (2256.23, 2913.69) cm² for the vaccine and control groups at 6 wpi, respectively. Based on these measurements, lambs in the vaccine group showed, on average, a >57% reduction in lesion size by 6 wpi compared with the control group, and a maximum reduction of 63% in lesion size at 3 wpi.

Mite numbers

The mean mite counts in both vaccinated and control groups in Trial 2 were statistically significantly higher than those in Trial 1 (p<0.001), but in both trials the vaccine group had statistically significantly lower mean mite counts per unit of log strip length at pm compared with the control group (Figure 2, /?=0.002). Estimated mean (95% lower, upper CI) mite counts per cm of skin strip for the vaccine group were 8 (6, 10) and 17 (13, 21) during Trial 1 and 2, respectively. In the control group mite counts were 12 (9, 15) and 26 (20, 34) in the two trials respectively. Accounting for the increased mean lesion perimeter at the leading edge of the lesion for the control group (202.80 cm) compared with the vaccine group (138.56 cm) across both trials, the estimated total mean (95% lower and upper CIs) mite numbers for the vaccine and control groups for Trial 1 were 1055 (835, 1333) and 2414 (1915, 3048), respectively. Similar estimates for Trial 2 were 2292 (1767, 2976) and 5251 (4045, 6811), respectively. Thus, across both trials the vaccinated lambs showed on average a >56% reduction in total mite numbers at the leading edge of the lesion compared with the control group.

Measurement of serum antibody (IgG) responses to recombinant P. ovis antigens

The model demonstrated a significant group effect for all 7 antigens and a trial effect for the following antigens: Pso o 2, Pso o 3 and cyclophilin; with non-significant trial effects for cathepsin L and Pso o 1. In addition, a significant group and trial interaction was observed for tropomysin and muGST. Figure 3 presents estimates of the mean antibody responses to all seven P. ovis recombinant antigens and corresponding 95% CIs for vaccine and control groups at weekly intervals across the entire experimental period (both pre- and post- infestation) for Trials 1 (2012 - Figure 3A) and 2 (2013 - Figure 3B) along with the observed antibody responses (OD450nm) for each individual. Encouragingly, the vaccinated animals all generated an IgG antibody response to the seven vaccine antigens. The estimated mean antibody levels and corresponding CIs showed strong evidence that the vaccine group had statistically significantly higher antibody levels compared with the control group during the entire experimental period. This response peaked 7-14 days after the final immunisation, with serum antibody levels then slowly declining.

Discussion

The data presented here demonstrate the efficacy of a recombinant subunit sheep scab vaccine based on a cocktail of seven recombinant P. ovis antigens. When administered subcutaneously to lambs, the vaccine resulted in highly significant reductions in both lesion size (>57%) and mite numbers (>56%) following challenge in repeated protection trials. The lesions in the immunised lambs were significantly smaller from 1 wpi until the end of the experiment at 6 wpi. In sheep scab, where disease is transmitted via direct contact or via fomites, even modest decreases in both lesion size and mite numbers may limit disease spread substantially [27-30]. Serum IgG responses were observed to all of the vaccine antigens prior to infestation in the vaccinates and no significant responses were observed to any of the antigens in the controls during this period.

The experimental challenge in the current model is highly likely to be much more severe than in a field outbreak, as the numbers of mites used in these trials (-50 mites per lamb) will usually be substantially higher than those commonly experienced during a natural infestation, where only small numbers of mites, or even a single ovigerous individual may be sufficient to establish a lesion [31-33]. Additionally, as a result of the limited numbers of mites encountered in a natural challenge, field infestations may develop more slowly over a longer period of time encompassing several months rather than the 6 week infestation described herein [31]. Hence the current infestation model may actually underestimate the field efficacy of this prototype sheep scab vaccine. Based on the estimates of slopes in conjunction with the line plots of lesion size for both vaccine and control groups presented here (Figure 1), it may be inferred that the lesion size would increase progressively with time, beyond the period investigated in the current experiment, and hence, the difference in lesion size between control and vaccine groups will become more pronounced at later time points. Therefore, the ultimate efficacy of this sheep scab vaccine may actually be greater than demonstrated in this study, however further studies in field conditions will be required to validate this hypothesis.

The subunit vaccine described herein represents the greatest reduction in lesion size with a recombinant sheep scab vaccine to date and provides real encouragement for future production of a commercially viable means of immunoprophylaxis. Previous attempts have been made to produce an effective vaccine for sheep scab. For example, Nisbet et al [14] produced a multi-protein recombinant vaccine based on P. ovis allergens, however the efficacy of this vaccine could not be determined due to the high degree of variability in the challenge infestation model. Other efforts have focused on the use of native extracts of P. ovis to generate protective immunity: A vaccine based on P. ovis soluble proteins was previously tested in cattle, with 8 out 14 vaccinated calves being free of palpable lesions by 8 wpi compared to 3/14 in the control group [8]. Unfortunately, native extract based sheep scab vaccines are not likely to be commercially feasible due to the absence of an in vitro culture system for P. ovis to supply sufficient material for commercial production and also the lack of reproducibility with which these extracts can be produced. The use of a cocktail vaccine is likely to be required for controlling complex eukaryotic parasites and may have advantages over single protein based vaccines [34]. Whereas single point mutations in drug targets can lead to drug resistance, this is far less likely in a vaccine relying on multiple epitopes and even less likely in a cocktail vaccine where an array of epitopes are exploited [29, 34]. The existing chemotherapeutics offer broad spectrum control, targeting multiple life cycle stages and in many cases multiple species of parasites [29]. In order for a vaccine to compete commercially it may also need to target different developmental stages of the parasite and, potentially, multiple ecto/endoparasite species [20,22,23]. Mathematical modelling has demonstrated that vaccines for the control of parasites may not need to achieve the same degree of efficacy as current chemical methods of control to achieve economic control of parasites [35-37]. In reality, sterile immunity against most ectoparasites may not be achievable via vaccination and unlike chemotherapeutics, an ectoparasite vaccine may not induce a rapid knockdown of parasite population and may not necessarily protect individuals from being parasitized [30, 37]. However, vaccination does have the potential to provide a longer duration of protection from re- infestation than that currently achievable with chemotherapeutic control, which ranges from low levels of protection with a single dose of Dectomax (Zoetis Ltd, USA) and up to 60 days for 2% Cydectin LA (Zoetis Ltd, USA). However, if used as part of an integrated control program, vaccines may reduce parasite populations over successive generations and, in the short term can mitigate the effects of parasitism by controlling population growth, limiting clinical pathology and alleviating the more extreme welfare symptoms [30]. Vaccination may therefore help to reduce disease impact by blocking or reducing the spread of disease within and between flocks [30]. As such, vaccines should not be considered as a single control measure for sheep scab but rather as an additional arm in a growing arsenal of tools available for coordinated control, i.e. diagnostic tools, existing chemical treatments and effective biosecurity.

However, to encourage producers to begin to switch from their current reliance on chemical control to a more coordinated approach involving anti-parasite vaccines, these products will have to demonstrate clear benefits, i.e. be efficacious, cost effective, environmentally friendly and sustainable [38]. One disadvantage of the cocktail based approach are the additional costs involved in commercial production of a vaccine based on multiple mite proteins and, although this is not necessarily a barrier to commercial success, it is important to ensure that the cost of the vaccine is reflective of the market. This may require further distillation of the number of antigens required for protection, or formulation of protective antigens and/or epitopes within a single fusion protein, as recently demonstrated with an Escherichia coli 0157:H7 subunit vaccine [39] and also by co-expression of multiple copies of a rabies virus glycoprotein using a foot and mouth disease virus expression system incorporating the 2A peptide [40]. It is also critical at this stage to develop effective strategies to use this vaccine in the field. This will require identifying the optimal methods of integrating the vaccine with existing control programmes. For example this may involve the combined use of diagnostic tests [24, 41] to identify and confirm outbreaks of sheep scab, treatment with existing chemotherapeutic compounds and administration of vaccine to neighbouring and surrounding flocks to block further transmission.

Tables

Table 1. Details of the recombinant P. ovis antigens used in the vaccine cocktail.

P. ovis Accession Referen Soluble Molecular Expression system

antigen No. ce in Weight

PBS* (kDa)

Cathepsin BQ834906. [23] No 25kDa E. coli BL21-Codon Plus - pET- L 1 22b(+) muGST AM991140. [14] Yes 25kDa E. coli BL21-Codon Plus - pET- 1 22b(+)

Pso o 1 AM269885. [21] Yes 25kDa P. pastoris-X-33 - pPICZaC

1

Pso o 2 AF187083. [24] No 14kDa E. coli BL21-Codon Plus - pET- 1 22b(+)

Pso o 3 No 25kDa E. coli BL21-Codon Plus - pET- 22b(+)

Pso o 10 AMI 14276. Yes 37kDa E. coli BL21-Codon Plus - pET- 1 22b(+)

Cyclophili AAP03083. Yes 38kDa E. coli BL21-Codon Plus - pET- n 1 SUMO

* Pso o 1, Pso o 10, Cyclophilin and muGST were soluble in PBS, whilst Pso o 2, Pso o 3 and Cathepsin L were formulated in Dialysis Buffer (DB). Predicted molecular weight in kilo Daltons.

^Λ Not yet assigned

^ΛΛ Unpublished data.

$ P. ovis cyclophilin identified as an EST from a P. ovis cDNA library. The following primer sequences were used to amplify full length P. ovis cyclophilin from cDNA derived from mixed stage P. ovis as described in [20] : Cyclophilin-For

5 ' ATGTCAACATGGACCCAAATTCAA' 3 , Cyclophilin-Rev

5 ' TTACATTTC ACC AC ATTGTGATATGAT3 ' . Cyclophilin was subsequently expressed in E. coli, confirmed by matrix assisted laser desorption ionisation mass spectroscopy and its peptidyl prolyl cis-trans isomerase (PPIase) activity confirmed by a coupled enzyme assay as described in [42]. § Pso o 3 identified as a homologue of the house dust mite allergen Der p 3 in an EST from a P. ovis cDNA library. The following primer sequences were used to amplify the coding region of Pso o 3, from cDNA derived from mixed stage P. ovis as described in [20], downstream of the predicted signal peptide sequence, and to allow subcloning into the expression vector (restriction sites underlined) :Pso o 3 -For 5' GATCCGAATTCGGC ATATCGAATGTTTCCACTTCC3 ' . Pso o 3-Rev-5' CCGCAAGCTTTACGATTCCGACAATCGTTTTAC3 ' .

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Claims

1. One or more P. ovis antigens selected from the group consisting

(i) Pso o 1 or an immunogenic fragment thereof;

(ii) Pso o 2 or an immunogenic fragment thereof;

(iii) Pso o 3 or an immunogenic fragment thereof;

(iv) muGST or an immunogenic fragment thereof;

(v) Cathepsin L or an immunogenic fragment thereof;

(vi) Pso o 10 or an immunogenic fragment thereof; and

(vii) Cyclophilin or an immunogenic fragment thereof;

for use in raising an immune response in an animal.

2. The antigen(s) of claim 1 for use of claim 1, wherein the Pso o 1 antigen comprises or is encoded by SEQ ID NOS: 4, 5 or an immunogenic fragment/antigen encoding fragment thereof; the Pso o 2 antigen comprises or is encoded by SEQ ID NOS: 6, 7 or an immunogenic fragment/antigen encoding fragment thereof; the Pso o 3 antigen comprises SEQ ID NO: 8 or an immunogenic fragment thereof; the muGST antigen comprises or is encoded by SEQ ID NOS: 2, 3 or an immunogenic fragment/antigen encoding fragment thereof; the Cathepsin L antigen is encoded by SEQ ID NO: 1 or an immunogenic antigen encoding fragment thereof; the Pso o 10 antigen comprises or is encoded by SEQ ID NOS: 9, 10 or an immunogenic fragment/antigen encoding fragment thereof; and the Cyclophilin antigen comprises SEQ ID NO: 11 or an immunogenic fragment thereof.

3. The antigen(s) of claim 1, for use of claim 1 wherein the antigen(s) comprise or are at least partially encoded by one or more of the sequences given as SEQ ID

NOS: 4, 5, 6, 7, 1-3 and 8-11.

4. The antigen(s) of any preceding claim for use of any preceding claim, wherein the antigen(s) is/are recombinant antigen(s)

5. The one or more antigens of any preceding claim, for use of any preceding claim, wherein the immune response is a protective immune response.

6. The one or more antigens of any preceding claim, for use of any preceding claim, wherein the animal is an ovine animal, a bovine animal or cattle.

7. The one or more antigens of any preceding claim, for use of any preceding claim wherein the use comprise two, three, four, five, six or seven of the antigens listed as (i)-(vii) above.

8. Pso o 1 or an immunogenic fragment thereof; Pso o 2 or an immunogenic fragment thereof; Pso o 3 or an immunogenic fragment thereof; muGST or an immunogenic fragment thereof; Cathepsin L or an immunogenic fragment thereof; Pso o 10 or an immunogenic fragment thereof; and Cyclophilin or an immunogenic fragment thereof:

for use in raising an immune response in an animal.

9. P. ovis antigen Pso o 1 or an immunogenic fragment thereof for use in raising an immune response in an animal.

10. P. ovis antigen Pso o 1 of claim 9, for use of claim 9, wherein the use further comprises one or more P. ovis antigens selected from the group consisting of:

(i) Pso o 2 or an immunogenic fragment thereof;

(ii) Pso o 3 or an immunogenic fragment thereof;

(iii) muGST or an immunogenic fragment thereof;

(iv) Cathepsin L or an immunogenic fragment thereof;

(iv) Pso o 10 or an immunogenic fragment thereof; and

(vi) Cyclophilin or an immunogenic fragment thereof;

11. P. ovis antigens Pso o 1 or an immunogenic fragment thereof and Pso 2 or an immunogenic fragment thereof, for use in raising an immune response in an animal.

12. An immunogenic composition or a vaccine comprising one or more P. ovis antigens selected from the group consisting of:

(i) Pso o 1 or an immunogenic fragment thereof;

(ii) Pso o 2 or an immunogenic fragment thereof;

(iii) Pso o 3 or an immunogenic fragment thereof; (iv) muGST or an immunogenic fragment thereof;

(v) Cathepsin L or an immunogenic fragment thereof;

(vi) Pso o 10 or an immunogenic fragment thereof; and

(vii) Cyclophilin or an immunogenic fragment thereof.

13. The immunogenic composition or vaccine of claim 9, wherein the Pso o 1 antigen comprises or is encoded by SEQ ID NOS: 4, 5 or an immunogenic fragment/antigen encoding fragment thereof; the Pso o 2 antigen comprises or is encoded by SEQ ID NOS: 6, 7 or an immunogenic fragment/antigen encoding fragment thereof; the Pso o 3 antigen comprises SEQ ID NO: 8 or an immunogenic fragment thereof; the muGST antigen comprises or is encoded by SEQ ID NOS: 2, 3 or an immunogenic fragment/antigen encoding fragment thereof; the Cathepsin L antigen is encoded by SEQ ID NO: 1 or an immunogenic antigen encoding fragment thereof; the Pso o 10 antigen comprises or is encoded by SEQ ID NOS: 9, 10 or an immunogenic fragment/antigen encoding fragment thereof; and the Cyclophilin antigen comprises SEQ ID NO: 11 or an immunogenic fragment thereof.

14. The immunogenic composition of claim 9 or 10, wherein the antigens comprise or are at least partially encoded by one or more of the sequences given as SEQ ID NOS: 4, 5, 6, 7, 1-3 and 8-11.

15. The immunogenic composition of claim 9, 10 or 11, wherein the antigen(s) is/are recombinant antigen(s).

16. The immunogenic composition of any one of claims 9, 10, 11 or 12, wherein the immunogenic composition or vaccine comprises two, three, four, five, six or seven of the antigens listed as 9(i)-9(vii) above.

17. The immunogenic composition or vaccine according to claim 9, 10, 11, 12 or 13 wherein the immunogenic composition or vaccine comprises one or more additional components selected from the group consisting of:

(i) an adjuvant;

(ii) other viral, fungal and/or other parasite antigens; and

(iii) excipients and/or diluents.

18. The immunogenic composition of vaccine according to any one of claims 9- 14, wherein the composition or vaccine is formulated for parenteral administration.

19. The immunogenic composition or vaccine according to any one of claims 9- 15, wherein the composition or vaccine is intended to be administered subcutaneously.

20. The immunogenic composition or vaccine according to any one of claims 9- 16, wherein the, or each, antigen is individually present in an amount of about 1 μg to about 100 μg.

21. The immunogenic composition or vaccine of any one of claims 9-17, for use in raising an immune response in an animal.

22. The immunogenic composition or vaccine of claim 18, for use of claim 18, wherein the immune response is a protective immune response.

23. The immunogenic composition or vaccine of claim 18 or 19, for use of claim 18 or 19, wherein the animal is an ovine animal, a bovine animal or cattle.

24. The immunogenic composition or vaccine of any one of claims 9-17, for use in a method of treating or preventing a Psoroptes ovis infection.

25. The immunogenic composition or vaccine of claim 21, for use of claim 21 wherein the method of treating or preventing a Psoroptes ovis infection treats or prevents psoroptic mange and/or one or more symptoms associated therewith.

26. The immunogenic composition or vaccine of claim 21 or 22, for use of claim 21 or 22, wherein the method of treating or preventing a Psoroptes ovis infection results in a reduced parasite burden and/or lesion size.

27. The immunogenic composition or vaccine of claim 21-23, for use of claim 21- 23, wherein the method of treating or preventing a Psoroptes ovis infection comprises administering an animal one or more doses of an immunogenic composition or vaccine according to claims 9-17.

28. An immunogenic composition or a vaccine comprising P. ovis antigen Pso o 1 or an immunogenic fragment thereof.

29. The immunogenic composition or vaccine of claim 28, further comprising one or more P. ovis antigens selected from the group consisting of:

(i) Pso o 2 or an immunogenic fragment thereof;

(ii) Pso o 3 or an immunogenic fragment thereof;

(iii) muGST or an immunogenic fragment thereof;

(iv) Cathepsin L or an immunogenic fragment thereof;

(v) Pso o 10 or an immunogenic fragment thereof; and

(vi) Cyclophilin or an immunogenic fragment thereof.

30. An immunogenic composition or vaccine comprising P. ovis antigen Pso o 1 or an immunogenic fragment thereof and p. ovis antigen Pso o 2 or an immunogenic fragment thereof.

31. A method of raising an anti-Psoroptes immune response in an animal, said method comprising the step of administering to an animal, an amount of one or more Psoroptes antigen(s) sufficient to induce an anti- Psoroptes immune response, wherein the Psoroptes antigen(s) are selected from the group consisting of:

(i) Pso o 1 or an immunogenic fragment thereof;

(ii) Pso o 2 or an immunogenic fragment thereof;

(iii) Pso o 3 or an immunogenic fragment thereof;

(iv) muGST or an immunogenic fragment thereof;

(v) Cathepsin L or an immunogenic fragment thereof;

(vi) Pso o 10 or an immunogenic fragment thereof; and

(vii) Cyclophilin or an immunogenic fragment thereof.