NZ723022B2 - Sheep nematode vaccine - Google Patents

Abstract

Discloses a method of raising an immune response in a non-human animal, the method comprising administering said animal Teladorsagia circumcincta antigen calcium-dependent apyrase-1 or an immunogenic fragment thereof.

Description

ation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT SHEEP NEMATODE VACCINE FIELD OF THE INVENTION The present invention relates to nematode antigens capable of g host immune responses. In particular, the invention provides vaccines for use in protecting t and/or reducing instances of Teladorsagia infections.

OUND OF THE ION Teladorsagia circumcincta (previously known as Ostertagia circumcincta) is the major cause of parasitic gastroenteritis in small ruminants in ate regions.

This nematode is controlled primarily by anthelmintics; however resistance is widespread and field es have often been found to be insensitive to a number of different anthelmintic classes (Bartley et al., 2004; Wrigley et al., 2006). T. cincta resides within the abomasum (or true stomach) of small nts and primarily causes disease in animals during their first year of grazing. It is a major cause of production losses, estimated to cost the UK sheep industry alone in excess of £80 M per annum (Nieuwhof & Bishop, 2005). The associated clinical signs range from suppressed appetite to diarrhoea, dehydration and death; however, the major impact of teladorsagiosis is its effect on lamb productivity via a reduction in weight gain (Gibson and Everett, 1976).

Protective immunity against challenge with T. circumcincta develops after continual (‘trickle’) infection over a number of weeks (Seaton et al., 1989). The degree of immunity that develops depends on a number of factors including, level of parasite challenge, age of animal and its genotype (Singleton et al., 2011). In ewes that have acquired immunity to T. cincta, resistance to the tes can lapse around the time of ition and early lactation (Houdijk et al., 2005). In terms of anti-parasite effects, the protective immune response has been shown to se the establishment of larvae in the abomasal mucosa, slow larval development in the gastric gland and to reduce the egg output of female worms in the abomasal lumen (Balic et al., 2003; Seaton et al., 1989; Smith et al., 1985, 1986; Stear et al., 2004).

Experiments that demonstrated successful adoptive transfer between immune and naive sheep using gastric lymph indicate the importance of local immune responses in protective mechanisms against T. circumcincta (Smith et al., 1986). The precise mechanisms remain to be defined, but roles for both immediate hypersensitivity reactions and local antigen specific IgA have been highlighted (Smith et al., 1986; 1987). Furthermore, antigen-specific IgA responses have been correlated with reductions in de length day et al., 2007; Smith et al. 2009), whereas IgE responses have been correlated with a reduction in faecal egg counts in grazing lambs (Huntley et al., 2001).

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT As sheep can acquire a protective immune response against T. circumcincta in l and experimental circumstances, vaccination represents a possible alternative for control.

Y OF THE INVENTION The present invention is based upon the identification of a number of antigens derived from species of the genus Teladorsagia, which can be used to raise immune responses in animals – particularly those animals susceptible or predisposed to infection by (or with) one or more Teladorsagia species. The antigens provided by this invention may be exploited to provide compositions and es for raising protective immune responses in animals – the protective immune responses serving to , prevent, treat or eliminate Teladorsagia infections/infestations.

In a first aspect, the t invention es one or more Teladorsagia antigen(s) or a fragment thereof, for use in g an immune response in an animal.

As stated, the inventors have discovered that the immune responses elicited by the Teladorsagia antigens of this invention protect animals against infection/infestation with nematode parasites belonging to the Teladorsagia genus.

An immune response which protects against infection/infestation by/with a pathogen may be a referred to as a “protective response”. In the context of this ion, the term “protective immune response” may e any immune response which facilitates or effects a reduction in host pathogen burden – i.e. the number of pathogenic organisms infecting a host. In other embodiments, and in the case of animals infected with Teladorsagia tes, a protective immune response elicited through use of the antigen(s) described herein may result in a reduction in the host faecal egg count (FEC: , the number of parasite eggs per gramme (EPG) of faeces – occurring as a result of suppression of egg output from female parasites in the abomasal lumen) and/or a decrease in the numbers of parasitic larvae ishing in the abomasal mucosa or a reduction in the numbers of adult worms (male and/or female) residing in the abomasal lumen. A protective immune response may also slow larval development.

One of skill would appreciate that any reduction in pathogen burden/FEC achieved h use of the n(s) bed herein, may be compared to the pathogen burden/FEC of an infected animal not exposed to (or administered) the antigen(s) provided by this invention – such animals being devoid of (or lacking) a protective immune response.

A second aspect of this invention provides a composition or vaccine composition comprising one or more of the Teladorsagia antigens described herein, for use in raising an immune response in an animal. In one embodiment, the immune response is a protective response.

Additionally, or alternatively, the immune response raised in the animal may prevent the occurrence of further (subsequent/secondary) Teladorsagia infections and may also have an effect on the development or survival of co-infecting nematodes of other genera.

In a third aspect, the invention provides a composition or vaccine composition comprising Teladorsagia cincta antigen calcium-dependent e-1 or an immunogenic fragment thereof when used in g an immune response in a non-human animal.

In a fourth aspect, the invention provides a vaccine ition comprising Teladorsagia circumcincta antigen m-dependent apyrase-1, or an immunogenic fragment thereof, and an adjuvant.

In a fifth aspect, the invention provides the use of one or more Teladorsagia antigens or a fragment(s) thereof for the manufacture of a ment for use in the ent and/or prevention of an infection/colonisation h a Teladorsagia pathogen.

In a sixth aspect, the invention provides a method of raising an anti-Teladorsagia immune se in an animal, said method comprising the step of administering to an animal, an amount of one or more Teladorsagia antigen(s) or fragment(s), sufficient to induce an anti-Teladorsagia immune response. ageously, the one or more Teladorsagia antigens (or fragments thereof) are derived from Teladorsagia circumcincta an ovine parasite infecting the abomasum and causing weight loss, diarrhoea and decreased wool production and, in some cases, death.

In a further aspect, the invention provides a method of raising an immune response in a non-human animal, the method sing administering said animal a Teladorsagia circumcincta antigen calcium-dependent apyrase-1 or an genic fragment thereof.

In a r aspect, the present invention provides for the use of Teladorsagia circumcincta antigen calcium-dependent apyrase-1 or an immunogenic fragment thereof for the manufacture of a medicament for raising a protective immune se against a Teladorsagia infection in a non-human animal.

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT The term “antigen” may relate to, for example, Teladorsagia proteins and/or peptides (including polypeptides and short peptide chains of one or more amino acids), glycoproteins and/or glycopeptides. In addition, the term “antigen” may relate to carbohydrate molecules. In one ment, antigens to be exploited in this invention may be ns which are present on the surface of Teladorsagia cells and/or exposed to the host (ovine) immune system during an infection. One of skill will appreciate that the term “antigens” may also encompass Teladorsagia proteins, polypeptides, peptides and/or carbohydrates which are otherwise known as “immunogens”.

In one embodiment, the antigens provided by this invention are antigens, which elicit host dy (for example, IgA and/or IgG) responses. In one embodiment, the ns are derived from post-infective larval stages of Teladorsagia species. The Teladorsagia antigens provided by this invention may include those secreted or excreted by Teladorsagia larvae in the gastric gland milieu during rapid growth phases within the mucosa or by adult worms in the abomasal lumen. In one embodiment, the antigens are derived from third and/or fourth stage Teladorsagia , but may also be secreted by adult stage parasites. Additionally or atively, the Teladorsagia ns described herein may comprise pathogen derived immunomodulatory nds.

In a further embodiment, the term “antigen” encompasses the exemplary T. circumcincta (Tci) antigens listed as (i)-(ix) below: (i) calcium-dependent e-1 (Tci-APY-1). (ii) astacin-like metalloproteinase-1 (Tci-MEP-1). (iii) excretory/secretory protein (unknown function: Tci-ES20). (iv) cathepsin F-1 (Tci-CF-1). (v) transforming growth protein 2-like protein (a TGFβ homologue: Tci- TGH-2). (vi) activation associated secretory protein (Tci-ASP-1). (vii) macrophage migration inhibitory factor (Tci-MIF-1). (viii) surface associated antigen (Tci-SAA-1). (ix) a fragment, mutant, t or derivative of any of (i)-(viii).

An exemplary Tci-SAA-1 ce is deposited under the accession number CAQ43040 and comprises the sequence given below as SEQ ID NO: 1.

SEQ ID NO: 1 mfcrvtvavl llavsahagf fddvsglasd vgdfftkqfn nvkdlfannq selekniqrv kdllmaikek akmlepmand aqkktisevn dafg geak feqnkakwqd ekgg lenvmklmnlk vmaal iapvilaftr [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT ed set by JXT An exemplary Tci-MIF-1 sequence is deposited under the accession number CBI68362 and comprises the ce given below as SEQ ID NO: 2.

SEQ ID NO: 2 Mpvfsfhtnv sadkvtpdll kqissvvari lhkpesyvcv hvvpdqqmif dgtdgpcgvg vlksiggvgg sknnehakal falikdhlgi agnrmyiefi digaadiafn srtfa An exemplary Tci-ASP-1 sequence is deposited under the accession number CBJ15404 and comprises the sequence given below as SEQ ID NO: 3.

SEQ ID NO: 3 mftpigiavl ylalvtphak agfccpadld qtdearkill nfhnevrrdv lnlt nvlg paknmykmdw dcnlekkale mispctvplp idtsipqnla qwllyrkmee tevlekapws wviaslrnlk ndteadlynw kirtisniln wrntkvgcah kvcqfptgtn mviscayggd klennevvwq kgptcecnay pdsyccnnlc dtkaaaalre epcksn An ary H-2 sequence is deposited under the accession number 78 and comprises the sequence given below as SEQ ID NO: 4.

SEQ ID NO: 4 mrllnsmgmq eppnvdsidl spstieemle slgendkleq dqeektfima vdpsdgidpd mlvarfpvsi ttmvrkvsra ylhvylhvse ivtv vvrerllngd tnpv eiqrsgkavl plrasdverw wksepilgly vvamlngeni avhpqqdhha rhtmfmsvil asdaksrgkr spsvcmpedq epgcclydli vdfqqigwkf iiaphkynay mcrgdcsvnh thvtrsghtk vaktgiitrq datgnqgmcc hpaeydavrm iymngdnqvt marvpgmiar kctcs An exemplary -1 sequence is deposited under the accession number ABA01328 and comprises the sequence given below as SEQ ID NO: 5.

SEQ ID NO: 5 llip hlfaatvkqq ysggvkplte dkkt kgsiefarlg qhispkdfga wnhftsfier hdkvyrnese alkrfgifkr nleiirsaqe ndkgtaiygi nqfadlspee fkkthlphtw kqpdhpnriv vdpk eplpesfdwr ehgavtkvkt eghcaacwaf svtgniegqw flakkklvsl saqqlldcdv vdegcnggfp ldaykeivrm gglepedkyp yeakaeqcrl vpsdiavyin gsvelphdee kmrawlvkkg pisigitvdd iqfykggvsr pttcrlssmi hgallvgygv wiik wged rgen acrinrfpts avvl An exemplary Tci-APY-1 sequence is deposited under the accession number CBW38507 and comprises the sequence given below as SEQ ID NO: 6.

SEQ ID NO: 6 mllyilslvl lidalppgyp dgkehgsrpt irslpdgste ykllivtdmd kdskagewtw ravtregrlt lspdmahvsi awdensernl tssmnikgra melsdlsvfh nriltpddrt gliseiknnk mipwvflnsg pgnttspfkc ewmtikddvl yvgghgnefr nkqgeivhrn nlwiktvtpe gevtnvdwtd vfnnlrnavg isepgylthe avqwsekqgh wyflprkesk tvyveeddek kgtdlliign pdldqfetkr igvlrpergy safdfipgtd dkiivalksk [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT tety vtvftidgei llddqkldgn ykfeglyfi An exemplary Tci-MEP-1 sequence is given below as SEQ ID NO: 7.

SEQ ID NO: 7 mrlavlllvlvvsaqaglldkvkdffkggnfgektktatlskfkklfektgilslgnklaemrskvmkk lelskakkaevdrklkeveermdntvenlkdtifeinavknvgeslfqsdilltkrqveevmdgveggr pkrqafkdqnypnttwqqgvfyrfddsadyytrkvfemgtkqweeatcidfkedkekkaensiiliked gcwsyvgqvggeqplslgdgceqvgiathelghalglfhtmsrydrddfitvvlenvvegfvdqyiket pqtttnygftydygsimhygassashnnkptmvandtryqesmgsqiisfidksmindhynckadcpka tsakcqnggfphprkcsecicpsgyggalcdqrptgcgqtlkakeskqflidklgfpsgvrdeftfcnh wieapegkkielkinsishgyahdgcilggveiktsedqtrtgfrfcspndrntvlvsasnrvpiitfn rsgqqqiileykvvs An exemplary Tci-ES20 sequence is given below as SEQ ID NO: 8.

SEQ ID NO: 8 lilvsasvyvsvqgqgngdmkkvelymgyakkdmekvreflklkderltkllsdlfryldktt fewmkdeatleqfiqtrgkfssalvhpdvqkrykdnrklwafryarlmnciggsdmgrataylpgvsvq ekeetlryslklertcaytyfr As such, one ment of this invention provides one or more of the T. circumcincta antigens selected from the group consisting of (i)-(ix) above or comprising one or more of the ces provided as SEQ ID NOS: 1-8 (or a fragment thereof), for raising immune responses in animals – in particular ovine animals such as sheep and goats.

Advantageously, the invention provides vaccine compositions sing one or more of the antigens provided as (i)-(ix) above, or one or more antigens comprising the sequences provided as SEQ ID NOS: 1-8 (or a fragment thereof), for use in raising immune responses (for example protective immune responses) in ovine animals.

SEQ ID NOS: 1-8 above (and any nts, variants or derivatives thereof), may be regarded as reference sequences – against which the sequences of the fragments, variants and derivatives described herein are compared. In other embodiments, the reference sequences may be the wild-type sequences of any of the ns given as (i)-(viii) In on to the definition provided above, the term “antigen” also encompasses fragments of any of the antigens described herein – this includes fragments of the antigens listed as (i)-(viii) above and antigens encoded by sequences comprising parts of SEQ ID NOS: 1-8. In particular, the term “antigen” encompasses nic or immunogenic fragments or epitopes capable of eliciting an immune response in an animal. Advantageously, the n fragments described ation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT herein are 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 ion are capable of providing protective immune responses against T. circumcincta in ovine animals.

In other embodiments, the term “antigen” or “antigen fragment” may encompass variants or derivatives of any of the antigen(s) bed herein – such antigens being referred to as “variant” or ative” antigens. Again, it should be understood that these terms include variants/derivatives of any of the ns given as (i)-(ix) above or encoded by any of SEQ ID NOS: 1-8. Further, the skilled man would understand that any variant or derivative antigen may elicit an immune response in an ovine animal 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 ses one or more nucleobase and/or amino acid substitutions, inversions, additions and/or ons relative to a reference sequence.

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 rvative 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 ties and which does not ntially 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 n 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 Teladorsagia antigens given as (i)-(viii) above) or sequences comprising SEQ ID NOS: 1-8 (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 se one or more es of a fragment or t described herein optionally in combination with one or more modifications to the structure of the antigen or one or more of the amino acid residues thereof.

The fragments, mutants, variants and/or derivatives provided by this invention 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 [Annotation] JXT None set by JXT ation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT encoding) the complete wild-type or native Teladorsagia (for example T. cincta) antigen, to about n-1 residues. In certain embodiments, the fragments, variants and/or tives provided by this invention comprise at least about 10, 15, , 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 n or the number (n) of amino acid residues comprising the primary ce of the antigen.

Additionally, or alternatively, the fragments, variants and/or derivatives ed by this invention are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homologous or cal to the various reference sequences provided herein.

The degree of (or percentage) “homology” n two or more (amino acid or nucleic acid) sequences may be determined by aligning two or more sequences and determining the number of d 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 d by a particular codon, or conservative amino acid substitutions.

A degree (or percentage) ity” between two or more (amino acid or nucleic acid) sequences may also be determined by aligning the ces and ascertaining the number of exact residue matches between the d 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.

In one embodiment, the invention provides multi-component compositions and vaccines for use in raising an immune response in an animal, the vaccine and/or composition comprising, consisting or substantially consisting of, each of the following T. circumcincta antigens: (i) calcium-dependent apyrase-1 (Tci-APY-1); (ii) astacin-like metalloproteinase-1 (Tci-MEP-1); (iii) excretory/secretory protein wn on: Tci-ES20); (iv) cathepsin F-1 (Tci-CF-1); (v) transforming growth protein 2-like protein (a TGFβ homologue: Tci- TGH-2); (vi) activation associated secretory protein (Tci-ASP-1); (vii) macrophage migration inhibitory factor (Tci-MIF-1); and (viii) surface associated antigen (Tci-SAA-1).

In one embodiment, one or more of the T. circumcincta antigens provided as (i)-(viii) above, is/are provided as a fragment or variant/derivative (as defined above).

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT The inventors have discovered that s (in particular sheep) administered a vaccine composition sing eight separate T. circumcincta antigens (for e, the antigens given as (i)-(viii) above) develop an immune response which confers a level of protection which is far higher than that observed following exposure to prior art es and vaccine compositions. For example, the vaccines provided by this invention have been ed to reduce host FECs and luminal parasite burdens by approximately %, 15%-85%, 20%-80%, 25%-75% or 30%-70%.

Without wishing to be bound by theory, the inventors hypothesise that the success of the vaccines and vaccine compositions bed herein is due to the use of antigens which elicit an immune response which mimics that occurring during a natural infection and which serves to prevent or suppress nematode-derived immunomodulation.

Antigens to be exploited in this ion may be obtained using inant technology. In one embodiment, an expression vector sing one or more nucleic acid sequences ng a T. circumcincta antigen (such as any of those described herein) may be used to produce one or more recombinant T. circumcincta antigens for use in raising immune responses in animals – particularly ovine animals.

Protocols for the recombinant preparation of any of the antigens provided by this invention are bed herein – see for example section entitled “Production of recombinant proteins for immunisation”. Nevertheless, one of skill will appreciate that other methods (for example methods utilising different primers and vectors etc.) may also be used.

In view of the above, the ion provides vectors, for example expression vectors, comprising nucleic acid sequence(s) encoding one or more of the T. circumcincta antigens described herein (or fragments thereof). By way of example, the vectors provided by this invention may comprise plasmid sion systems such as those known as pET, pPICZ, pSUMO and/or pGST. Vectors according to this ion 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 ar 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 ent, use of one or more als (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT ation] JXT Unmarked set by JXT regarding transformation/transfection techniques may be found in Current Protocols in lar y, Ausuble, F.M., ea., John Wiley & Sons, N.Y. (1989) which is orated herein by reference.

In one embodiment, the host cell is a bacterial cell such as, for e, an Escherichia coli cell.

In view of the above, the present invention r provides a process for the production of a inant Teladorsagia antigen d 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 a T. circumcincta 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 sed recombinant protein or peptide from the cell culture and/or the culture supernatant.

Recombinant proteins/peptides produced according to the method described above may be partially purified from the host cell before being used in a vaccine or vaccine composition. 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 tography.

In one embodiment, the invention es a composition (for e a vaccine composition) comprising, consisting or substantially consisting of, each of the following inant T. circumcincta antigens: (i) calcium-dependent apyrase-1 (Tci-APY-1); (ii) astacin-like metalloproteinase-1 (Tci-MEP-1); (iii) excretory/secretory protein (unknown on: Tci-ES20); (iv) cathepsin F-1 (Tci-CF-1); (v) transforming growth protein 2-like protein (a TGFβ homologue: Tci- TGH-2); (vi) activation associated secretory protein (Tci-ASP-1); (vii) macrophage migration inhibitory factor (Tci-MIF-1); and (viii) surface associated antigen (Tci-SAA-1); for use in g an immune response in an animal (for example an ovine species – including sheep and goats).

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT In one embodiment, any of the Teladorsagia antigens described herein may be admixed with r component, such as another polypeptide and/or an adjuvant, diluent or excipient. In one embodiment, the vaccine compositions provided by this ion may comprise a QuilA adjuvant. Additionally, or alternatively, vaccines or e itions provided by this invention may, for example, contain viral, fungal, bacterial or other parasite antigens used to control other es/infections or infestations. For example, the vaccine or vaccine composition may be included within a multivalent vaccine, which includes antigens against other ovine (for example, sheep) es.

In a still further , the present invention provides an ovine population, for example a farmed population of sheep and/or goats, treated, vaccinated or sed with a vaccine or composition described herein, said vaccine or composition comprising one or more of the Teladorsagia antigens bed herein.

One of skill will appreciate that the vaccines described in this invention may take the form of subunit-type vaccines whereby one or more Teladorsagia antigens are used to inoculate an animal. Additionally or alternatively, the vaccine may comprise a nucleic acid molecule (known as a DNA e) encoding one or more antigens encoded by SEQ ID NOS: 1-8 above or an immunogenic nt f, to be expressed by the cells of an animal to be vaccinated. In this way, constitutive expression of Teladorsagia antigens in a vaccinated host (such as, for example a vaccinated ovine subject (sheep or goat)) may elicit a constitutive protective immune response.

The compositions, including the vaccine compositions, provided by this invention may be formulated as sterile pharmaceutical compositions comprising one or more of the antigens described herein and a pharmaceutical excipient, carrier or diluent. These composition may be formulated for oral, topical (including dermal and sublingual), parenteral (including subcutaneous, intradermal, intramuscular and intravenous), transdermal and/or mucosal administration.

The (vaccine) compositions described herein, may comprise a te 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 T. circumcincta antigens described herein with liquid carriers or finely divided solid rs or both.

Compositions (the term “composition” including a vaccine compositions), suitable for oral administration n the r is a solid are most ably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of one or more of the Teladorsagia antigens of [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT this invention. A tablet may be made by compression or moulding, ally 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 T. circumcincta n(s)) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surfaceactive 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 nd, 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 nd may also be formulated as dispersible granules, which may for example be ded in water before administration, or sprinkled on food. The es 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 ueous 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 Teladorsagia 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 ons or suspensions of an active compound (for e one or more Teladorsagia antigens) in aqueous or oleaginous vehicles.

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

Compositions comprising one or more Teladorsagia 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 e, 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) [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT ed set by JXT immune response, such as single or double emulsions of oil in water. Such longacting compositions are particularly convenient for prophylactic use.

Compositions suitable (or formulated) for mucosal stration e 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 itions 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 on 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 ients such as diluents, buffers, ring agents, binders, surface active , thickeners, lubricants, preservatives ding 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 ueous solvents are propylene glycol, polyethylene glycol, ble 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 ed 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 le 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 ce, conventional soluble excipients, such as lactose or sucrose, may be orated 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 T. circumcincta [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT ation] JXT Unmarked set by JXT antigens) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary ents. 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 T. circumcincta antigens and may optionally further include an acceptable watermiscible solvent for veterinary use, for example polyethylene , 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 T. circumcincta ns 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 bed herein may be administered on about 2 to about 5 occasions over a period of about 1 to about 10 weeks or on an annual boost basis. In one embodiment, each animal may be administered about 10, , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100µg of each (or a predetermined selection of) the one or more antigens described herein. As such, where the vaccine comprises 8 antigens, the total protein content may range from about 80µg to about 800µg. Furthermore, each animal may be stered the antigen(s) on 2, 3, 4 or 5 ons 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 T. circumcincta antigen(s) on each administration occasion.

In one embodiment, a vaccine formulated for administration to sheep may comprise approximately 50µg of each Teladorsagia (for example T. circumcincta) antigen. As such, where the e comprises, for example, 8 T. circumcincta antigens, the total n (antigen) content may be in the region of 400µg. Further, the vaccine may be administered three times with a three week gap n each administration.

In addition to providing T. circumcincta antigens for use in raising immune responses in animals, the t invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have ty or specificity for) any of the Teladorsagia antigens provided by this invention.

Production and ion of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example “Basic s in Antibody production and characterisation” Howard & Bethell, 2000, Taylor & Francis Ltd. Such antibodies may be used in diagnostic procedures, to, for example detect or se T. circumcincta infection/infestations in animal (for example ovine) species, as well as for passive immunisation.

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT ation] JXT Unmarked set by JXT The present invention further provides a vaccine for use in ting or controlling T. circumcincta ion/infestation and associated diseases in ovine hosts. The vaccine may be a polypeptide or polynucleotide vaccine.

The invention further provides a method for immunising an ovine t against T. cincta infection/infestation and associated disease (for example secondary infections etc.), said method comprising the step of administering a vaccine of the invention to the ovine subject.

While this invention predominately concerns antigens derived from the nematode organism T. cincta and their use in vaccine compositions for raising immune responses in animals (particularly ovine animals), owing to a high degree of homology between the T. circumcincta antigens described herein and specific antigens from other, closely related, nematode species, the utility of the antigens provided by this invention is not arily limited to raising immune responses which are protective against Teladorsagia ions/infestations. In particular, the antigens described herein exhibit significant homology/identity to certain antigens derived from the bovine pathogen, agia ostertagi. Details of these antigens and an indication of the level of identity exhibited between the disclosed T. circumcinta antigens and certain related O. ostertagi antigens are given in the table below.

Teladorsagia Accession Function* Closest % % O. ost circumcincta number Ostertagia identity Cover- reference Antigen ostertagi -age homologue A-1 CAQ43040 L3-enriched BQ098696.1a,b 94% 100% Unpublished surface (aa1- associated 162) antigen Tci-MIF-1 CBI68362 L3-enriched BQ457770.1a 99% 91% Unpublished macrophage (aa11- migration 115) inhibitory factor Tci-ASP-1 CBJ15404 L4-enriched CAD23183.1 76% 97% Mol Biochem activation- (aa5- tol associated 235) 2003; 126, [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT secretory 201-208 protein Tci-TGH-2 ACR27078 Transforming No significant hit - - - growth in NCBI, EMBL protein or e 4 2-like protein Tci-CF-1 ABA01328 L4-enriched BQ457843.1a 73% 59% Unpublished ** Secreted (aa12- cathepsin F 229) Tci-ES20 Not yet Excretory/ CAC44259.1 35% 100% Mol Biochem submitted* secretory (ES) (aa1- Parasitol ** protein 140) 2003; 126, 201-208 Tci-MEP-1 Not yet Astacin-like 95.2 69% 100% Parasitology submitted* ES (aa1- 2002; 125, ** metalloprotein 498) 383-391 Tci-APY-1 CBW3850 L4-enriched ADG63133.1 92% 96% Parasitology 7 ES calcium- (aa12- 2011; 138, activated 339) 333-343 apyrase *Putative or inferred on **Tci-CF-1 is highly polymorphic, the clone used for vaccine production had following amino acid substitutions compared to hed ce. In each case the amino acid in the published sequence is in italics, that in the vaccine isoform sequence is in normal font and the amino acid on in the hed ce is in subscript: I44⇒T44, N101⇒D101, T129⇒A129, R137⇒Q137, R305⇒K305, L306⇒P306, S307⇒Y307 *** Full length sequences not yet ted. a From translated EST sequence b with following caveat from authors: “WARNING: uent examination of these samples has revealed the presence of an additional Trichostrongyloidea cattle nematode, Cooperia oncophora.

Sequences in this library may derive from either Ostertagia or Cooperia.” In view of the above, it should be understood that the various aspects and embodiments of this ion (as applying to T. circumcincta antigens and their use in raising immune responses in animals, especially ovines) may further apply to one or more of the O. ostertagi antigens described above.

Moreover, in view of the levels of ty exhibited between the T. circumcincta antigens described herein and the O. ostertagi antigens identified above, one or more of the T. circumcincta antigens described herein may be used to raise immune responses in bovine subjects, the immune responses being protective and serving to reduce, prevent, treat or eliminate Ostertagia (for example O.

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT ostertagi) infections/infestations. One of skill will appreciate that the T. circumcincta antigens ed by this invention may be used individually or together (for e 2, 3, 4, 5, 6, 7 or all 8 of the T. circumcincta antigens) to raise immune responses in bovine hosts.

Alternatively, the present invention may extend to the use of one or more (for example 2, 3, 4, 5, 6 or all 7) of the O. ostertagi antigens presented in the table above, optionally in combination with one or more of the T. circumcincta antigens described herein, for use in raising immune ses in bovine subjects. Again, such immune responses may be protective against Ostertagia infections/infestations.

In one embodiment, this invention extends to compositions or vaccine compositions sing one or more of the Ostertagia antigens bed above optionally in combination with one or more the T. circumcincta antigens described herein, for use in raising immune responses in bovine subjects.

The invention may further provide uses of one or more of the Ostertagia antigens optionally in combination with one or more of the Teladorsagia antigens for the manufacture of medicaments for use in the treatment and/or prevention of an infection/colonisation by/with an agia pathogen in a bovine host. Similarly, the invention may also embrace methods of raising stertagia responses in bovine hosts, the methods comprising administering to a bovine subject, an amount of one or more of the Ostertagia antigens described above, sufficient to induce an anti- Ostertagia immune se.

One of skill will appreciate that references to the Ostertagia antigens described above not only include antigens comprising or ting of the sequences identified by the Accession numbers ted in the table above, but fragments thereof – in particular, fragments which are capable of raising immune ses (for example protective immune responses) in bovine animals (i.e. the fragments are antigenic and/or immunogenic) as well as mutants, ts and/or derivatives thereof. It should be understood that the definitions of fragments, mutants, variants and/or derivatives provided in relation to the Teladorsagia antigens of this invention, also apply to the Ostertagia antigens described above. As such, the agia antigen fragments, variants and/or derivatives encompassed by this invention may exhibit at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% homology or identity to the various Ostertagia sequences described above.

ED DESCRIPTION The present invention will now be bed in detail with reference to the following Figures which show: [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT Figure 1: Effects of immunization of sheep with recombinant antigens derived from Teladorsagia circumcincta on faecal worm egg counts (FWEC) after nge infection. Panels A and C: FWECs of sheep challenged with 2000 T. circumcincta three times per week for 4 weeks following immunization with an 8-protein cocktail in the context of Quil A (dashed line) or with Quil A only (solid line). Each data point represents the arithmetic mean FWEC ± SEM. Panel A represents data from Trial 1; Panel C ents data from Trial 2. Panels B and D show cumulative FWECs, for each animal in each group in Trial 1 (Panel B) and Trial 2 (Panel D). “Imm” represents sheep immunized with the 8-protein cocktail; “Con” represents those administered with Quil A nt only. Note that, in panel D, for Groups 1 and 2 in Trial 2, tive FWEC is calculated over 84 days, whereas for Groups 3 and 4 cumulative FWEC is calculated over 112 days. One er” animal in Group 1 of Trial 2, sheep number 675J, is indicated.

Figure 2. Trial 1: Lumenal T. cincta burdens of sheep in Group 1 and Group 2. Each data point ents the mean number (± SEM) of T. circumcincta enumerated in lumenal ts of seven sheep in each group. Panel A depicts counts categorised into developmental stage and the gender of the adult worms harvested. Panel B depicts the counts as l burdens (all stages and genders). “*” denotes a significant difference between the means (P < 0.05), “**” denotes a highly significant difference between mean (P < 0.01).

Figure 3. Trial 1: Mucosal T. circumcincta s of sheep in Group 1 and Group 2. Each data point represents the mean number (± SEM) of nematodes harvested from the mucosal contents of seven sheep in each group. “*” denotes a significant difference between the means (P < 0.05).

Figure 4: Effects of immunization of sheep with recombinant antigens derived from Teladorsagia circumcincta on abomasal nematode burden after challenge infection (Trial 1). Panels A-C represent the number of T. cincta enumerated in the abomasum. Panel A depicts the total nematode burden, panel B the adult nematode burden and panel C the juvenile nematode burden of each of seven sheep in Group 1 (immunized) or Group 2 (control, adjuvant only). Horizontal bars represent the mean value.

Figure 5: Weight gain of sheep in Group 1 and Group 2 from Day 0 to Day 84 of the experiment (Trial 1).

Figure 6: Effects of immunization of sheep with recombinant antigens derived from Teladorsagia circumcincta on juvenile nematode burden distribution after challenge infection (Trial 2; Group 1 and Group 2). Numbers of juvenile T. circumcincta enumerated in the abomasal lumen and the abomasal mucosa of each [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT of these sheep are shown. “Imm” represents sheep immunized with the 8-protein il; “Con” represents those immunized with Quil A adjuvant only.

Figure 7: Effects of zation of sheep with recombinant antigens derived from Teladorsagia circumcincta on abomasal nematode burden after challenge infection (Trial 2; Group 3 and Group 4). Data shown represent the total numbers of T. circumcincta ated in the abomasum of each of seven sheep in Group 3 (immunized) or Group 4 (control, adjuvant only).

Figure 8: Serum antibody responses of sheep to the recombinant ns used to immunize Group 1 in Trial 1. Each data point represents the mean value derived from 7 sheep. Standard errors in panels A and B have been omitted to aid interpretation. Panels A and B show serum antibody responses for vaccinated sheep (Group 1). Panel A shows data for IgG, panel B shows data for IgA. “Imm” represents dates on which sheep were immunized; “Trick” represents the e infection; “PM” is the post mortem date.

Figure 9: Serum antibody responses of sheep to the inant proteins used to immunize Groups 1 and 3 in Trial 2. Each data point represents the mean value derived from 14 sheep until day 84, after which each data point ents the mean of 7 sheep necropsied later in the trial. Standard errors have been omitted to aid interpretation. Panel A shows data for IgG, panel B shows data for IgA. “Imm” represents dates on which sheep were immunized; “Trick” represents the trickle infection; “PM” is the post-mortem date.

Figure 10: Serum antibody responses of sheep to L4 excretory/secretory ts of Teladorsagia circumcincta. ‘Imm’ represents the days on which animals were immunized with recombinant antigen cocktail (Immunized group) or adjuvant only (Control group).

Figure 11: Immunoblots to investigate serum IgG (Panel A) and IgA (Panel B) binding to components of somatic extracts and excretory/secretory ts of Teladorsagia circumcincta. Lanes 1 and 5 contain L3 somatic extract, lanes 2 and 6 contain L4 somatic extract, lanes 3 and 7 contain L4 ES material and lanes 4 and 8 contain adult somatic extract. Blots were incubated with sera pooled from 7 immunized sheep (Lanes 1-4, sheep from Group 3, Trial 2) or non-immunized sheep (Lanes 5-8, sheep from Group 4, Trial 2). Sera had been collected from the animals on the date of the third immunization immediately prior to the initiation of trickle infection. * represents molecular mass (kDa).

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT Figure 12: Serum IgG responses of control, adjuvant only recipients to recombinant Tci-MEP-1 and Y-1. Each data point represents the mean value (± SEM) derived from 7 (Trial 1, panel A) or 14 (Trial 2, Panel B) sheep until day 84, after which each data point represents the mean of 7 sheep in Trial 2.

Figure 13: l antibody titres to the recombinant proteins used to immunize sheep in Trial 1. Each bar represents the mean value derived from 7 sheep (± SEM). Panel A shows data for IgG, panel B shows data for IgA. Asterisks indicate mean values which are tically significantly higher than those for the remaining antigens within the same treatment group.

Figure 14: Mucosal antibody titres to the recombinant proteins used to immunize sheep in Trial 2. Each bar represents the mean value derived from 7 sheep (± SEM). Panel A shows data for IgG, panel B shows data for IgA. Asterisks indicate mean values which are statistically significantly higher than those for the remaining antigens within that Group.

Figure 15: Mucosal antibody levels to the recombinant proteins used to ze sheep in Trials 1 and 2. Panels A and B show data for IgG, panels C and D show data for IgA. All graphs show correlation biplots y representing sheep s) and their n-specific antibody responses (axes). The arrows indicate directions of higher antigen-specific antibody response. The onal projection of points onto each axis approximates the relative responses by sheep. The correlations between responses to ic antigens are represented by the angle between the corresponding vectors for each antigen. Open circles represent immunized sheep, closed circles represent control, non-immunized sheep. In Trial 2 (Panels B and D), blue circles and pink circles represent Groups 1 and 3 (immunized) respectively. Green and orange circles represent control Group 2 and 4 respectively.

Materials and s Production of recombinant proteins for immunisation Eight recombinant proteins were used in combination to immunise 6 month- old lambs. Details of these proteins are given in Table 1. Three proteins, macrophage migration inhibitory factor-1 (Tci-MIF-1), calcium-dependent apyrase-1 (Tci-APY-1) and a TGFβ homologue (Tci-TGH-2) were ed e of their putative immunoregulatory function (McSorley et al., 2009; Nisbet et al., 2010a; Nisbet et al., 2011). The remaining five proteins were selected using a combined immunoscreening/proteomics approach: cathepsin F-1 F-1), n-like metalloproteinase-1 (Tci-MEP-1), a 20 kDa protein of unknown function (Tci-ES20) and activation-associated secretory protein-1 (Tci-ASP-1) (Redmond et al., 2006; ation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT Smith et al., 2009; Nisbet et al., 2010b). A final protein was chosen because of its gy to known vaccine ate antigens of other parasitic nematodes. This protein is known as surface-associated antigen (Tci-SAA-1, Nisbet et al., 2009).

Cloning and sequencing of the cDNA encoding Tci-SAA-1, Tci-MIF-1 and Y-1 and production of recombinant ns of each of these proteins in a bacterial expression system have been described previously (Nisbet et al., 2009; Nisbet et al., 2010a; Nisbet et al., 2011). Identical tion and purification parameters were employed in the current study. For Tci-MEP-1, ucleotide primers for use in the rapid ication of cDNA ends (RACE) were designed from the EST sequence CB036707 and RACE performed using the SMART™ RACE kit (Clontech) according to the manufacturer’s instructions, using total RNA extracted from L4 stage T. circumcincta (prepared as described in Nisbet et al., 2008) as a template.

Amplification of the full coding sequence (CDS) of Tci-mep-1 was performed using oligonucleotide primers incorporating the initiation and termination codons from the contigs generated by 5’ and 3’ RACE, cDNA generated from L4 as template (prepared as described in Redmond et al., 2006) and the Advantage® 2 PCR Kit (Clontech) according to the cturer’s instructions. Following confirmatory sequencing, oligonucleotide primers were designed to amplify the CDS of Tci-mep-1, omitting the ce encoding the signal peptide (bases 1-48 of the CDS) and the termination codon. Using these primers, plasmid containing the full-length CDS as a template and the Advantage® 2 PCR Kit (Clontech), Tci-mep-1 was amplified and sub-cloned into the expression vector pET SUMO (Invitrogen). The resulting plasmid was used to transform Escherichia coli BL21-CodonPlus (DE3)-RIL competent cells (Stratagene). Recombinant protein expression was d in the presence of 1 mM isopropyl β-Dthiogalactopyranoside (IPTG). ble recombinant Tci-MEP-1 was ed from inclusion bodies solubilised in 8M urea, ed by nickel column affinity chromatography using HisTrap™ HP columns (GE Healthcare) and a stepwise imidazole gradient in the presence of 8M urea in 20mM phosphate buffer, pH 7.6. Purified Tci-MEP-1 was then dialysed t 2M urea in 20mM phosphate buffer, pH 7.6. The full CDS of the cDNA encoding Tci-TGH-2 (accession number FJ410914) was ied by PCR using ucleotide primers incorporating the initiation codon, but omitting the termination codon. Plasmid containing the full CDS in a cloning vector was used as a template (kindly supplied by Prof Rick Maizels, University of Edinburgh) and the Advantage® 2 PCR Kit (Clontech) was employed according to the cturer’s instructions. Tci-tgh-2 was sub-cloned into the expression vector pET SUMO (Invitrogen) and recombinant protein expression performed as described above. Soluble recombinant Tci-TGH-2 was purified from [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT cell lysates by nickel column affinity chromatography using p™ HP columns (GE Healthcare). Next, rTci-TGH-2 was eluted in 500 mM imidazole, 20mM phosphate , pH 7.6 and then dialysed t 20mM phosphate buffer, pH 7.6 at RT for 3 hrs. Sub-cloning of the CDS of Tci-asp-1 (after removal of the bases encoding the signal e) from a pET22b(+) vector (described in Nisbet et al., 2010b) into pET SUMO, using the conditions outlined above for Tci-tgh-2, permitted the expression of soluble recombinant Tci-ASP-1 which was sed and then ed by nickel column affinity chromatography as described, above, for Tci-TGH- 2. For the expression of Tci-CF-1 protein, oligonucleotide primers were designed to amplify the CDS of Tci-cf-1, omitting the ce encoding the signal peptide (bases 1-42 of the CDS) and the termination codon. Using these primers, cDNA generated from L4 as template (prepared as described in Redmond et al., 2006) and the Advantage® 2 PCR Kit (Clontech), Tci-cf-1 was sub-cloned into the vector pPICZαC (Invitrogen) and used to transform the yeastPichia pastoris [X-33 Mut+ strain (Invitrogen)] following linearisation with PmeI (New England Biolabs).

Recombinant protein expression was induced in the presence of 0.5% methanol, as described in Nisbet et al. (2007) and soluble recombinant Tci-CF-1 was purified from culture atant by nickel column affinity chromatography as bed above for Tci-TGH-2. Tci-ES20, a homologue of a 20kDa ory/secretory (ES) n of Ostertagia agi, was identified during an immunoscreening/proteomic analysis of genic T. circumcincta ES molecules (Smith et al., 2009). The complete coding sequence was determined by obtaining the putative full-length cDNA via polymerase chain reaction (PCR) amplification from a cDNA library. This SMART™ cDNA library was ucted [using T. circumcincta L4 (8 days post infection, dpi) RNA] in λTriplEx2 by long-distance PCR following manufacturer’s instructions (Clontech). It was packaged using Gigapack Gold III packaging t (Stratagene) and amplified in E. coli XL1-Blue cells (Stratagene). A gene-specific oligonucleotide primer (incorporating the putative termination codon identified from EST CB043664) was used in conjunction with a vector-specific primer to amplify the Tci-es20 CDS directly from a heat-denatured phage lysate preparation of the library. The resultant amplicon was column-purified (QIAquick PCR purification kit, Qiagen) and ligated into pGEM®-T ga). Constructs were transformed into E. coli JM109 (Promega), colonies with Tci-es20-containing plasmids were isolated and propagated and the plasmids subjected to automated sequencing (Eurofins MWG operon). The cDNA ng Tci-ES20 was then subcloned into the vector pPICZαC (Invitrogen) and used to transform P. pastoris [X-33 (Mut+) strain (Invitrogen)] following [Annotation] JXT None set by JXT ation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT linearisation with PmeI (New England Biolabs). Recombinant protein expression and purification were as described, above, for Tci-CF-1. Protein concentrations were determined using the Pierce BCA™ (bicinchoninic acid) assay (Thermo Scientific) with bovine serum albumin (BSA) standards and stability and integrity of each recombinant protein were monitored using SDS-PAGE. Tci-MIF-1; Tci-APY-1; Tci- SAA-1; Tci-CF-1; Tci-ES20 and Tci-MEP-1 were stored in solution at +4°C and Tci- ASP-1 and Tci-TGH-2 were stored at -20°C.

Table 1 Table 1. Recombinant proteins used in Teladorsagia circumcincta vaccine trial Name Accession on* Expression nce number system Tci-SAA-1 CAQ43040 L3-enriched (+) Nisbet et al., surface associated E. coli BL21 2009 antigen (DE3)-RIL Tci-MIF-1 CBI68362 L3-enriched pET22b(+) Nisbet et al., macrophage E. coli BL21 2010a migration inhibitory (DE3)-RIL factor Tci-ASP-1 CBJ15404 L4-enriched pET SUMO Nisbet et al., activation- E. coli BL21 2010b associated (DE3)-RIL ory protein Tci-TGH-2 ACR27078 Transforming pET SUMO McSorley et growth protein 2- E. coli BL21 al., 2010 like n (DE3)-RIL Tci-CF-1 28** L4-enriched pPICZαC Redmond et ed cathepsin Pichia pastoris al., 2006 F X33 strain Tci-ES20 Not yet Excretory/secretory pPICZαC Smith et al., submitted*** (ES) protein Pichia pastoris 2009 X33 strain Tci-MEP-1 Not yet Astacin-like ES pET SUMO Smith et al., submitted*** metalloproteinase E. coli BL21 2009 (DE3)-RIL Tci-APY-1 CBW38507 L4-enriched ES pSUMO Nisbet et al., [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT calcium-activated E. coli BL21 2011 e (DE3)-RIL ive or inferred on **Tci-CF-1 is highly polymorphic, the clone used for vaccine production had following amino acid substitutions compared to hed sequence. In each case the amino acid in the published sequence is in s, that in the vaccine isoform sequence is in normal font and the amino acid position in the published sequence is in subscript: I44⇒T44, N101⇒D101, T129⇒A129, R137⇒Q137, R305⇒K305, L306⇒P306, S307⇒Y307 *** Full length sequences not yet deposited. These molecules have been derived from EST data in the public domain: Tci-ES20 based on 64, Tci-MEP-1 based on CB036707 Immunisation trial en, Texel crossbred male/female sheep which had been raised in conditions to minimise helminth infection risk, were housed in two groups of 7 animals in separate pens within the same building. The sheep were 204-206 days old at the initiation of the ment. Faecal egg counts (FEC, Christie and n 1982), performed prior to initiation of the experiment, confirmed that all s had negative FECs. Sheep in Group 1 were immunised by subcutaneous injection using a 400 µg recombinant protein mix (incorporating 50 µg each Tci-ASP-1; Tci-MIF-1; Tci-TGH-2; Tci-APY-1; Tci-SAA-1; Tci-CF-1; Tci-ES20; Tci-MEP-1 in PBS) plus 5 mg total Quil A (Brenntag Biosector). Seven of the 8 recombinant proteins were PBS- soluble and were stered as a mixture in a single injection with 2.5 mg Quil A.

Tci-MEP-1 was insoluble in PBS and was therefore formulated with 100 mM urea in PBS plus 2.5 mg Quil A. The two preparations were injected subcutaneously, one immediately following the other, at two sites on the neck of each sheep. Each sheep received three immunisations of the recombinant protein mix with an interval of 3 weeks between each immunisation. Sheep in the control group (Group 2) each received three immunisations with urea/PBS/Quil A only, at the same time as the sheep in Group 1. On the day of the third immunisation, an oral trickle challenge was initiated whereby each sheep in both groups was administered with 2000 T. circumcincta L3. This was continued three times per week (Monday, Wednesday and Friday) for 4 weeks. Blood samples were taken prior to each immunisation and weekly samples taken from the day of the third immunisation onwards to determine antigen-specific serum IgA and IgG responses and serum pepsinogen levels (Lawton et al., 1996). FECs were performed tie and Jackson 1982) three times per [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT week y, Wednesday and ) from 14 days after the start of the trickle challenge until the end of the experiment 5 weeks later. All sheep were weighed weekly. For both groups, al swab samples were collected at post-mortem (Smith et al., 2009) to determine levels of antigen-specific IgA and IgG antibody at the abomasal mucosal surface. At necropsy, lumenal and mucosal nematode burdens (adult and larval parasites) were enumerated ing standard techniques.

The percentage of stunted or “inhibited” larvae was determined, based on size, as bed previously (Halliday et al., 2010). The experiment was performed under the regulations of a UK Home Office Project Licence.

Trial 2 Twenty-eight, Texel crossbred male/female sheep were raised as described for Trial 1 and were housed in four groups of 7 animals. The sheep were 172-178 days old and were not ing helminth eggs at the start of the experiment. Groups 1 and 3 were immunized by subcutaneous injection using the recombinant protein mix exactly as described for Trial 1, with each sheep receiving three immunizations with an interval of 3 weeks between each. Sheep in Groups 2 and 4 each received three immunizations with BS/Quil A, at the same time as Groups 1 and 3. At the final immunization, the oral trickle challenge commenced in all Groups and all biological samples were obtained as described above, for Trial 1. Sheep in Groups 1 and 2 were euthanized 7 weeks after the start of the infection period (as for Trial 1) and those in Groups 3 and 4 were euthanized 4 weeks later. For all groups, lumenal and mucosal nematode burdens were enumerated as described for Trial 1. Trial 1 and Trial 2 were performed under the strict tions of a UK Home Office Project Licence and the experimental design was ratified by the Moredun Research Institute Experiments and Ethics Committee.

Measurement of dy responses to recombinant antigens Following initial antibody:antigen titrations to ensure optimisation of the que, antigen-specific antibody levels in serum and abomasal mucus s were assessed by ELISA. High binding microtitre plates (Greiner Bio-One) were coated overnight at 4°C with 50 µl antigen (5 μg ml-1 in 50 mM carbonate buffer, pH 9.6). Plates were washed six times with wash buffer [phosphate buffered saline (PBS), 0.05% v/v Tween-20], then blocked with 5% soya milk powder in 0.5% (v/v) Tween 20 in Tris Buffered Saline (TTBS), pH 7.4, for 1 h at room temperature. After g, 50 µl abomasal mucus (diluted 1:4 in TTBS) from individual animals or 50 µl serum [diluted at 1:10 (IgA) or 1:1000 (IgG) in TTBS], were added and incubated for [Annotation] JXT None set by JXT [Annotation] JXT ionNone set by JXT [Annotation] JXT Unmarked set by JXT 1 h at room temperature. Wells were re-washed and 50 µl horseradish daseconjugated polyclonal mouse anti sheep/goat IgG (A9452, Sigma) at 1:1000 or 50 µl mouse anti-bovine/ovine IgA monoclonal dy (Serotec, MCA628) at 1:250 in TTBS, were added for 1 h at room temperature. After a further wash, the IgG ELISA was developed by the addition of 50 µl ylenediamine dihydrochloride substrate (OPD, Sigma) to each well. After 15 min in darkness, the reaction was stopped by addition of 25 µl 2.5M H2SO4 and OD values read at 490 nm. For the IgA ELISA, 50 µl horseradish peroxidase-conjugated polyclonal rabbit anti-mouse IgG (P0260, DakoCytomation), at 1:1,000 were added for 1 h at room temperature prior to a final wash and development with OPD as described above. Each sample was assayed in triplicate. OD values were corrected against a reagent blank and all test plates had a positive and negative serum control to account for plate to plate variation.

Measurement of antibody responses to native T. circumcincta antigens Antigen-specific IgG levels in the sera of sheep which had been immunized with the recombinant antigen il, or the non-immunized control sheep, were assessed by ELISA. The native antigens used to coat ELISA plates were somatic extracts of T. circumcincta L3, prepared as described previously (Nisbet et al., 2009), along with L4 ES products, prepared as described in Smith et al., (2009). Antigen- specific IgG levels were ed in all sera from s in Trial 1 and from four, randomly selected, animals from Groups 1 and 2 of Trial 2. All experimental ions were as described, above, for the determination of recombinant antigenspecific IgG levels in serum by ELISA. blotting of nematode somatic extracts Somatic extracts of T. circumcincta L3, L4 and adult worms, prepared as described previously (Nisbet et al., 2009), along with L4 ES products, prepared as described in Smith et al., (2009), were subjected to blotting using serum, collected on the date of the third (final) immunization immediately prior to the initiation of trickle infection, from immunized or non-immunized sheep.

Immunoblotting, to determine serum IgG and IgA binding to ents of each extract, was performed as described previously (Nisbet et al., 2009) using pools of serum from 7 immunized (Group 3) and 7 non-immunized sheep (Group 4).

Statistical analysis A generalised ve mixed modelling (GAMM) approach was adopted for the analysis of longitudinal FEC data. A GAMM model on log(FEC+1) was specified with Gaussian error structure and identity link function, with group as a fixed effect and animal effects introduced as random. The model included separate smoothing [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT curves to model the nonlinear relationship of the response with time by group and non-homogenous within-group variances were allowed. A first order autoregressive residual correlation structure was incorporated. Serum and mucosal antibody responses to individual antigens were modelled using linear mixed models (LMMs) with group as a fixed effect and animal as a random effect. For serum antibody data, ed measures over time were modelled by random intercept and slope LMMs also including time and its interaction with group as a fixed effect. geneous within-group variances were allowed in all cases. Linear contrasts were set up to compare subsets of antigen-specific responses in abomasal mucus at post mortem.

In Trial 2, the 28 animals were housed in 4 separate groups (pens) of 7 animals for logistical reasons. Two groups (14 animals) were immunized (Group 1 and Group 3), and the other two (14 animals) were used as adjuvant-only controls (Group 2 and Group 4). Pen effects between the two immunized groups (1 and 3) and between the two adjuvant-only groups (2 and 4) were tested. No tically significant pen effects were found for any of the above response types, so Groups 1 and 3 were combined and Groups 2 and 4 were combined for data modelling. For analysis of worm burden data, generalised linear models (GLMs) were used. Data overdispersion was detected and it was generally accounted for by specifying a ve binomial error bution. Where necessary, overdispersion was incorporated using n GLMs correcting the rd errors by specifying the mean and variance relationship. de burdens were assessed at post mortem in Groups 1 and 2 four weeks before those of Groups 3 and 4, so data were analysed separately.

Model selection was based on the Akaike’s information criterion (AIC) and likelihood ratio tests (LRT) (Akaike, 1974). The mixed models were fitted by residual maximum likelihood (REML; Smouse and , 1972). Throughout the data analysis some animal measurements were identified as rs. Their nce on parameter estimates was considered in each case. The Cook’s distance with a 4/n cut-off value was used to support decisions in relation to outlying values (Cook, 1977). Statistically significant terms were determined at the level of 0.05. All statistical analyses were conducted using R n 2.13.

Results FECs analysis Trial 1: FEC data is shown in Figure 1A and B. Sheep in both immunised and control groups began to excrete strongyle type eggs in their faeces from 16-19 days after the start of the trickle challenge. In both groups, FECs rose until 23 days [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT after the start of challenge. Thereafter, sheep in Group 1 ed substantially fewer eggs than those in Group 2. By the end of the ment, at day 42 of the trickle challenge, Group 1 sheep were producing a mean of 8.7 (± 5.5) eggs per gramme (EPG) of faeces, whereas sheep in Group 2 were producing 107.6 (± 50.8) EPG, representing a reduction of 92% in mean FEC at that time-point. REML (GAMM) analysis identified an overall effect of treatment (immunization) (P = 0.003) and time (P < 0.001), and a significant treatment × time interaction (P = 0.20). The mean cumulative FECs for the on of the experiment, estimated by taking the sum of all egg counts on each sampling date, were 252 (± 132) EPG in Group 1 and 890 (± 231) EPG in Group 2, representing an overall mean FEC ion of 72% in the immunised versus the control group. FEC Mean cumulative FECs for the duration of the challenge period, calculated using the area under the curve (AUC, Taylor et al., 1997) technique were 595 (± 316) EPG in Group 1 and 1975 (± 532) EPG in Group 2, representing an overall reduction of 70% in the immunized versus the control (adjuvant only) group (Figure 1B).

In Trial 2, sheep began to excrete nematode eggs from 14-16 days after challenge e 1C). At peak egg shedding, on day 86, mean FECs in the extant immunized group (Group 3) were 251 ± 75 EPG, whereas in the control group (Group 4) they were 908 ± 158 EPG, representing a 73% reduction in mean FEC.

Mean cumulative FECs, ated using the area under the curve (AUC, Taylor et al., 1997) technique, in Trial 2 were 4998 (±) 2233 EPG in Group 1 (immunized) and 4127 (±) 803 EPG in Group 2 (adjuvant only, Figure 1D). The high mean FECs, and associated SEM, in Group 1 were attributable to the influence of data from a single outlier animal (sheep 675J, Figure 1D). Influence was ed using Cook’s distance criterion (Cook, 1977): 675J was regarded as a “highly influential” case (Cook’s distance = 0.3129 based on a LMM . For Groups 3 and 4, which were necropsied 4 weeks after Groups 1 and 2, mean cumulative FECs were 7005 (±) 681 EPG in Group 3 (immunized) and 16727 (±) 2,699 EPG in Group 4 (control, adjuvant only), enting an overall mean FEC reduction of 58% in the immunized versus the control group (Figure 1D). GAMM analysis indicated a statistically-significant effect of immunization (data from Groups 1 and 3 combined vs. Groups 2 and 4 combined as detailed in Materials and Methods) on FEC over the course of the experiment (P = 0.0237).

Abomasal parasite burdens Trial 1: inary analysis Abomasal T. circumcincta enumerations were subdivided into lumenal and mucosal burdens. Within the lumen, Group 1 sheep had significantly fewer adult male ation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT 04) and female T. circumcincta (P = 0.011, Figure 2A) than was ed in the Group 2 sheep. There was no significant difference in parasite gender ratio between the two groups. Taking all developmental stages and genders into account (Figure 2B) Group 1 harboured significantly fewer luminal parasites than the sheep in Group 2 (P = 0.0037) - sheep in Group 1 had 72% less nematodes in the abomasal lumen than those in Group 2. Within the mucosa, the numbers of adult female worms in Group 1 were significantly less than those observed in Group 2 (P = 0.016) (Figure 3). There was no significant difference between the s of male worms or larval stages enumerated in the mucosa in the two groups, although fewer male worms were enumerated in Group 1 sheep and fewer larval stages in Group 2 sheep (Figure 3).

Trial 1: supplementary analysis of total worms numbers (lumenal plus mucosal) Immunized sheep (Group 1) harboured 55% fewer T. circumcincta (total of adults and larvae) at necropsy than control, nt only (Group 2) sheep (P = 0.011, Figure 4A). Group 1 sheep had statistically-significantly lower mean adult de burdens than sheep in Group 2 (75% reduction, P = 0.0066, Figure 4B). Comparison of juvenile nematode s in the abomasum indicated no significant differences between the two groups (Figure 4C). No significant differences were observed in the length of worms recovered from the different groups (data not shown).

Liveweight gain The average increase in weight from Day 0-Day 84 of sheep in Group 1 was 2.1 kg more than that observed in sheep in Group 2 (p = 0.10) (Figure 5).

Trial 2 Groups 1 and 2 (post mortem at day 84): The total abomasal de burdens (adults and larvae) in immunized sheep were not statistically significantly different to the l, adjuvant only group (mean total nematode burdens: Group 1; 6843 ± 1144, Group 2; 6250 ± 966). When adult nematode burdens and juvenile nematode burdens were analysed separately, the adult nematode burdens in immunized sheep were not statistically significantly different to the control, adjuvant only group. ison of the juvenile nematode burdens indicated that zed sheep had fewer juvenile nematodes than control, adjuvant only sheep in the abomasal lumen (Group 1: 50 ± 42; Group 2: 218 ± 81), (Figure 6). Because of the preponderance of “zero” values in the counts from the immunized sheep, statistical analysis using models was unreliable in this case.

Conversely, there were more juvenile stages in the abomasal mucosa of Group 1 than Group 2 (Group 1: 643 ± 198; Group 2: 114 ± 70; P=0.0367, Figure 6).

[Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT Groups 3 and 4 (post mortem at day 112): sed sheep (Group 3) harboured 57% fewer T. circumcincta total nematodes at necropsy than did the control, adjuvant only (Group 4) recipients (P = 0.0199, Figure 7). In both Groups 3 and 4, adult worms comprised 99% of the total nematode burden and no significant difference in the s of juvenile stages was observed between the two Groups.

Measurement of serum antibody ses to T. cincta antigens In both trials, following tertiary immunization, serum IgG levels against all recombinant proteins reached peak levels, which declined slowly thereafter (Figures 8A and 9A). Serum IgA levels peaked after secondary immunization and, for all recombinants, with the exception of Tci-MIF-1, levels remained relatively constant until the end of the experiment es 8B and 9B). Following immunization with the recombinant antigens, sheep produced serum IgG, prior to parasite challenge, which bound native L4 ES components (Figure 10). The nature of the immunoreactive antigens in this ES material, and other T. cincta extracts, was investigated further by immunoblotting: IgG bound to parasite ents, in somatic extracts of L4 and adult T. circumcincta as well as L4 ES, of the expected size range for the following vaccine components, Tci-CF-1 (23.9 kDa), Tci-APY-1 (38.6 kDa) and Tci- MEP-1 (55.6 kDa) (Figure 11A). IgA also bound parasite components, in somatic extracts of L4 and adult T. circumcincta and L4 ES, of the expected size range for the vaccine components, Tci-CF-1, Y-1 (Adult only) and Tci-MEP-1 (Figure 11B).

In addition IgA bound an unknown parasite component of ca. 43 kDa in L3 c extract.

In both Trials 1 and 2, from 14 days after initiation of challenge, control, adjuvant only recipients ted serum IgG that bound recombinant Tci-MEP-1 and Tci-APY-1 (Figure 12). Antigen-specific serum IgA which bound to the recombinant proteins was not observed in the control, adjuvant only recipients (data not shown).

Measurement of antibody responses to recombinant antigens in abomasal mucus In Trial 1 and 2, mean recombinant antigen-specific mucosal IgG levels in abomasal mucus of the immunized sheep were significantly higher than in the control, nt only recipients for each protein (Figures 13A and 14A). In Trial 1, mean Tci-APY, P, and Tci-CFspecific IgG levels were significantly higher than those measured against the other five inants (P <0.0001), whereas in Trial 2, mean Tci-MEPspecific IgG levels were icantly higher than responses to the [Annotation] JXT None set by JXT ation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT remaining antigens (Day 84 necropsy) while Tci-MEP and Tci-APYspecific IgG levels were significantly higher at the Day 112 necropsy. A joint biplot representation of animals and antigen-specific mucosal IgG responses (Figures 15A and 15B) illustrates the relationships between treatments, between animals within groups, with respect to IgG ses to the different antigens and l differences between immunized and control, adjuvant only sheep.

Mucosal Tci-APY and Tci-MEPspecific IgA levels were significantly higher than those directed t the other six recombinant antigens in Trial 1 and 2 (Figures 13B and 14B). The overall differences between zed sheep and nt only recipients are represented in joint biplots of animals and antigenspecific mucosal IgA responses in Figures 15C and 15D.

Discussion Here, we demonstrated that sation of sheep with a cocktail of eight recombinant T. circumcincta proteins results in significant levels of protection in terms of FECs and parasite burdens when compared to challenge control sheep. As far as we are aware, this is the first published report of successful vaccination t this nematode species using a inant vaccine. Indeed, the levels of protection are higher than observed in any other system using a recombinant vaccine against a parasitic nematode in the definitive ruminant host. The level of protection achieved, in terms of FEC and abomasal luminal burden, is similar to the highest reported levels following ation with detergent extracts of T. circumcincta L3 (Wedrychowicz et al., 1992;1995). In those experiments immune anti-parasite responses were variable, but parasite burdens were significantly d (by up to 72%) and FECs were reduced by more than 70%. The antigens that stimulated protection in the previous trials (Wedrychowicz et al., 1992; 1995) were not characterised in detail and their identity remains elusive.

Other attempts to protect sheep t T. circumcincta using native antigen preparations, for example lectin-binding integral membrane glycoproteins, have not been successful (Smith et al., 2001). This l lack of success in immunisation against T. circumcincta is in contrast to the situation in other, closely related, parasitic nematode species. For example, the closest homologues of Tci-ASP-1, the N-type single domain ASPs, Oo-ASP-1 and Oo-ASP-2, are the principal components of an riched native extract of adult Ostertagia ostertagi which has been used with success in vaccination trials in cattle (Geldhof et al., 2002, 2004; Meyvis et al., 2007).

However, vaccination with a recombinant version of Oo-ASP-1 has failed to induce either protective immunity or native-antigen specific antibodies in vaccinated calves (Geldhof et al., 2008). This reflects the outcomes of many nematode vaccine trials [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT ed set by JXT using recombinant versions of native proteins/complexes where the native molecules show great promise, but where recombinant versions fail to induce protective immunity (Geldhof et al., 2007). This atic” approach to antigen identification, where protective native extracts are identified by an iterative process of fractionation and vaccination and recombinant versions of single (or multiple e.g. see Cachat et al., 2010) protective antigens are produced and tested in vivo, therefore appeared to be of limited value for the development of a vaccine against T. circumcincta.

The approach to antigen identification described herein was ntially different to the pragmatic approach, and followed a more ed approach by attempting to mimic and exploit elements of the natural, successful immune response to T. cincta in infected sheep. First, we identified potential vaccine candidate molecules by immunochemical and proteomic analyses; this was done by screening immunoblots of T. circumcincta ES material with IgA from infected, immune sheep and ing these responses to those observed in infected, non-immune sheep or non-infected sheep (Smith et al., 2009). We also identified a homologue of a known protective antigen A-1 (Zhan et al., 2004)] using bioinformatic analysis of stage-specific cDNA libraries (Nisbet et al., 2008; 2009). Finally, using a combination of these technologies, we identified a suite of potentially suppressive molecules produced by the parasite (McSorley et al., 2010, Nisbet et al., 2010a; 2011). We produced recombinant versions of each of these molecules, examined that they were s of IgA present in mucus derived from immune sheep and then combined them into a multi-component vaccine which aimed to e the host immune system to d to potentially immunostimulatory molecules (Tci-CF- 1,Tci-MEP-1,Tci-ES20,Tci-ASP-1 and Tci-SAA-1) and to produce a possible neutralising effect on putatively immunosuppressive components. The rationale behind using a combination of recombinant molecules, as opposed to single antigens, is as follows: previous ation trials using single recombinant n preparations of homologues of some of the les described herein, in different nematode/host models, have failed. In O. ostertagi, for example, the astacin-like metalloproteinase MET-1, which shares >50% amino acid identity with Tci-MEP-1, was selected by immunoscreening but failed to give any protection when used as a single recombinant antigen in a vaccine trial (De Maere et al., 2005). Similarly, recombinant Oo-ASP1, which shares >75% sequence identity with Tci-ASP-1 (Nisbet et al., 2010b), has failed to induce protective immunity in vaccinated calves (Geldhof et al., 2008) and a recombinant version of the Necator americanus orthologue of Tci- SAA-1 (Na-SAA-1 , 71% amino acid ty) failed to induce icant protection against L3 challenge in a hamster model (Xiao et al., 2008).

[Annotation] JXT None set by JXT ation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT The ism of action of the vaccine used herein is not yet clear. These nematodes are acquired by ingestion of L3 from e. Thereafter, the developing parasites (L3 and L4) and adult worms reside in the host’s abomasum. Protective immunity t T. circumcincta in sheep exposed to continuous field or experimental trickle challenge has been associated with decreased larval establishment (L3) and development (L3 and L4) in the mucosa and reduced egg output from female worms in the lumen (Balic et al., 2003; Seaton et al., 1989; Smith et al., 1985, 1986; Stear et al., 2004). In the current study, in Trial 2, adult worm s in vaccinated and adjuvant only groups were similar at day 84, so it seems unlikely that exclusion and expulsion of ng L3 or death/delayed development of L4 worms was responsible for the observed reduction in adult parasite numbers at day 112 of that trial. The reduction in the numbers of adult worms may therefore be ed to either a direct effect anti-parasitic effect of the induced immune response against the adult worms or a cumulative fitness-reducing effect throughout the life of the worm, culminating in the lower level, or shorter duration, of adult survival.

The immune mechanisms responsible for the ed effects on the parasites are likely to be complex: In naturally-acquired immunity to T. circumcincta in sheep roles for immediate hypersensitivity reactions and for larval antigen-specific IgA in gastric ions have been indicated (Smith et al., 1986; 1987; Stear et al., 1995; 1999; Halliday et al., 2007; Smith et al. 2009). Cellular effectors of the immune response, e.g. γδTCR+ T cells, CD4+ T cells, eosinophils, globular leukocytes and mast cells may also play a role in immunity against T. circumcincta in naturally- or experimentally-exposed sheep (e.g. Stear et al., 2002; 2009, Balic et al., 2003; Halliday et al., 2010, Williams 2012).

In conclusion, we have developed a multi-component vaccine against T. circumcincta which, in experimental circumstances, reduced mean FECs and mean luminal parasite burdens by >70%. It should be noted that, according to Barnes et al., (1995) it is not essential for a vaccine against parasitic nematodes to be 100% effective in sheep, and antial benefits” can be gained by using a vaccine that is 60% ive in 80% of the flock, if the vaccine is based on the stimulation of al immunity’. On this basis, the results of this study would clearly indicate that the vaccine used here holds much potential. It is not yet clear whether all of the eight recombinant protein components of the vaccine are required for this level of cy and further work will seek to clarify this and also to confirm the anti-parasite effects of the 8-protein cocktail vaccine.

References [Annotation] JXT None set by JXT [Annotation] JXT MigrationNone set by JXT [Annotation] JXT Unmarked set by JXT Balic, A., Bowles, V.M., Liu, Y.S. & Meeusen, E.N. Local immune responses in sensitized sheep ing challenge infection with Teladorsagia circumcincta. te Immunol. 25, 371-381 (2003).

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Claims (16)

Claims

1. A method of raising an immune response in a non-human animal, the method sing administering said animal rsagia circumcincta antigen m-dependent apyrase-1 or an immunogenic fragment thereof.

2. The method of claim 1, wherein the method further comprises administering one or more T. circumcincta antigens selected from the group consisting of: (i) cathepsin F-1; (ii) excretory/secretory protein; (iii) transforming growth protein 2-like protein; (iv) activation associated ory protein; (v) macrophage migration inhibitory factor; (vi) surface associated antigen; and (vii) an immunogenic fragment of any of (i)-(vi).

3. A composition or vaccine composition comprising Teladorsagia cincta n calcium-dependent apyrase-1 or an immunogenic fragment thereof when used in raising an immune response in a non-human animal animal.

4. The composition or vaccine composition of claim 3, wherein the composition or vaccine composition further comprises one or more T. circumcincta antigen(s) selected from the group consisting of: (i) cathepsin F-1; (ii) excretory/secretory protein; (iii) transforming growth protein 2-like protein; (iv) activation associated secretory protein; (v) macrophage migration inhibitory factor; (vi) surface associated n; and (vii) an genic fragment of any of (i)-(vi).

5. The method, composition or vaccine composition of any preceding claim, wherein the Teladorsagia circumcincta antigen calcium-dependent e-1 ses a sequence exhibiting 70% identity to the sequence of SEQ ID NO: 6.

6. The method, ition or vaccine composition of any preceding claim, wherein the animal is an ovine , sheep or goat.

7. The method, composition or vaccine composition of any preceding claim, wherein the antigen(s) is/are recombinant antigens.

8. The method, composition or vaccine composition of any preceding claim, wherein the immune response is a protective immune response and/or the immune response reduces host T. cincta FECs and luminal T. circumcincta burdens.

9. The method, composition or vaccine composition of any preceding claim, wherein the antigen(s) are admixed with another vaccine, polypeptide, adjuvant, diluent or excipient.

10. The method, composition or e composition of any preceding claim, wherein the T. circumcincta antigen m-dependent apyrase-1 is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen ADG63133.1.

11. The method, composition or vaccine composition of any one of claims 2-11, n: (i) surface ated n is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen BQ098696.1; (ii) hage migration inhibitory factor is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen 70.1; (iii) activation associated secretory protein is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen CAD23183.1; (iv) cathepsin F-1 is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen BQ457843.1; and/or (v) excretory/secretory protein is an antigen having a sequence which is at least 70% cal to Ostertagia ostertagi antigen CAC44259.1.

12. The , composition or vaccine of claim 10 or 11, wherein the immune response is raised in a bovine host.

13. A e composition comprising Teladorsagia circumcincta antigen mdependent apyrase-1, or an immunogenic fragment thereof, and an adjuvant.

14. The vaccine composition of claim 13, wherein the Teladorsagia circumcincta n calcium-dependent apyrase-1 comprises a sequence at least 70% identical to the sequence of SEQ ID NO: 6.

15. The vaccine composition of claim 13 or 14, further comprising one or more other Teladorsagia circumcincta ns selected from the group consisting of; (i) cathepsin F-1; (ii) excretory/secretory protein; (iii) transforming growth protein 2-like protein; (iv) tion associated secretory protein; (v) macrophage migration inhibitory factor; (vi) surface associated antigen; and (vii) an immunogenic fragment of any of i).

16. The vaccine composition of claims 13, 14 or 15, wherein: (i) calcium-dependent apyrase-1 is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen ADG63133.1; (ii) surface associated antigen is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen BQ098696.1; (iii) macrophage migration inhibitory factor is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi n BQ457770.1; (iv) activation associated secretory protein is an antigen having a sequence which is at least 70% identical to Ostertagia ostertagi antigen CAD23183.1; (v) cathepsin F-1 is an antigen having a sequence which is at least 70% identical to Ostertagia agi antigen 43.1; and/or (vi) excretory/secretory protein is an antigen having a sequence which is at least 70% cal to Ostertagia ostertagi antigen CAC44259.1. [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk [Annotation] jyk None set by jyk [Annotation] jyk MigrationNone set by jyk [Annotation] jyk Unmarked set by jyk ......... -..... ......... CD T l 84 T "'"�'"' �''l'' .. 82 I '"l ''' ''-" ..,,. 79 � I \I"'., -.: "'' 77 \ � " 75 l .,/ l- , � immunisation 72 to first 1-· - 70 68 i \ ' • 65 , ,, 1 : \ . i $: 63 61 experiment relative 58 Day 42, begins 56 Day of 3rd nisation, trickle infection -200 150 50 O 1 2 � (LI E E l"a i.. C) ..... GJ c. V> C) C) (LI - (I) - c: 100 ::i: 0 u C) C) (LI � u (LI � u.