Abstract:
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 may be exploited to provide compositions and vaccines for raising protective immune responses in animals—the protective immune responses serving to reduce, prevent, treat or eliminate  Teladorsagia  infections/infestations.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to nematode antigens capable of raising host immune responses. In particular, the invention provides vaccines for use in protecting against and/or reducing instances of  Teladorsagia  infections. 
       BACKGROUND OF THE INVENTION 
       [0002]      Teladorsagia circumcincta  (previously known as  Ostertagia circumcincta ) is the major cause of parasitic gastroenteritis in small ruminants in temperate regions. This nematode is controlled primarily by anthelmintics; however resistance is widespread and field isolates have often been found to be insensitive to a number of different anthelmintic classes (Bartley et al., 2004; Wrigley et al., 2006).  T. circumcincta  resides within the abomasum (or true stomach) of small ruminants 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 &amp; 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). 
         [0003]    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. circumcincta , resistance to the parasites can lapse around the time of parturition and early lactation (Houdijk et al., 2005). In terms of anti-parasite effects, the protective immune response has been shown to decrease 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 nematode length (Halliday 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). 
         [0004]    As sheep can acquire a protective immune response against  T. circumcincta  in natural and experimental circumstances, vaccination represents a possible alternative for control. 
       SUMMARY OF THE INVENTION 
       [0005]    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 vaccines for raising protective immune responses in animals—the protective immune responses serving to reduce, prevent, treat or eliminate  Teladorsagia  infections/infestations. 
         [0006]    In a first aspect, the present invention provides one or more  Teladorsagia  antigen(s) or a fragment thereof, for use in raising an immune response in an animal. 
         [0007]    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 invention, the term “protective immune response” may embrace 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  parasites, 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: namely, 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 establishing 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. 
         [0008]    One of skill would appreciate that any reduction in pathogen burden/FEC achieved through use of the antigen(s) described 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. 
         [0009]    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. 
         [0010]    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. 
         [0011]    In a third aspect, the invention provides the use of one or more  Teladorsagia  antigens or a fragment(s) thereof for the manufacture of a medicament for use in the treatment and/or prevention of an infection/colonisation by/with a  Teladorsagia  pathogen. 
         [0012]    In a fourth aspect, the invention provides a method of raising an anti- Teladorsagia  immune response 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. 
         [0013]    Advantageously, 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. 
         [0014]    The term “animal” encompasses animals collectively known as ovine animals. As such, the invention provides antigens and compositions for use in raising immune responses in ovine subjects such as sheep and goats—hosts of the  Teladorsagia circumcincta  parasite. 
         [0015]    As such, one embodiment of this invention provides:
       (i) one or more antigens derived from  T. circumcincta;      (ii) compositions and medicaments comprising one or more antigens derived from  T. circumcincta ; and   (iii) methods exploiting one or more antigens derived from  T. circumcincta;          
 
         [0019]    for use in raising immune responses in ovine animals (including sheep and/or goats). 
         [0020]    It should be understood that all references to “antigen” encompass immunogenic components or compounds derived from  Teladorsagia , and in particular,  T. circumcincta . In one embodiment, the term “antigen” encompasses  Teladorsagia  antigens which elicit or mimic immune responses occurring during a natural infection. The term natural infection may encompass environmentally/community acquired infections. 
         [0021]    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 embodiment, antigens to be exploited in this invention may be antigens 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”. 
         [0022]    In one embodiment, the antigens provided by this invention are antigens, which elicit host antibody (for example, IgA and/or IgG) responses. In one embodiment, the antigens 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  larvae, but may also be secreted by adult stage parasites. Additionally or alternatively, the  Teladorsagia  antigens described herein may comprise pathogen derived immunomodulatory compounds. 
         [0023]    In a further embodiment, the term “antigen” encompasses the exemplary  T. circumcincta  (Tci) antigens listed as (i)-(ix) below:
       (i) calcium-dependent apyrase-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, variant or derivative of any of (i)-(viii).       
 
         [0033]    An exemplary Tci-SAA-1 sequence is deposited under the accession number CAQ43040 and comprises the sequence given below as SEQ ID NO: 1. 
       SEQ ID NO: 1 
       [0034]      
         [0000]    
       
         
               
               
             
           
               
                   
                 mfcrvtvavl llaysahagf fddvsglasd vgdfftkqfn 
               
               
                   
                   
               
               
                   
                 nvkdlfannq selekniqrv kdllmaikek akmlepmand 
               
               
                   
                   
               
               
                   
                 aqkktisevn nymqqvdafg aqvkrdgeak feqnkakwqd 
               
               
                   
                   
               
               
                   
                 mlnnifekgg lenvmklmnlk satqctvmaal iapvilaftr 
               
             
          
         
       
     
         [0035]    An exemplary Tci-MIF-1 sequence is deposited under the accession number CB168362 and comprises the sequence given below as SEQ ID NO: 2. 
       SEQ ID NO: 2 
       [0036]      
         [0000]    
       
         
               
               
             
           
               
                   
                 Mpvfsfhtnv sadkvtpdll kqissvvari lhkpesyvcv 
               
               
                   
                   
               
               
                   
                 hvvpdqqmif dgtdgpcgvg vlksiggvgg sknnehakal 
               
               
                   
                   
               
               
                   
                 falikdhlgi agnrmyiefi digaadiafn srtfa 
               
             
          
         
       
     
         [0037]    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 
       [0038]      
         [0000]    
       
         
               
               
             
           
               
                   
                 mftpigiavl ylalvtphak agfccpadld qtdearkill 
               
               
                   
                   
               
               
                   
                 nfhnevrrdv ssaspllnlt gavlmrnvlg paknmykmdw 
               
               
                   
                   
               
               
                   
                 dcnlekkale mispctvplp idtsipqnla qwllyrkmee 
               
               
                   
                   
               
               
                   
                 tevlekapws wviaslrnlk ndteadlynw kirtisniln 
               
               
                   
                   
               
               
                   
                 wrntkvgcah kvcqfptgtn mviscayggd klennevvwq 
               
               
                   
                   
               
               
                   
                 kgptcecnay pdsyccnnlc dtkaaaalre epcksn 
               
             
          
         
       
     
         [0039]    An exemplary Tci-TGH-2 sequence is deposited under the accession number ACR27078 and comprises the sequence given below as SEQ ID NO: 4. 
       SEQ ID NO: 4 
       [0040]      
         [0000]    
       
         
               
               
             
           
               
                   
                 mrllnsmgmq eppnvdsidl spstieemle slgendkleq 
               
               
                   
                   
               
               
                   
                 dqeektfima vdpsdgidpd mlvarfpvsi ttmvrkvsra 
               
               
                   
                   
               
               
                   
                 ylhvylhvse plpepeivtv vvrerllngd vgdivatnpv 
               
               
                   
                   
               
               
                   
                 eiqrsgkavl plrasdverw wksepilgly vvamlngeni 
               
               
                   
                   
               
               
                   
                 avhpqqdhha rhtmfmsvil asdaksrgkr spsvcmpedq 
               
               
                   
                   
               
               
                   
                 epgcclydli vdfqqigwkf iiaphkynay mcrgdcsvnh 
               
               
                   
                   
               
               
                   
                 thvtrsghtk vaktgiitrq datgnqgmcc hpaeydavrm 
               
               
                   
                   
               
               
                   
                 iymngdnqvt marvpgmiar kctcs 
               
             
          
         
       
     
         [0041]    An exemplary Tci-CF-1 sequence is deposited under the accession number ABA01328 and comprises the sequence given below as SEQ ID NO: 5. 
       SEQ ID NO: 5 
       [0042]      
         [0000]    
       
         
               
               
             
           
               
                   
                 msllflllip hlfaatvkqq ysggvkplte lrtdlidkkt 
               
               
                   
                   
               
               
                   
                 kgsiefarlg qhispkdfga wnhftsfier hdkvyrnese 
               
               
                   
                   
               
               
                   
                 alkrfgifkr nleiirsaqe ndkgtaiygi nqfadlspee 
               
               
                   
                   
               
               
                   
                 fkkthlphtw kqpdhpnriv dlaaegvdpk eplpesfdwr 
               
               
                   
                   
               
               
                   
                 ehgavtkvkt eghcaacwaf svtgniegqw flakkklvsl 
               
               
                   
                   
               
               
                   
                 saqqlldcdv vdegcnggfp ldaykeivrm gglepedkyp 
               
               
                   
                   
               
               
                   
                 yeakaeqcrl vpsdiavyin gsvelphdee kmrawlvkkg 
               
               
                   
                   
               
               
                   
                 pisigitvdd iqfykggvsr pttcrlssmi hgallvgygv 
               
               
                   
                   
               
               
                   
                 eknipywiik nswgpnwged gyyrmvrgen acrinrfpts 
               
               
                   
                   
               
               
                   
                 avvl 
               
             
          
         
       
     
         [0043]    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 
       [0044]      
         [0000]    
       
         
               
               
             
           
               
                   
                 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 evtdeptety vtvftidgei 
               
               
                   
                   
               
               
                   
                 llddqkldgn ykfeglyfi 
               
             
          
         
       
     
         [0045]    An exemplary Tci-MEP-1 sequence is given below as SEQ ID NO: 7. 
       SEQ ID NO: 7 
       [0046]      
         [0000]    
       
         
               
             
           
               
                 mrlavlllylvvsaqaglldkvkdffkggnfgektktatlskfkklfek 
               
               
                   
               
               
                 tgilslgnklaemrskvmkklelskakkaevdrklkeveermdntvenl 
               
               
                   
               
               
                 kdtifeinavknvgeslfqsdilltkrqveevmdgveggrpkrqafkdq 
               
               
                   
               
               
                 nypnttwqqgvfyrfddsadyytrkvfemgtkqweeatcidfkedkekk 
               
               
                   
               
               
                 aensiilikedgcwsyvgqvggeqplslgdgceqvgiathelghalglf 
               
               
                   
               
               
                 htmsrydrddfitvvlenvvegfvdqyiketpqtttnygftydygsimh 
               
               
                   
               
               
                 ygassashnnkptmvandtryqesmgsqiisfidksmindhynckadcp 
               
               
                   
               
               
                 katsakcqnggfphprkcsecicpsgyggalcdqrptgcgqtlkakesk 
               
               
                   
               
               
                 qflidklgfpsgvrdeftfcnhwieapegkkielkinsishgyahdgci 
               
               
                   
               
               
                 lggveiktsedqtrtgfrfcspndrntvlvsasnrvpiitfnrsgqgqi 
               
               
                   
               
               
                 ileykvvs 
               
             
          
         
       
     
         [0047]    An exemplary Tci-ES20 sequence is given below as SEQ ID NO: 8. 
       SEQ ID NO: 8 
       [0048]      
         [0000]    
       
         
               
             
           
               
                 mlrsillilvsasvyvsvqgqgngdmkkvelymgyakkdmekvreflkl 
               
               
                   
               
               
                 kderltkllsdlfryldkttfewmkdeatleqfiqtrgkfssalvhpdv 
               
               
                   
               
               
                 qkrykdnrklwafryarlmnciggsdmgrataylpgvsvqekeetlrys 
               
               
                   
               
               
                 lklertcaytyfr 
               
             
          
         
       
     
         [0049]    As such, one embodiment 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 sequences 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. 
         [0050]    Advantageously, the invention provides vaccine compositions comprising 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. 
         [0051]    SEQ ID NOS: 1-8 above (and any fragments, 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 antigens given as (i)-(viii) 
         [0052]    In addition 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 antigenic or immunogenic fragments or epitopes capable of eliciting an immune response in an animal. Advantageously, the antigen fragments described 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 invention are capable of providing protective immune responses against  T. circumcincta  in ovine animals. 
         [0053]    In other embodiments, the term “antigen” or “antigen fragment” may encompass variants or derivatives of any of the antigen(s) described herein—such antigens being referred to as “variant” or “derivative” antigens. Again, it should be understood that these terms include variants/derivatives of any of the antigens 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 comprises one or more nucleobase and/or amino acid substitutions, inversions, additions and/or deletions relative to a reference sequence. 
         [0054]    One of skill will appreciate that the term “substitution” may encompass one or more conservative substitution(s). One of skill in this field will understand that the term “conservative substitution” is intended to embrace the act of replacing one or more amino acids of a protein or peptide with an alternate amino acid with similar properties and which does not substantially alter the physico-chemical properties and/or structure or function of the native (or wild type) protein. 
         [0055]    In the context of this invention, a variant/derivative antigen may comprise or be encoded by a mutant sequence which when compared to a reference sequence (such as for example a wild type sequence (including sequences encoding any of the specific  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. 
         [0056]    An antigen which may be regarded as a derivative may further comprise one or more features of a fragment or variant described herein optionally in combination with one or more modifications to the structure of the antigen or one or more of the amino acid residues thereof. 
         [0057]    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 encoding) the complete wild-type or native  Teladorsagia  (for example  T. circumcincta ) antigen, to about n-1 residues. In certain embodiments, the fragments, variants and/or derivatives provided by this invention comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300 residues—the upper limit (n−1) depending upon the size (n) of the nucleic acid encoding the complete antigen or the number (n) of amino acid residues comprising the primary sequence of the antigen. 
         [0058]    Additionally, or alternatively, the fragments, variants and/or derivatives provided 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 identical to the various reference sequences provided herein. 
         [0059]    The degree of (or percentage) “homology” between two or more (amino acid or nucleic acid) sequences may be determined by aligning two or more sequences and determining the number of aligned residues which are identical or which are not identical but which differ by redundant nucleotide substitutions (the redundant nucleotide substitution having no effect upon the amino acid encoded by a particular codon, or conservative amino acid substitutions. 
         [0060]    A degree (or percentage) “identity” between two or more (amino acid or nucleic acid) sequences may also be determined by aligning the sequences and ascertaining the number of exact residue matches between the aligned sequences and dividing this number by the number of total residues compared—multiplying the resultant figure by 100 would yield the percentage identity between the sequences. 
         [0061]    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 (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); and   (viii) surface associated antigen (Tci-SAA-1).       
 
         [0070]    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). 
         [0071]    The inventors have discovered that animals (in particular sheep) administered a vaccine composition comprising eight separate  T. circumcincta  antigens (for example, 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 vaccines and vaccine compositions. For example, the vaccines provided by this invention have been observed to reduce host FECs and luminal parasite burdens by approximately 10%-90%, 15%-85%, 20%-80%, 25%-75% or 30%-70%. 
         [0072]    Without wishing to be bound by theory, the inventors hypothesise that the success of the vaccines and vaccine compositions described 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. 
         [0073]    Antigens to be exploited in this invention may be obtained using recombinant technology. In one embodiment, an expression vector comprising one or more nucleic acid sequences encoding a  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. 
         [0074]    Protocols for the recombinant preparation of any of the antigens provided by this invention are described 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. 
         [0075]    In view of the above, the invention 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 expression systems such as those known as pET, pPICZ, pSUMO and/or pGST. Vectors according to this invention may otherwise be referred to as “nucleic acid constructs”. 
         [0076]    In a further aspect, the present invention provides host cells transfected or transformed with a vector as described herein. Eukaryotic or prokaryotic cells, such as, for example plant, insect, mammalian, fungal and/or bacterial cells, may be transfected with one or more of the vectors described herein. One of skill in this field will be familiar with the techniques used to introduce heterologous or foreign nucleic acid sequences, such as expression vectors, into cells and these may include, for example, heat-shock treatment, use of one or more chemicals (such as calcium phosphate) to induce transformation/transfection, the use of viral carriers, microinjection and/or techniques such as electroporation. Further information regarding transformation/transfection techniques may be found in Current Protocols in Molecular Biology, Ausuble, F. M., ea., John Wiley &amp; Sons, N.Y. (1989) which is incorporated herein by reference. 
         [0077]    In one embodiment, the host cell is a bacterial cell such as, for example, an  Escherichia coli  cell. 
         [0078]    In view of the above, the present invention further provides a process for the production of a recombinant  Teladorsagia  antigen encoded by any of the sequences described herein (or an immunogenic fragment thereof), which recombinant antigen (or immunogenic fragment thereof) is for use in raising an immune response in an animal (for example an ovine), said method comprising the step of (a) transforming a host cell with a nucleic acid sequence according to this invention (e.g. a nucleic acid encoding 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 expressed recombinant protein or peptide from the cell culture and/or the culture supernatant. 
         [0079]    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 chromatography. 
         [0080]    In one embodiment, the invention provides a composition (for example a vaccine composition) comprising, consisting or substantially consisting of, each of the following recombinant  T. circumcincta  antigens:
       (i) calcium-dependent apyrase-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); and   (viii) surface associated antigen (Tci-SAA-1);       
 
         [0089]    for use in raising an immune response in an animal (for example an ovine species—including sheep and goats). 
         [0090]    In one embodiment, any of the  Teladorsagia  antigens described herein may be admixed with another component, such as another polypeptide and/or an adjuvant, diluent or excipient. In one embodiment, the vaccine compositions provided by this invention may comprise a QuilA adjuvant. Additionally, or alternatively, vaccines or vaccine compositions provided by this invention may, for example, contain viral, fungal, bacterial or other parasite antigens used to control other diseases/infections or infestations. For example, the vaccine or vaccine composition may be included within a multivalent vaccine, which includes antigens against other ovine (for example, sheep) diseases. 
         [0091]    In a still further aspect, the present invention provides an ovine population, for example a farmed population of sheep and/or goats, treated, vaccinated or immunised with a vaccine or composition described herein, said vaccine or composition comprising one or more of the  Teladorsagia  antigens described herein. 
         [0092]    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 vaccine) encoding one or more antigens encoded by SEQ ID NOS: 1-8 above or an immunogenic fragment thereof, 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. 
         [0093]    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. 
         [0094]    The (vaccine) compositions described herein, may comprise a discrete dosage unit and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing into association one or more of the  T. circumcincta  antigens described herein with liquid carriers or finely divided solid carriers or both. 
         [0095]    Compositions (the term “composition” including a vaccine compositions), suitable for oral administration wherein the carrier is a solid are most preferably presented as unit dose formulations such as boluses, capsules or tablets each containing a predetermined amount of one or more of the  Teladorsagia  antigens of this invention. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine an active compound (for example one or more  T. circumcincta  antigen(s)) in a free-flowing form such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, lubricating agent, surface-active agent or dispersing agent. Moulded tablets may be made by moulding an active compound with an inert liquid diluent. Tablets may be optionally coated and, if uncoated, may optionally be scored. Capsules may be prepared by filling an active compound, either alone or in admixture with one or more accessory ingredients, into the capsule shells and then sealing them in the usual manner. Cachets are analogous to capsules wherein an active compound together with any accessory ingredient(s) is sealed in a rice paper envelope. An active compound may also be formulated as dispersible granules, which may for example be suspended in water before administration, or sprinkled on food. The granules may be packaged, e.g., in a sachet. Formulations suitable for oral administration wherein the carrier is a liquid may be presented as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water liquid emulsion. 
         [0096]    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. 
         [0097]    Composition and vaccine compositions formulated for parenteral administration include sterile solutions or suspensions of an active compound (for example one or more  Teladorsagia  antigens) in aqueous or oleaginous vehicles. 
         [0098]    Injectable compositions and vaccines may be adapted for bolus injection or continuous infusion. Such preparations are conveniently presented in unit dose or multi-dose containers, which are sealed after introduction of the formulation until required for use. Alternatively, an active compound (for example one or more  T. circumcincta  antigens) may be in powder form that is constituted with a suitable vehicle, such as sterile, pyrogen-free water or PBS before use. 
         [0099]    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 example, suitable polymeric or hydrophobic materials, or ion-exchange resins. They may also include preparations or adjuvants known to enhance the affinity and/or longevity of an animal (for example ovine) immune response, such as single or double emulsions of oil in water. Such long-acting compositions are particularly convenient for prophylactic use. 
         [0100]    Compositions suitable (or formulated) for mucosal administration include compositions comprising particles for aerosol dispersion, or dispensed in drinking water. When dispensed such compositions should desirably have a particle diameter in the range 10 to 200 microns to enable retention in, for example, the nasal cavity; this may be achieved by, as appropriate, use of a powder of a suitable particle size or choice of an appropriate valve. Other suitable compositions include coarse powders having a particle diameter in the range 20 to 500 microns, for administration by rapid inhalation through the nasal passage from a container held close up to the nose, and nasal drops comprising 0.2 to 5% w/v of an active compound in aqueous or oily solution or suspension. 
         [0101]    It should be understood that in addition to the carrier ingredients mentioned above, the various compositions described herein may include, an appropriate one or more additional (pharmaceutically acceptable) carrier ingredients such as diluents, buffers, flavouring agents, binders, surface active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like, and substances included for the purpose of rendering the formulation isotonic with the blood of the intended recipient. 
         [0102]    Pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.1 M and preferably 0.05 M phosphate buffer or 0.8% saline. Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer&#39;s dextrose, dextrose and sodium chloride, lactated Ringer&#39;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. 
         [0103]    Compositions suitable for topical formulation may be provided for example as gels, creams or ointments. 
         [0104]    Compositions for veterinary use may conveniently be in either powder or liquid concentrate form. In accordance with standard veterinary formulation practice, conventional water-soluble excipients, such as lactose or sucrose, may be incorporated in the powders to improve their physical properties. Thus, particularly suitable powders of this invention comprise 50 to 100% w/w and preferably 60 to 80% w/w of the active ingredient(s) (for example one or more  T. circumcincta  antigens) and 0 to 50% w/w and preferably 20 to 40% w/w of conventional veterinary excipients. These powders may either be added to, for example, animal feed—perhaps by way of an intermediate premix, or diluted in animal drinking water. 
         [0105]    Liquid concentrates of this invention suitably contain one or more  T. circumcincta  antigens and may optionally further include an acceptable water-miscible solvent for veterinary use, for example polyethylene glycol, propylene glycol, glycerol, glycerol formal or such a solvent mixed with up to 30% v/v of ethanol. The liquid concentrates may be administered to the drinking water of animals. 
         [0106]    In general, a suitable dose of each the  T. circumcincta  antigens provided by this invention may be in the range of about 10 to about 100 μg protein per animal. Furthermore, the one or more antigens described herein may be administered on about 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, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg of each (or a predetermined selection of) the one or more antigens described herein. As such, where the vaccine comprises 8 antigens, the total protein content may range from about 80 μg to about 800 μg. Furthermore, each animal may be administered the antigen(s) on 2, 3, 4 or 5 occasions over a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 week period. It should be understood that each animal may receive the same or a different dose of the  T. circumcincta  antigen(s) on each administration occasion. 
         [0107]    In one embodiment, a vaccine formulated for administration to sheep may comprise approximately 50 μg of each  Teladorsagia  (for example  T. circumcincta ) antigen. As such, where the vaccine comprises, for example, 8  T. circumcincta  antigens, the total protein (antigen) content may be in the region of 400 μg. Further, the vaccine may be administered three times with a three week gap between each administration. 
         [0108]    In addition to providing  T. circumcincta  antigens for use in raising immune responses in animals, the present invention may also provide polyclonal and/or monoclonal antibodies (or antigen binding fragments thereof) that bind (or have affinity or specificity for) any of the  Teladorsagia  antigens provided by this invention. Production and isolation of polyclonal/monoclonal antibodies specific for protein/peptide sequences is routine in the art, and further information can be found in, for example “Basic methods in Antibody production and characterisation” Howard &amp; Bethell, 2000, Taylor &amp; Francis Ltd. Such antibodies may be used in diagnostic procedures, to, for example detect or diagnose  T. circumcincta  infection/infestations in animal (for example ovine) species, as well as for passive immunisation. 
         [0109]    The present invention further provides a vaccine for use in preventing or controlling  T. circumcincta  infection/infestation and associated diseases in ovine hosts. The vaccine may be a polypeptide or polynucleotide vaccine. 
         [0110]    The invention further provides a method for immunising an ovine subject against  T. circumcincta  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. 
         [0111]    While this invention predominately concerns antigens derived from the nematode organism  T. circumcincta  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 necessarily limited to raising immune responses which are protective against  Teladorsagia  infections/infestations. In particular, the antigens described herein exhibit significant homology/identity to certain antigens derived from the bovine pathogen,  Ostertagia 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. 
         [0000]    
       
         
               
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 Closest 
                   
                   
                   
               
               
                 
                   Teladorsagia 
                 
                   
                   
                 
                   Ostertagia 
                 
               
               
                 
                   circumcincta 
                 
                 Accession 
                   
                 
                   ostertagi 
                 
                 % 
                 % 
                 O. ost 
               
               
                 Antigen 
                 number 
                 Function* 
                 homologue 
                 identity 
                 Coverage 
                 reference 
               
               
                   
               
             
             
               
                 Tci-SAA-1 
                 CAQ43040 
                 L3-enriched 
                 BQ098696.1 a,b   
                 94% 
                 100%  
                 Unpublished 
               
               
                   
                   
                 surface 
                   
                   
                 (aa1- 
               
               
                   
                   
                 associated 
                   
                   
                 162) 
               
               
                   
                   
                 antigen 
               
               
                 Tci-MIF-1 
                 CBI68362 
                 L3-enriched 
                 BQ457770.1 a   
                 99% 
                 91% 
                 Unpublished 
               
               
                   
                   
                 macrophage 
                   
                   
                 (aa11- 
               
               
                   
                   
                 migration 
                   
                   
                 115) 
               
               
                   
                   
                 inhibitory 
               
               
                   
                   
                 factor 
               
               
                 Tci-ASP-1 
                 CBJ15404 
                 L4-enriched 
                 CAD23183.1 
                 76% 
                 97% 
                 Mol Biochem 
               
               
                   
                   
                 activation- 
                   
                   
                 (aa5- 
                 Parasitol 
               
               
                   
                   
                 associated 
                   
                   
                 235) 
                 2003; 126, 
               
               
                   
                   
                 secretory 
                   
                   
                   
                 201-208 
               
               
                   
                   
                 protein 
               
               
                 Tci-TGH-2 
                 ACR27078 
                 Transforming 
                 No significant hit 
                 — 
                 — 
                 — 
               
               
                   
                   
                 growth 
                 in NCBI, EMBL 
               
               
                   
                   
                 protein 
                 or Nembase 4 
               
               
                   
                   
                 2-like protein 
               
               
                 Tci-CF-1 
                 ABA01328** 
                 L4-enriched 
                 BQ457843.1 a   
                 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 
                 CAD19995.2 
                 69% 
                 100%  
                 Parasitology 
               
               
                   
                 submitted*** 
                 ES 
                   
                   
                 (aa1- 
                 2002; 125, 
               
               
                   
                   
                 metalloproteinase 
                   
                   
                 498) 
                 383-391 
               
               
                 Tci-APY-1 
                 CBW38507 
                 L4-enriched 
                 ADG63133.1 
                 92% 
                 96% 
                 Parasitology 
               
               
                   
                   
                 ES calcium- 
                   
                   
                 (aa12- 
                 2011; 138, 
               
               
                   
                   
                 activated 
                   
                   
                 339) 
                 333-343 
               
               
                   
                   
                 apyrase 
               
               
                   
               
               
                 *Putative or inferred function 
               
               
                 **Tci-CF-1 is highly polymorphic, the clone used for vaccine production had following amino acid substitutions compared to published sequence. 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 position in the published sequence is in subscript: I 44             T 44 , N 101             D 101 , T 129             A 129 , R 137             Q 137 , R 305             K 305 , L 306              P 306 , S 307             Y 307   
               
               
                 ***Full length sequences not yet deposited. 
               
               
                   a From translated EST sequence 
               
               
                   b with following caveat from authors: “WARNING: Subsequent 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 .” 
               
             
          
         
       
     
         [0112]    In view of the above, it should be understood that the various aspects and embodiments of this invention (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. 
         [0113]    Moreover, in view of the levels of identity 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. ostertagi ) infections/infestations. One of skill will appreciate that the  T. circumcincta  antigens provided by this invention may be used individually or together (for example 2, 3, 4, 5, 6, 7 or all 8 of the  T. circumcincta  antigens) to raise immune responses in bovine hosts. 
         [0114]    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 responses in bovine subjects. Again, such immune responses may be protective against  Ostertagia  infections/infestations. 
         [0115]    In one embodiment, this invention extends to compositions or vaccine compositions comprising one or more of the  Ostertagia  antigens described above optionally in combination with one or more the  T. circumcincta  antigens described herein, for use in raising immune responses in bovine subjects. 
         [0116]    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  Ostertagia  pathogen in a bovine host. Similarly, the invention may also embrace methods of raising anti- Ostertagia  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 response. 
         [0117]    One of skill will appreciate that references to the  Ostertagia  antigens described above not only include antigens comprising or consisting of the sequences identified by the Accession numbers presented in the table above, but fragments thereof—in particular, fragments which are capable of raising immune responses (for example protective immune responses) in bovine animals (i.e. the fragments are antigenic and/or immunogenic) as well as mutants, variants 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  Ostertagia  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. 
     
    
     
       DETAILED DESCRIPTION 
         [0118]    The present invention will now be described in detail with reference to the following Figures which show: 
           [0119]      FIG. 1 : Effects of immunization of sheep with recombinant antigens derived from  Teladorsagia circumcincta  on faecal worm egg counts (FWEC) after challenge 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 represents 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 adjuvant only. Note that, in panel D, for Groups 1 and 2 in Trial 2, cumulative FWEC is calculated over 84 days, whereas for Groups 3 and 4 cumulative FWEC is calculated over 112 days. One “outlier” animal in Group 1 of Trial 2, sheep number 675J, is indicated. 
           [0120]      FIG. 2 . Trial 1: Lumenal  T. circumcincta  burdens of sheep in Group 1 and Group 2. Each data point represents the mean number (±SEM) of  T. circumcincta  enumerated in lumenal contents 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 overall burdens (all stages and genders). “*” denotes a significant difference between the means (P&lt;0.05), “**” denotes a highly significant difference between mean (P&lt;0.01). 
           [0121]      FIG. 3 . Trial 1: Mucosal  T. circumcincta  burdens 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&lt;0.05). 
           [0122]      FIG. 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. circumcincta  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. 
           [0123]      FIG. 5 : Weight gain of sheep in Group 1 and Group 2 from Day 0 to Day 84 of the experiment (Trial 1). 
           [0124]      FIG. 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 of these sheep are shown. “Imm” represents sheep immunized with the 8-protein cocktail; “Con” represents those immunized with Quil A adjuvant only. 
           [0125]      FIG. 7 : Effects of immunization 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  enumerated in the abomasum of each of seven sheep in Group 3 (immunized) or Group 4 (control, adjuvant only). 
           [0126]      FIG. 8 : Serum antibody responses of sheep to the recombinant proteins 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 trickle infection; “PM” is the post mortem date. 
           [0127]      FIG. 9 : Serum antibody responses of sheep to the recombinant 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 represents 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. 
           [0128]      FIG. 10 : Serum antibody responses of sheep to L4 excretory/secretory products of  Teladorsagia circumcincta . ‘Imm’ represents the days on which animals were immunized with recombinant antigen cocktail (Immunized group) or adjuvant only (Control group). 
           [0129]      FIG. 11 : Immunoblots to investigate serum IgG (Panel A) and IgA (Panel B) binding to components of somatic extracts and excretory/secretory products 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). 
           [0130]      FIG. 12 : Serum IgG responses of control, adjuvant only recipients to recombinant Tci-MEP-1 and Tci-APY-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. 
           [0131]      FIG. 13 : Mucosal 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 statistically significantly higher than those for the remaining antigens within the same treatment group. 
           [0132]      FIG. 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. 
           [0133]      FIG. 15 : Mucosal antibody levels to the recombinant proteins used to immunize 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 jointly representing sheep (points) and their antigen-specific antibody responses (axes). The arrows indicate directions of higher antigen-specific antibody response. The orthogonal projection of points onto each axis approximates the relative responses by sheep. The correlations between responses to specific 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 METHODS 
     Production of Recombinant Proteins for Immunisation 
       [0134]    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 TGF6 homologue (Tci-TGH-2) were selected because 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 (Tci-CF-1), astacin-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; Smith et al., 2009; Nisbet et al., 2010b). A final protein was chosen because of its homology to known vaccine candidate 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 Tci-APY-1 and production of recombinant versions 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 production and purification parameters were employed in the current study. For Tci-MEP-1, oligonucleotide primers for use in the rapid amplification of cDNA ends (RACE) were designed from the EST sequence CB036707 and RACE performed using the SMART™ RACE kit (Clontech) according to the manufacturer&#39;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 manufacturer&#39;s instructions. Following confirmatory sequencing, oligonucleotide primers were designed to amplify the CDS of Tci-mep-1, omitting the sequence 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 induced in the presence of 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Insoluble recombinant Tci-MEP-1 was purified from inclusion bodies solubilised in 8M urea, followed by nickel column affinity chromatography using HisTrap™ HP columns (GE Healthcare) and a step-wise imidazole gradient in the presence of 8M urea in 20 mM phosphate buffer, pH 7.6. Purified Tci-MEP-1 was then dialysed against 2M urea in 20 mM phosphate buffer, pH 7.6. The full CDS of the cDNA encoding Tci-TGH-2 (accession number FJ410914) was amplified by PCR using oligonucleotide 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 manufacturer&#39;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 cell lysates by nickel column affinity chromatography using HisTrap™ HP columns (GE Healthcare). Next, rTci-TGH-2 was eluted in 500 mM imidazole, 20 mM phosphate buffer, pH 7.6 and then dialysed against 20 mM 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 peptide) 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 expressed and then purified 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 sequence 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 pPICZaC (Invitrogen) and used to transform the yeast  Pichia 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 supernatant by nickel column affinity chromatography as described above for Tci-TGH-2. Tci-ES20, a homologue of a 20 kDa excretory/secretory (ES) protein of  Ostertagia ostertagi , was identified during an immunoscreening/proteomic analysis of immunogenic  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 constructed [using  T. circumcincta  L4 (8 days post infection, dpi) RNA] in XTriplEx2 by long-distance PCR following manufacturer&#39;s instructions (Clontech). It was packaged using Gigapack Gold III packaging extract (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 (Promega). 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 encoding Tci-ES20 was then subcloned into the vector pPICZaC (Invitrogen) and used to transform  P. pastoris  [X-33 (Mut+) strain (Invitrogen)] following 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. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Table 1. Recombinant proteins used in  Teladorsagia circumcincta  vaccine trial 
               
             
          
           
               
                   
                 Accession 
                   
                 Expression 
                   
               
               
                 Name 
                 number 
                 Function* 
                 system 
                 Reference 
               
               
                   
               
               
                 Tci-SAA-1 
                 CAQ43040 
                 L3-enriched 
                 pET22b(+) 
                 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 
               
               
                   
                   
                 secretory protein 
               
               
                 Tci-TGH-2 
                 ACR27078 
                 Transforming 
                 pET SUMO 
                 McSorley et al., 
               
               
                   
                   
                 growth protein 2- 
                   E. coli  BL21 
                 2010 
               
               
                   
                   
                 like protein 
                 (DE3)-RIL 
               
               
                 Tci-CF-1 
                 ABA01328** 
                 L4-enriched 
                 pPICZαC 
                 Redmond et 
               
               
                   
                   
                 Secreted cathepsin F 
                 
                   Pichia pastoris 
                 
                 al., 2006 
               
               
                   
                   
                   
                 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., 
               
               
                   
                   
                 calcium-activated 
                   E. coli  BL21 
                 2011 
               
               
                   
                   
                 apyrase 
                 (DE3)-RIL 
               
               
                   
               
               
                 *Putative or inferred function 
               
               
                 **Tci-CF-1 is highly polymorphic, the clone used for vaccine production had following amino acid substitutions compared to published sequence. 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 position in the published sequence is in subscript: I 44             T 44 , N 101             D 101 , T 129             A 129 , R 137             Q 137 , R 305             K 305 , L 306             P 306 , S 307             Y 307   
               
               
                 ***Full length sequences not yet deposited. These molecules have been derived from EST data in the public domain: Tci-ES20 based on CB043664, Tci-MEP-1 based on CB036707 
               
             
          
         
       
     
       Immunisation Trial 
       [0135]    Fourteen, 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 experiment. Faecal egg counts (FEC, Christie and Jackson 1982), performed prior to initiation of the experiment, confirmed that all animals 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 administered 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 (Christie and Jackson 1982) three times per week (Monday, Wednesday and Friday) 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, abomasal 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 following standard techniques. The percentage of stunted or “inhibited” larvae was determined, based on size, as described previously (Halliday et al., 2010). The experiment was performed under the regulations of a UK Home Office Project Licence. 
       Trial 2 
       [0136]    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 excreting 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 urea/PBS/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 regulations of a UK Home Office Project Licence and the experimental design was ratified by the Moredun Research Institute Experiments and Ethics Committee. 
       Measurement of Antibody Responses to Recombinant Antigens 
       [0137]    Following initial antibody:antigen titrations to ensure optimisation of the technique, antigen-specific antibody levels in serum and abomasal mucus samples 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 washing, 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 1 h at room temperature. Wells were re-washed and 50 μl horseradish peroxidase-conjugated polyclonal mouse anti sheep/goat IgG (A9452, Sigma) at 1:1000 or 50 μl mouse anti-bovine/ovine IgA monoclonal antibody (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 o-phenylenediamine dihydrochloride substrate (OPD, Sigma) to each well. After 15 min in darkness, the reaction was stopped by addition of 25 μl 2.5M H 2 SO 4  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. 
         [0000]    Measurement of Antibody Responses to Native  T. circumcincta  Antigens 
         [0138]    Antigen-specific IgG levels in the sera of sheep which had been immunized with the recombinant antigen cocktail, 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 assessed in all sera from animals in Trial 1 and from four, randomly selected, animals from Groups 1 and 2 of Trial 2. All experimental conditions were as described, above, for the determination of recombinant antigen-specific IgG levels in serum by ELISA. 
       Immunoblotting of Nematode Somatic Extracts 
       [0139]    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 immunoblotting 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 components 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 
       [0140]    A generalised additive 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 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, repeated measures over time were modelled by random intercept and slope LMMs also including time and its interaction with group as a fixed effect. Heterogeneous 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. 
         [0141]    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 statistically 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 negative binomial error distribution. Where necessary, overdispersion was incorporated using Poisson GLMs correcting the standard errors by specifying the mean and variance relationship. Nematode 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. 
         [0142]    Model selection was based on the Akaike&#39;s information criterion (AIC) and likelihood ratio tests (LRT) (Akaike, 1974). The mixed models were fitted by residual maximum likelihood (REML; Smouse and Kojina, 1972). Throughout the data analysis some animal measurements were identified as outliers. Their influence on parameter estimates was considered in each case. The Cook&#39;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 version 2.13. 
       Results 
     FECs Analysis 
       [0143]    Trial 1: FEC data is shown in  FIG. 1A  and B. Sheep in both immunised and control groups began to excrete trichostrongyle type eggs in their faeces from 16-19 days after the start of the trickle challenge. In both groups, FECs rose until 23 days after the start of challenge. Thereafter, sheep in Group 1 excreted substantially fewer eggs than those in Group 2. By the end of the experiment, 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&lt;0.001), and a significant treatment×time interaction (P=0.20). The mean cumulative FECs for the duration 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 reduction 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 ( FIG. 1B ). 
         [0144]    In Trial 2, sheep began to excrete nematode eggs from 14-16 days after challenge ( FIG. 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, calculated 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,  FIG. 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,  FIG. 1D ). Influence was assessed using Cook&#39;s distance criterion (Cook, 1977): 675J was regarded as a “highly influential” case (Cook&#39;s distance=0.3129 based on a LMM model). 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), representing an overall mean FEC reduction of 58% in the immunized versus the control group ( FIG. 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: Preliminary Analysis 
       [0145]    Abomasal  T. circumcincta  enumerations were subdivided into lumenal and mucosal burdens. Within the lumen, Group 1 sheep had significantly fewer adult male (P=0.004) and female  T. circumcincta  (P=0.011,  FIG. 2A ) than was observed 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 ( FIG. 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) ( FIG. 3 ). There was no significant difference between the numbers 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 ( FIG. 3 ). 
       Trial 1: Supplementary Analysis of Total Worms Numbers (Lumenal Plus Mucosal) 
       [0146]    Immunized sheep (Group 1) harboured 55% fewer  T. circumcincta  (total of adults and larvae) at necropsy than control, adjuvant only (Group 2) sheep (P=0.011,  FIG. 4A ). Group 1 sheep had statistically-significantly lower mean adult nematode burdens than sheep in Group 2 (75% reduction, P=0.0066,  FIG. 4B ). Comparison of juvenile nematode burdens in the abomasum indicated no significant differences between the two groups ( FIG. 4C ). No significant differences were observed in the length of worms recovered from the different groups (data not shown). 
       Liveweight Gain 
       [0147]    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) ( FIG. 5 ). 
       Trial 2 
     Groups 1 and 2 (Post Mortem at Day 84): 
       [0148]    The total abomasal nematode burdens (adults and larvae) in immunized sheep were not statistically significantly different to the control, 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. Comparison of the juvenile nematode burdens indicated that immunized sheep had fewer juvenile nematodes than control, adjuvant only sheep in the abomasal lumen (Group 1: 50±42; Group 2: 218±81), ( FIG. 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,  FIG. 6 ). 
       Groups 3 and 4 (Post Mortem at Day 112): 
       [0149]    Immunised sheep (Group 3) harboured 57% fewer  T. circumcincta  total nematodes at necropsy than did the control, adjuvant only (Group 4) recipients (P=0.0199,  FIG. 7 ). In both Groups 3 and 4, adult worms comprised 99% of the total nematode burden and no significant difference in the numbers of juvenile stages was observed between the two Groups. 
         [0000]    Measurement of Serum Antibody Responses to  T. circumcincta  Antigens 
         [0150]    In both trials, following tertiary immunization, serum IgG levels against all recombinant proteins reached peak levels, which declined slowly thereafter ( FIGS. 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 ( FIGS. 8B and 9B ). Following immunization with the recombinant antigens, sheep produced serum IgG, prior to parasite challenge, which bound native L4 ES components ( FIG. 10 ). The nature of the immunoreactive antigens in this ES material, and other  T. circumcincta  extracts, was investigated further by immunoblotting: IgG bound to parasite components, 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) ( FIG. 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, Tci-APY-1 (Adult only) and Tci-MEP-1 ( FIG. 11B ). In addition IgA bound an unknown parasite component of ca. 43 kDa in L3 somatic extract. 
         [0151]    In both Trials 1 and 2, from 14 days after initiation of challenge, control, adjuvant only recipients generated serum IgG that bound recombinant Tci-MEP-1 and Tci-APY-1 ( FIG. 12 ). Antigen-specific serum IgA which bound to the recombinant proteins was not observed in the control, adjuvant only recipients (data not shown). 
         [0152]    Measurement of Antibody Responses to Recombinant Antigens in Abomasal Mucus 
         [0153]    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, adjuvant only recipients for each protein ( FIGS. 13A and 14A ). In Trial 1, mean Tci-APY-1-, Tci-MEP-1-, and Tci-CF-1-specific IgG levels were significantly higher than those measured against the other five recombinants (P&lt;0.0001), whereas in Trial 2, mean Tci-MEP-1-specific IgG levels were significantly higher than responses to the remaining antigens (Day 84 necropsy) while Tci-MEP-1- and Tci-APY-1-specific IgG levels were significantly higher at the Day 112 necropsy. A joint biplot representation of animals and antigen-specific mucosal IgG responses ( FIGS. 15A and 15B ) illustrates the relationships between treatments, between animals within groups, with respect to IgG responses to the different antigens and overall differences between immunized and control, adjuvant only sheep. 
         [0154]    Mucosal Tci-APY-1- and Tci-MEP-1-specific IgA levels were significantly higher than those directed against the other six recombinant antigens in Trial 1 and 2 ( FIGS. 13B and 14B ). The overall differences between immunized sheep and adjuvant only recipients are represented in joint biplots of animals and antigen-specific mucosal IgA responses in  FIGS. 15C and 15D . 
       DISCUSSION 
       [0155]    Here, we demonstrated that immunisation 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 against this nematode species using a recombinant 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 vaccination 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 reduced (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. 
         [0156]    Other attempts to protect sheep against  T. circumcincta  using native antigen preparations, for example lectin-binding integral membrane glycoproteins, have not been successful (Smith et al., 2001). This general 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 ASP-enriched 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 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 “pragmatic” 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.    
         [0157]    The approach to antigen identification described herein was substantially different to the pragmatic approach, and followed a more targeted approach by attempting to mimic and exploit elements of the natural, successful immune response to  T. circumcincta  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 comparing 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 [Ac-SAA-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 immunosuppressive 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 targets of IgA present in mucus derived from immune sheep and then combined them into a multi-component vaccine which aimed to provoke the host immune system to respond 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 vaccination trials using single recombinant antigen preparations of homologues of some of the molecules described herein, in different nematode/host models, have failed. In  O. ostertagi , for example, the astacin-like metalloproteinase MET-1, which shares &gt;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 &gt;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 identity) failed to induce significant protection against L3 challenge in a hamster model (Xiao et al., 2008). 
         [0158]    The mechanism of action of the vaccine used herein is not yet clear. These nematodes are acquired by ingestion of L3 from pasture. Thereafter, the developing parasites (L3 and L4) and adult worms reside in the host&#39;s abomasum. Protective immunity against  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 burdens in vaccinated and adjuvant only groups were similar at day 84, so it seems unlikely that exclusion and expulsion of incoming 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 ascribed 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. 
         [0159]    The immune mechanisms responsible for the observed 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 secretions 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). 
         [0160]    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 &gt;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 “substantial benefits” can be gained by using a vaccine that is 60% effective in 80% of the flock, if the vaccine is based on the stimulation of ‘natural 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 efficacy and further work will seek to clarify this and also to confirm the anti-parasite effects of the 8-protein cocktail vaccine. 
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