Patent Application: US-201013516909-A

Abstract:
the present invention provides one or more antigens from the fourth stage larvae of non - blood feeding parasitic nematodes , for raising an immune responses in an animals , in particular bovines . the invention further provides methods of making immunogenic and / or vaccine compositions .

Description:
the present invention will now be described in detail with reference to the following figures which show : fig1 . flow chart of the methods used to prepare the antigen fractions for the calf vaccination trials . fig2 . sds - page and immunoblot analysis of cona binding membrane proteins from h . contortus and o . ostertagi . a , coomassie blue stained . b , immunoblot probed with sera from calves immunised with cona binding membrane proteins from adult o . ostertagi ( smith 29 ). m . molecular weight markers . lanes 1 and 4 , adult h contortus ; lanes 2 and 5 , adult o . ostertagi ; lanes 3 and 6 , l4 o . ostertagi . lanes 1 - 3 , non reducing sds - page . lanes 4 - 6 reducing sds - page . fig3 . kinetics of the antibody response following vaccination of calves with o . ostertagi l4 antigens or adjuvant alone in experiment i . closed squares = vaccinates ; open triangles = controls . fig4 . group mean faecal egg counts of vaccinated and control calves in experiment 1 . closed squares = vaccinated animals ; open triangles controls . epg = eggs per gram . fig5 . group mean worm counts of vaccinated and control calves in experiment 1 . fig6 . mass spectrometry fingerprint analysis of the protective cona binding fraction used in trial 1 . fig7 . sds - page of the preparations used as antigens in trial 2 . lane 1 = cona binding fraction ; lane 2 = pool 1 ; lanes 3 and 4 were combined to provide pool 2 and lane 5 = pool 3 . the remaining lanes contain molecular weight markers . fig8 . kinetics of the antibody response following vaccination of calves with o . ostertagi l4 fractions or adjuvant alone in trial 2 . closed squares = group 2 . 1 . open squares group 2 . 2 . closed circles = group 2 . 3 . open circles = group 2 . 4 . open triangles = group 2 . 5 ( controls ). fig9 . group mean faecal egg counts of vaccinated and control calves in trial 2 . closed squares = group 2 . 1 . open squares = group 2 . 2 . closed circles = group 2 . 3 . open circles = group 2 . 4 . open triangles group 2 . 5 ( controls ). epg = eggs per gram . fig1 . group mean worm counts of the calves in trial 2 . *, p & lt ; 0 . 05 . fig1 . chromatography and gel profiles of the preparations used as antigens in trial 3 . fig1 . group mean faecal egg counts of vaccinated and control calves in trial 3 . epg = eggs per gram . fig1 . group mean cumulative faecal egg counts of vaccinated and control calves in trial 3 . epg = eggs per gram . fig1 . mass spectrometry fingerprint analysis of the protective peak 3 fraction used in trial 3 . fig1 . chromatography and gel profiles of the preparations used as antigens in trial 4 . fig1 , group mean faecal egg counts of vaccinated and control calves in trial 4 . epg = eggs per gram . all calves were reared and housed indoors in conditions designed to exclude accidental infection with nematode parasites . those used as donors for o . ostertagi eggs or fourth stage larvae were of various breeds and aged between 3 and 12 months at the time of infection . those used in the vaccine trials were castrated holstein - fresian crosses aged between 6 and 12 months at the start of each trial . infective larvae were from strains of o . ostertagi which have been maintained at moredun research institute for several years . the methods for faecal egg counting and enumeration of worm burdens have been described before ( 31 , 32 ) . fourth stage o . ostertagi larvae were harvested from donor calves which had been infected with a single dose of approximately 200 , 000 l3 seven days earlier . soon after the animals had been killed by captive bolt and pithing , the abomasums were removed and the contents discarded . after a brief rinse in warm saline , each abomasum was pinned mucosal surface uppermost to a block of polystyrene which was then inverted and floated in a large baermann funnel containing warm saline . following four hours at 37 ° c ., fourth stage larvae were drained from the base of the funnel . the funnels were then incubated at 4 ° c . overnight by which time any larvae still in suspension had settled out and could be drawn off . all larvae were frozen at − 70 ° c . until required for antigen extraction . prior to sds - page samples were heated at 100 ° c . for 3 min in an equal volume of 63 mm tris - hcl ph6 . 8 containing 5 % ( w / v ) sds , ± 10 mm dtt under non - reducing or reducing ( 10 mm dtt ) conditions and separated on 4 - 12 % gradient acrylamide gels ( biorad , hercules , calif ., usa ). molecular weight markers ( fermentas , burlington , ontario , canada ) were run on each gel and the gels were either stained with coomassie blue r250 ( sigma , st . louis , mo ., usa ) ( 0 . 025 % in 40 % methanol / 10 % acetic acid ) and destained in 20 % methanol / 10 % acetic acid , or silver stained as follows . after sds - page the gels were washed 3 times in distilled water , and then fixed overnight in 40 % methanol / 10 % glacial acetic acid . this was followed by incubation for 20 min in 20 % methanol / 5 % acetic acid then 4 × 15 min washes in distilled water . gels were then incubated in 50 ml 5 mg / l dtt for 45 min , then for 40 min in 50 ml 0 . 1 % w / v agno 3 , followed by 2 rapid washes in water and 2 washes in 25 ml 3 % na 2 co 3 . the gels were then developed in 50 ml 3 % na 2 co 3 with the addition of 25 μl formalin , and the development stopped after 15 min by adding 20 ml 2 . 3 m citric acid . sds - page separated proteins were transferred to pvdf membrane ( millipore , billerica , mass ., usa ) using a semi - dry apparatus . membranes were blocked in 10 % marvel ( premier foods international , spalding , lincs ., uk ) in 10 mm tris , 0 . 5m nacl , 0 . 05 % ( v / v ) tween - 20 , 0 . 02 % ( w / v ) thimerosal ( tntt ), the assay diluent and wash buffer , overnight at 4 ° c . periodate treatment was carried out by washing the membrane twice , for 20 minutes , in 50 mm naac ph 4 . 5 , then incubating for 1 h in 50 mm nalo 4 / 50 mm naac , in the dark at room temperature . after further washes of 2 × 10 minutes in 50 mm naac , then 2 × 10 minutes in tntt , the membrane was incubated for 30 minutes in 50 mm nabh 4 , after which it was washed for 3 × 10 minutes in tntt . membrane strips were incubated with pooled serum samples from each group , diluted 1 / 300 in tntt , for 2 h at room temperature . they were then washed 3 × 5 minutes in tntt , then incubated with rabbit anti - bovine immunoglobulin horseradish peroxidise conjugated antibody diluted 1 / 1000 in tntt ( p0159 , dakocytomation , glostrup , denmark ). these were estimated by the bicinchoninic protein assay reagent according to the manufacturer &# 39 ; s instructions ( pierce , thermo fisher scientific inc ., waltham , mass ., usa ). triton x - 100 extracts of ostertagia l4 membranes were prepared as detailed for haemonchus ( 33 ) , and diluted four - fold with 10mm tris - hc 1 , 0 . 5m nacl , 0 . 05 % nan 3 , 10μm mncl 2 , 100μm cac 1 2 , ph 7 . 4 ( lectin wash buffer , lwb ). the solution was pumped ( 8ml / h ) at 4 ° c . through concanavalina ( cona ) lectin cross linked to agarose beads ( vector laboratories , burlingame , ca , usa ) contained in a column . after thorough washing in lwb / 0 . 5 % reduced triton x - 100 the column was eluted with lwb / 0 . 25 % chaps / 0 . 2m methylmannopyranoside / 0 . 2m methylglucopyranoside ( fig1 flow chart ). for elution , sufficient sugar solution was pumped onto each column to cover the beads , then the flow was stopped for approximately one hour . the pump was re - started and the peak monitored at od 280 was retained as the “ 1 hour eluate ”. the elution process was then repeated exactly , except that the flow was stopped overnight to produce an “ overnight eluate ”. the eluates were pooled and passed through a column of sephadex g - 25 to remove the sugar and exchange the buffer to 10mm tris - hc 1 , 0 . 1 % chaps , ph 7 . 4 and stored at - 70 ° c . before use as immunogens . the cona eluate was fractionated on a monoq anion exchange column , 1 ml bed volume ( pharmacia , pfizer , kent , uk ) equilibrated in 10 mm tris / 0 . 1 % chaps ph7 . 4 . the cona eluate was applied to the column ( 1 ml / min ), and unbound proteins were collected . the bound proteins were eluted by a linear gradient increase in nacl from 0 m to 1 m over 20 ml , with 10 × 2 ml fractions being collected . the fractions were then pooled as follows :— pool 1 = unbound material and proteins eluted with up to 0 . 1m nacl ; pool 2 = fractions eluted between 0 . 1 and 0 . 5m nacl and pool 3 = fractions eluted between 0 . 5 and 1 . 0m nacl ( fig flow chart ). further batches of pool 2 and 3 material were prepared and fractionated by gel filtration using a superose 12 column equilibrated with 10 mm tris , 0 . 1 % chaps , 0 . 5m nacl ph 7 . 4 and flowing at 0 . 5 ml / min . two hundred ul of pool 2 containing 0 . 75 μg of protein was separated in 2 runs was fractionated on a single 30 cm column . pool 3 was fractionated under identical conditions except two 30 cm columns were coupled in series to improve the resolution . microtitre plates were coated overnight at 4 ° c . with 50 μl coating protein per well ( cona eluate ), at 0 . 5 μg / ml in 50 mm sodium bicarbonate buffer , ph 9 . 6 . the plates were washed six times with wash buffer ( pbs , 0 . 05 % v / v tween - 20 ), then incubated with 200 μl 10 % ( w / v ) infasoy ( cow and gate , trowbridge , wiltshire , uk ) in tntt overnight at 4 ° c . after washing , 50 μl serum per well , diluted 1 : 2000 in tntt , were added for 1 h at room temperature . the wells were re - washed and 50 μl peroxidase conjugated rabbit anti - bovine immunoglobulin diluted 1 : 1000 in tntt added for 1 h at room temperature . after a final wash , 50 μl o - phenylenediamine dihydrochloride substrate ( sigma ) were added to each well . after 10 min in the dark , the colour reaction was stopped by addition of 25 μl 2 . 5 m sulphuric acid per well and od values read at 490 nm . each test sample was assayed in triplicate . pooled serum taken at the time of challenge from the group of calves in experiment 2 immunised with the unfractionated cona eluate was included on each plate as a reference sample , and od values expressed relative to this value . the cona binding fraction used in trial 1 and peak 3 employed in trial 3 were fractionated by 1 - dimensional sds - page under reducing conditions . each sample ( approximately 10 μg ) was mixed with 10 μl , sds - page sample buffer ( 0 . 05 m tris , ph 6 . 8 , containing 5 % ( w / v ) sds , 20 % ( v / v ) glycerol , 0 . 01 % ( w / v ) bromophenol blue and 10 mm dtt ), boiled for 5 mm before loading onto 10 % gels with a 3 % stacking gel . after protein separation , gels were stained with colloidal coomassie blue ( simplyblue ™ safestain , invitrogen ), destained in water and the image of each track captured . mass spectrometry analysis was performed at the moredun research institute &# 39 ; s proteomics facility . each gel track was sliced horizontally into about 27 equal gel slices of approximately 2 . 5 mm each and individual slices were finely chopped ( approximately 1 mm3 ), transferred to clean 0 . 5 ml eppendorf tubes and processed using standard in - gel reduction , alkylation and trypsinolysis steps ( 15 ) . digest supernatants of 20 μl final volume were transferred to hplc sample vials and stored at 4 ° c . until required for liquid chromatography - electrospray ionization - tandem mass spectrometry ( lc - esi - ms / ms ) analysis . liquid chromatography was performed using an ultimate 3000 nano - hplc system ( dionex ) comprising a wps3000 well - plate micro auto - sampler , a flm - 3000flow manager and column compartment , a uvd - 3000 uv detector , an lpg - 3600 dual - gradient micropump and an srd - 3600 solvent rack controlled by chromeleon chromatography software . samples of 4 μl were applied to the column by direct injection . peptides were eluted by the application of a 15 - mm linear gradient from 8 % to 45 % solvent b ( 80 % acetonitrile , 01 % formic acid ) and directed through a 3 - nl uv detector flow cell . lc was interfaced directly with a 3 - d high capacity ion trap mass spectrometer ( esquire hctplus ™, bruker daltonics ) utilizing a low - volume ( 50 μl / min maximum ) stainless steel nebuliser ( agilent , catalogue number g1946 - 20260 ) and esi . ms / ms analysis was performed as previously described ( 16 ) . a peak list file was generated from the resultant data and submitted to a local database server using the mascot search engine for protein database searching against ncbinr and nembase databases . the modifications used in these searches were a global modification of carbamidomethyl ( c ) and a variable modification of oxidation ( m ). the tolerances used were ; for ms data , 1 . 5 da , and for ms / ms data , 0 . 5 da . matches achieving a significant molecular weight search ( mowse ) score were considered significant if two peptides matched for each protein , each of which had to contain an unbroken b or y ion series of a minimum of four amino acid residues . the other criterion considered in assigning a positive identification for each protein was a concordance between the calculated theoretical molecular mass value of the protein and the observed position of the peptide on 1 - d gel electrophoresis . ostertagia ostertagi l4 cdna library construction and validation this was made in lambda triplex ( clontech ) and amplified x 1 according to the manufacturer &# 39 ; s instructions . ten - fold dilutions of the unamplified primary library were made in sm buffer over the dilution range 10 − 1 - 10 − 5 . a 10 μl aliquot of each dilution was mixed with 200 μl of xl 1 - blue plating cells ( od . 600 = 0 . 5 ) and incubated for 30 min at 37 ° c . to allow the phagemids to bind to the cells . after incubation , 4 ml of nzy top agarose at 48 ° c . was added and the mixture was plated onto pre - warmed 100 mm diameter lb - agar plates . after the top agarose solidified the plates were incubated overnight at 37 ° c . the 10 − 1 dilution plate had 293 plaques , therefore the primary library contained 2 . 93 × 10 5 pfu / ml . the amplified cdna library was titrated as above except that the top nzy agarose was supplemented with 100 μl of 100 mm iptg and 80 μl of 50 mg / ml x - gal to allow the selection of a blue “ wild type ” phagemid plaque . several blue plaques were identified and agar plugs containing individual plaques taken into 0 . 5 ml sm buffer containing 20 μl chcl 3 , to prevent bacterial growth , and stored at + 4 ° c . the sm buffer supernatant , prepared as above , containing a wild type lambda triplex phagemid was titrated and a dilution that gave near confluent plaques was selected . this clone was plated as above and grown overnight at 37 ° c . after overnight incubation the plates were flooded with 5 ml of sm buffer and agitated gently on an orbital rocker for 5 h . the resulting suspension of e . coli / phagemid was divided into 1 ml aliquots and subjected 3 rounds of freezing and thawing (− 80 ° c . for 30 min followed by 37 ° c . for 5 min ) to lyse both the e . coli and phagemid . the resulting lysate was stored at − 80 ° c . until required . the unamplified primary library was diluted 10 − 1 in sm buffer and plated in nzy top agarose onto lb agar plates as above and incubated at 42 ° c . for 6 h . a nitrocellulose filter , pre - treated with 10 mm iptg , was placed on top of the top agarose of each plate and incubated at 37 ° c . overnight . after overnight incubation the plates were transferred to + 4 ° c . for 1 h . the filters were marked to ensure correct orientation later and carefully lifted off the plates . the filters were washed extensively ( several changes of tntt buffer over ˜ 6 h ) and blocked overnight in a solution of 1 % w / v gelatine in tntt at + 4 ° c . when immunoscreening using sera from trial 3 the filters were blocked in tntt alone , as this was shown to give a lower level of background staining when the blots were developed . filters were probed for 1 h at room temperature with pooled serum from the best protected groups ( groups 2 . 4 and 3 . 3 see table 5 and fig1 ) in trial2 and 3 ( diluted to 1 / 400 in tntt buffer ). the serum had been pre - absorbed as follows :— 500 μl was mixed with 500 μl of e . coli / lambda triplex freeze / thaw lysate at + 4 ° c . overnight , centrifuged and the supernatant retained for subsequent use . bovine igg was detected with a biotin - labelled monoclonal antibody to bovine igg ( dako ) diluted to 1 / 4000 in tntt for 1 h at room temperature . in trial 3 bovine igg was detected with a similar antibody ( sigma ) diluted to 1 / 2000 biotin was detected with streptavidin - hrpo conjugate ( sigma ) diluted 1 / 2000 or 1 / 5000 in tntt for 1 h at room temperature . the filters were washed between each step with 3 × 5 min washes in tntt finally , hrpo activity was revealed with 3 , 3 - diaminobenzidine ( sigmafast , sigma ) prepared as per the manufacturer &# 39 ; s instructions . immuno - positive plaques were picked into 0 . 5 ml sm buffer with 20 μl chcl 3 and subjected to a second round of screening to obtain clones of each positive plaque . the o . ostertagi dna encoded in the immuno positive clones was amplified by per using primers directed at the ptriplex vector sequence flanking the cloning site . the primer sequences were ; the pcr reaction mixture contained 2 μl of a freeze / thaw lysate prepared from individual immuno - positive clones , as template and 23 μl of a reaction mix containing 1 × reaction buffer ( bioline ), 5 mm mgcl2 , 200 μm dntps , 1 μm of each primer and 1 u taq polymerase per reaction . pcr products were purified , using a proprietary clean up kit ( qiagen ) and sequenced using the pyrosequencer or sent to eurofins ( mwg ) for sequencing four immunisation - challenge trials were conducted with weight balanced groups of calves . the number of animals assigned to each group and the dose of antigen each group received is laid out in table 5 . fig1 shows a flow chart of how the different immunogen fractions were prepared . all groups were immunised three times at three week intervals and challenged with 50 , 000 o . ostertagi l3 one week later . immunogens were diluted with cold phosphate buffered saline , ph 7 . 4 , ( pbs ) and mixed with quila ( superfos biosector ) so that each calf received either 20 mg ( trial 1 ) or 5 mg ( trials 2 , 3 and 4 ) of adjuvant at each immunisation . control immunogen was prepared identically , except that pbs was substituted for antigen , and administered to all challenge control animals . one ml of immunogen was injected intramuscularly into each side of the neck . all animals were bled at approximately weekly intervals to monitor the kinetics of the antibody response . details of antigens , dose and numbers of animals are given in table 5 . arithmetic group means are shown throughout with their standard errors . significant differences between groups were calculated by the t test in trial 1 and by analysis of variance followed by tukey &# 39 ; s test in trials2 , 3 and 4 . to satisfy bartlett &# 39 ; s test for equal variances the egg data i was log transformed prior to analysis . recovery of fourth stage o . ostertagi larvae from donor calves ranged from 5 % to 20 % of the dose given . the yield of cona binding membrane proteins was approximately 0 . 3 mg per 100 , 000 fourth stage larvae . cona binding integral membrane proteins , prepared in the same way from adult haemonchus conforms or fourth and adult stages of o . ostertagi were compared by gel analysis and western blotting . coomassie stained gels indicated differences in the profiles of all three fractions ( fig2 a ), although additional bands present in the l4 but not in the adult o . ostertagi preparations were of most interest in this case . when the three cona binding fractions were probed with anti - sera from calves which had been immunised with material obtained in the same way from adult ostertagia , additional bands were still detected in the l4 fraction ( fig2 b ). digesta from a worm free calf was treated in exactly the same way as the l4s , but no protein peak was detected when the cona column was eluted with sugar ( not shown ). the gel profile of the preparation used to immunise the vaccinated calves in trial 1 was very similar to that shown in fig2 lane 3 . serum antibody titres in the control group remained at background concentrations throughout ( fig3 ). in contrast , a marked response was observed in the vaccinated group by week 5 , two weeks after the second vaccination . this response reached a peak on week 8 two weeks after the third immunisation . mean egg counts of the immunised calves were always lower than controls throughout the experiment , although the difference was not statistically significant on days 28 and 30 ( fig4 ). however the group means of the cumulative eggs per gram over days 19 to 30 were significantly different ( p = 0 . 01 ), with the vaccinated animals shedding 60 % fewer eggs . significantly ( p & lt ; 0 . 01 ) fewer worms were recovered from the vaccinates ( 1909 ± 252 ) compared to the controls ( 3621 ± 414 and fig4 ). small numbers of early fourth stage larvae were found in some calves but no difference between vaccinates and controls was observed . 1d ) identity of components in the protective fraction by mass spectrometry . trial 2 : immunisation with sub fractions of the cona lectin binding glycoproteins . this trial was done partly to determine whether the level of protection detected in the first trial could be improved if fractions were prepared which were more enriched for the protective components but also to find out whether simpler fractions could be equally protective . a flow chart depicting how these preparations were made is shown in fig1 , the details relating to which calves received which fraction and the dose of protein administered are presented in table 5 whereas the sds - page profiles of the immunogens are shown in fig7 . the kinetics of the antibody responses of each group is shown in fig8 . all vaccinated groups showed a similar antibody response to o . ostertagi l4 antigen , and had a significantly ( p & lt ; 0 . 01 ) higher antibody titres compared to the control group from one week after the second immunisation until the end of the experiment . all four vaccinated groups showed significantly reduced egg counts compared to the adjuvant only control group from day 20 to day 29 ( fig9 ). the group means of the cumulative egg counts over days 19 to 34 were significantly lower in each of the vaccinated groups compared to the controls , with the corresponding percentage protection ranging from 70 to 85 % as detailed in table 5 . only group 4 showed a significant reduction in worm burden at necropsy , with 64 % fewer worms than the control group ( fig1 and table 5 ). 2d ) identification of components in the best protected group by cdna library screening . the sequences and , where possible , the corresponding identities of 135 immuno positive clones selected by the calves immunised with the pool 2 fraction are shown in tables 1 and 2 trial 3 : immunisation with sub - fractions of the 0 . 1 to 0 . 5m monoq pool . the object of this trial was to separate the protective antigens identified by group 2 . 2 by gel filtration in order to narrow the identity of the candidate protective polypeptides . ( no attempt was made to do this for the unbound fraction as too little protein was available for the task ) the peaks separated by gel filtration together with an sds page analysis of the polypeptides present in each of the three antigen pools used to immunise the calves are shown in fig1 . details of the groups and doses of protein administered are laid out in table 5 . the kinetics of the group mean egg counts of the calves in trial 3 are shown in fig1 and the cumulative counts are presented in fig1 . the mean egg output of the calves immunised with peak 3 was consistently lower than the control calves with an overall reduction of 83 % ( table 5 ). however , there was a large variance in the egg output of the control calves in this trial so this difference just failed to be statistically significant ( p = 0 . 056 ). the number of worms recovered from any of the vaccinated groups and the control group was very similar ( table 5 ). 3c ) identity of the components in peak 3 , the most protective of the pool3 sub - fractions . ten μg of the peak 3 fraction was separated by sds - page and subjected to mass spectrometry as described in the methods . about 16 polypeptide bands were visible fig1 . twelve significant identities were obtained ranging from about 18 to 110 kda in molecular weight and these are listed in fig1 . 3 d ) identification of components in the best protected group by cdna library screening . the sequences and , where possible , the corresponding identities of 46 immuno positive clones selected by the calves immunised with the pool 3 fraction are shown in tables 3 and 4 . trial 4 : immunisation with sub fractions of the 0 . 1 to 0 . 5m monoq pool as before the object of this trial was to determine whether the components responsible for the protection in group 3 . 3 could be separated with a view to simplifying their identity . the peaks separated by gel filtration together with an sds page analysis of the polypeptides present in each of the three antigen pools used to immunise the calves are shown in fig1 . details of the groups and doses of protein administered are laid out in table 5 . the kinetics of the group mean egg counts of the calves in trial 4 are shown in fig1 and the overall protective effects are summarised in table 5 . the mean egg outputs of all four groups of immunised calves were lower than the controls but there was little to choose between the efficacy of the different fractions . there was little doubt that the cona binding fraction of fourth stage o . ostertagi membrane extracts contained protective antigens , since all the groups vaccinated with this antigen or one of its derivatives had lower egg counts than their respective controls . as a crude measure , the mean percent reduction in egg output of all 11 groups immunised with this preparation or sub - fractions of it was 62 %. more impressively , three of these fractions reduced cumulative egg counts by more than 80 % ( table 5 ). the effect against worm numbers was more variable however , and did not necessarily correlate with the degree of egg reduction ( table 5 ). however , the two best fractions did reduce worm numbers by 50 % or more ( table 5 ). these protection figures were better than those achieved with the same cona binding extract of adult o . ostertagi where eggs were only reduced by between 30 and 50 % and there was no measurable effect against worm numbers ( smith et al 2000 ). these results support the hypothesis developed in the introduction that fourth stage o . ostertagi were likely to be more susceptible to the gut antigen approach to vaccination than their adult counterparts and that gut membrane antigens sourced from this developing stage are likely to be more efficacious . this idea does not seem to have been mooted before and could have general applicability to various other non - blood feeding nematode parasite genera across a range of hosts . obtaining large numbers of fourth stage o . ostertagi is a laborious and expensive procedure . the trials reported here were made possibly by a regular supply of donor calves which were scheduled to be culled anyway after having been the subject of unrelated studies at the institute . because it was not possible to obtain the l4s without some contaminating digesta , the possibility existed that plant material was the source of some of the bands present in the antigen preparations . this possibility was discounted when attempts to make similar preparations from worm free abomasal digesta did not yield any protein . presumably the cellulose cell walls of the plant cells which make up the bulk of the digesta are resistant to triton extraction . another possibility was that some of the l4 preparation polypeptides , which were additional to those observed in similar preparations from adult worms , were bovine in origin — perhaps from small pieces of abomasal tissue leaching from the mucosa when it was being incubated at 37 c to recover the larvae . however , an immunoblot developed with serum from calves immunised with o , ostertagi proteins revealed that several of these bands could not have been bovine proteins . this discovery of apparently novel bands in the l4 fraction prompted a protection trial . the encouragingly positive result from the first vaccine experiment lead onto 3 further “ fractionate and vaccinate ” trials where the overall objective was to determine whether simpler fractions containing fewer components would be just as if not more efficacious . it was striking how little native protein was actually required to achieve a good level of protective immunity ( table 5 ), but because of the difficulty and expense of obtaining large numbers of ostertagia l4s , synthetic antigens , probably derived by recombinant dna techniques , will be essential for a commercial vaccine . obviously , the cdnas of the protective polypeptides are required to do this and some progress was made in that direction through a combination of mass spectrometry and cdna library immunoscreening . much remains to be done however before a single protective antigen can be identified . 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