Immune response of lambs to vaccination with Ostertagia circumcincta surface antigens eliciting bile antibody responses

Inrernatiod

Journalfor

Pamitology,

Pergamon 0020-7519(95)00028-3

Vol. 25, No. 9, pp. 1111-l 121, 1995 Austraiii Society for Parasitology Elaevier science Ltd Printed in Great Britain 020&7519/95 $9.50 + 0.00

Immune Response of Lambs to Vaccination with Ostertagia circumcincta Surface Antigens Eliciting Bile Antibody Responses HALINA

WEDRYCHOWICZ,*t PETER

*Department

KENNETH H. HOLMES4

BAIRDENJ and ANDREW

ELIZABETH TAITS

M. DUNLOP,5

of Veterinary Parasitology,

Warsaw Agricultural University, Grochowska 272, 03-849 Warsaw, Poland SDepartment of Veterinary Parasitology, and $Department of Veterinary Physiology, University of Glasgow, Bearsden Road, Glasgow, G61 lQH, U.K. (Received 21 September 1994; accepted 7 April 1995)

Aketract-Wedryckowicz H., Bat&o K., Dunlop E. M., Holmes P. H. & Teit A. 1995. immune reqonse of 1lImbB to vecein&m with oswhlgia eirc-a surface aatigem elidtiag bile 8mtibody rs!qlma znh?rll&onal JlmrnaI f&r Parasitology 25: 1111-1121. hmme tn snrfaee ant&gem of infective larvae of Oatertagh circamchcta e by bile m&hod&e ofsbeepimmunetoch8hgewerestodiedin ~FiabDonretlanleIrrabe.Tbeshecpwerevrcciarrted~ueouayrrlth2~of25~kg body weight of smfece proteh immwnoprecipitated by bile &&od&e derived from protehd lambs. Tkese antigens were puritkd from immune complexes by afihity cbromatogrqiby end then injected witk bery%un hydroxide as an adjuvent. Tke immunhd lemba were ckelhged wits 5 x 10’ L3 end tke worm burdeos evaluated on day 21 poet ckellenge. These were eig&ca&y (P < 0.01) lower in the vaccineted group tken in the challenged emtrols (72% protection). The mucosel eml,bile IgM, reqpdhg the L3 sorfece, &owed sigdcantly higher levels in the vacehated lambs compared to the tzbabqs co&ok Mucoeel a14 bile IgA antibody levels agaimt the same antigens were low and no sign&ant ditTerencea were obsewed between v&I&ted and control lambs. Key words: Ostertagia circumcincta; nematode; surface antigens; western blotting; antibody izotypes; immunofluorescence; biotinylation; non-specific stimulation.

INTRODUCTION

It is well establishedthat various isotypic forms of antibody may have distinct effector functions and that the immune systemcan regulate the expression of the antibody isotype elicited to a given epitope,in responseto differencesin the macromolecularform of the antigen. The sameparasite-derivedmolecule may elicit both effector and blocking isotypes of antibodies (Fisch & Manser 1987, Capron h Dessaint1988).In a previous paper (Wedrychowicz tTo whom correspondence should be addressed.

et al., 1992a)it wasreported that significantlevelsof serumand bile IgA reactingwith the surfaceextracts of infective larvae of 0. circumcincta were only detectedin lambsvaccinatedwith L3 surfaceextracts and beryllium hydroxide and that these lambs appeared to be resistant to challenge infection. Serum IgA of this group reacted with the whole surface of exsheathedlarvae and with the site of opening of the excretory pore, while IgG antibodies from the samegroup of animals reacted strongly with the anterior and posterior parts of the infective larvae. Both IgA and IgG responsesto surface

1111

1112

H. Wedrychowin et al. Group

r

Sheepno.

Table l--Experimental protocols Antigen Adjuvant 25 pg/kg of L3 surface Beryllium proteins* hydroxide on DO and D14 MOHM

Challenge5 x 104L3 on D35

174 175 190 204 II 173 1 mg/kg of egg albumin Sxio‘J Li Be(OHh 176 on DO and D14 on D35 188 189 III 177 5xlO4L3 187 on D35 201 202 203 D, refers to the days on which immunization and challengewere undertaken relative to the first immunization. *, L3 surface antigen precipitated by bile IgA antibodies from lambs immunized with crude surface extract of 0. circumcinc~aL3 and resistant to challengeinfection. (Wedrychowicz ef al., 19928)

antigenswere stage (L3) specific. It has also been shownthat bile IgA of the challenge-protectedIambs recognizeda specificsetof polypeptidescomparedto IgA derivedfrom naivelambs,susceptible to infection. (wedrychowicz et al., 1992a,Wedrychowicz, Holmes & Tait, 1994a). The role of IgA antibodiesin helminth immunity is still not fully understood however, it has heen suggestedthat IgA antibodiesmay facilitate parasite survival rather than play an important role in the protective response(Befus & Bienenstock, 1982, Wedrychowicz, MacLean & Holmes, 1984, 1985). The importance of non-specificstimulation of the host immunesystemin conferring protection against infection with gastrointestinal nematodeshas not heen fully recognized. It has been reported that a single subcutaneousvaccination with 100 ug of Coryne-bacterium parvum proteins provoked a 53% reduction of the challengeinfection of T. colubriformis in rabbits while vaccination with serum proteins from normal animalsplus beryllium hydroxide asadjuvant elicited a 99% reduction of infection on challenge with the nematode (Wedrychowicz, Bezubik & Trojanczuk, 1989). Thus, at the present time, it is unclear from the data available what the relative roles of non-specific immunemechanisms and those specificto particular parasiteantigensare in the induction of a protective immuneresponseto gastrointestinalnematodes.The aim of the work presentedhere was twofold: firstly to investigate the role of infective larval surface antigens recognized by bile IgA from immune animals,in eliciting a protective response.Secondly to investigate the role of non-antigen-specificimmune mechanismsin the induction of immunity to 0. circumcincta.

MATRRIALS AND MR’I’M@DS Experimental design. Thirteen5-month-old, mate,Fin--

Dorsetlambs,weighingapproximateiy3.5kg, wbicbbad beenrearedunderparasite-free conditions, wereallocatedto 3groups.GroupsI andII (of 4 lambxeaeb)werevaeclaated at 2-week-intervals, as shownin Table 1. All vaccinated animalsand 5 naive lambs(Group III) werecbalimged orally with 5x iti infective larvae (L3) 21 days after administration of the secondimmunizing dose.All experimentalsheepweren-p&d on day21after c e. Pmwsitohgid

and smn+g

metim&. 7% m

Used

throughouttheexp&nant weree&r&i&y tfrasantoasde+ cribedin our previouspaper(Wedry&owia et‘a3.,f992a). L3 surface biotinykatim Approlrmiltely4 x I@’ L3 were purifiedandexsheathed (Wedrycbowicz et d, 1@2a)then resuspended at a concentration of 3 x if? larvaeper ml in 0.05M carbonate/bicarbonate buf&r pH 1.2andincubated with 0.3 mgof Sulfo~i~dyl-2~~ido~~l-l,3ditiopropioniate (NITS-SS)biotin for 1 h at roomtemperature in the dark. Biotinylationwasterminate4by washing extensivelyin 0.15M NaCl,andthelarvaehomogenbred in buffercontaining0.2M TRIS,0.15M NaCl,0.02M EDTA and proteaseinhibitors:13.5pM of Na+Tesyl-L-Lysine CblorometbylKetone(TLCK), 7 pM of N-Tosyl-~+phenylalaninechlorometbyl(TPCK), 0.5 pM pbertyimethyi~tdphony1liuoride(PMSF)and2 pM of antipain@@a) u&g a glasshomogenizer in an ice-waterbath.Thehomagenate wascentrifuged at 14,OOOg, ~es~~~ov~, t&red through0.45gmMillipore&ers and the gmtein cmmhtion estimated by themctbodof Lowry e?Irl. (1951).

centrifugation,dissolvedin 1.0 M EDTA, pH 8, and the biotinyiated L3 surface antigenswere isolatedusing streptavidinafEnitychromatography (seebelow).

Surface antigens of 0. circumcintn Afinity

chromatography

of

biotinylated

polypeptides.

NHS-SS-biotin labelled components were isolated from the immune complexes by affinity chromatography on streptavidin-agarose (Wedrychowicz, 1994) and used for vaccination. AC&ants and immunization. Beryllium hydroxide was freshly precipitated from BeSO solution according to the method of Hall (1984). The equivalent of 4 mg/kg body weight of BeSO was injected subcutaneously a few seconds prior to injection of 1 ml of sterile PBS solution containing the equivalent of 25 ug/kg of infective larval surface protein. Adjuvant controls (Group II) received the same dose of Be(OH)z and 1 mg/kg body weight of ovalbumin. Evaluation bile. ELISA

of antibody

levels in sera, abomasal

mucosa and

and immunofluorescence tests were undertaken as previously described (Wedrychowicz et al., 1992, Wedrychowicz et al., 1994b) using sera at 1 in 200 dilution and bile or abomasal proteins at 1 in 20 dilution. Western blot analysis. Solubilized surface or somatic extracts were analysed on SDS-polyacrylamide gels comprized of a 6% stack and 10% resolving region using the discontinuous buffer system of Laemmli (1970) and 29.2 to 0.8 weight ratio of acrylamide to bis-acrylamide. Western blotting was carried out as described earlier (Wedrychowicz et al., 1994a). Statistical analysis. Variations around the means of egg, larval or adult counts were expressed as the standard error (S.E.M.). Students t-test was used to assess the statistical significance of differences between means from each group of lambs.

1113

In order to assess the role of non-specificimmune mechanisms inducedby the adjuvant, a secondgroup of lambs(Group II) were immunizedwith adjuvant and an irrelevant protein (ovalbumin). The resultsof this experiment, in terms of the adult worm burden after challenge,are also shownin Table 2. As with the Group I animalsthere is a considerable(54%) reduction relative to the control group (Group III) and this difference is significant at the 0.01 level. However, the difference in worm numbersbetween Group II and Group I is not significant (P~0.1). Clinical

effects

Changesin body weight of vaccinatedand control lambs were monitored and the challenge controls

66 -

RESULTS Protection

In order to establishthe role of the surfaceantigens recognized by bile IgA from immune animals, the surfaceproteinsprecipitatedby theseantibodieswere used to immunize Group 1 lambs (Table 1). The surfacepolypeptidesrecognizedby theseantibodies were analysed by immunoprecipitation of biotinylated surface polypeptides followed by purification using streptavidin affinity chromatography and characterization by SDS-PAGE. The surfacepolypeptidesrecognizedby immunebile IgA areshownin Fig. 1 together with those recognizedby bile IgA antibodiesfrom non-immuneanimals.Preparations of thesepolypeptides(illustrated in Fig. 1, track 4) were used to immunize lambs (Group I) using beryllium hydroxide as an adjuvant, and after immunization, the lambs were challenged with 5 x lo4 infective larvae and the level of protection estimatedrelative to a control non-immunizedgroup (Group III) by counting the number of adult worms in the abomasumat slaughter21 daysafter challenge. The results(Table 2) show that the worm burden is reducedby 72% in the vaccinated group (Group I) relative to the controls (Groups III) and that the differenceis significantat the 0.01 level.

45

29 -

20 -

Fig. 1. SDS-PAGE and western blot analysis of L3 surface the biotinylated extract and the surface extract fraction used for immunization. Track I-unfractionated biotinylated surface polypeptides; track 2-biotiylated surface polypeptides ptipitated by bile IgA of unprotected lambs;3-

biotinylatedsurface polypeptides precipitated by bileIgA of

sheep resistant to infection; &SDS-PAGE of polypeptide mixture showed in track 3 and used for vaccination of Group I (silver staining).

H. Wedrychowin et al.

Ill4

Group 1

II

111

Table 2---Abomasal worm burdens Sheepno. % protection No. of worms 174 175 190 204 X+/-S.E.M 173 176 188 189 X+i-S.E.M 177 187 201 202 203 X+/-S.E.M

10550 4700 10950 3525 7431+/1932 13750 10250 9550 14850 12100+/1297 32750 32800 19750 25300 22650 26650+/-2657

(a) 0.5

m

GFOUPI

a

GrcupIl

72.1

54.6

0

Statistical significanceof differences (Student’s f-test): I v III-PiO.01; II v III--P
Fig. 2. LocalIgG, IgA andIgM antibodylev&sagainstthe infective larval surfaceantigensusedfor imrmmization assessed by ELISA. A. Bile antibodiesfrom d56. B. Mucosalantibodiesfrom d56.Group I-immunized with bile IgA surfacepolypeptides; Group II-immunized with ovalbumin;Group III--challenge controls. Histograms represent means of 4-5 sheep and the bars depict standard errors.

extracts waslow in all experimentalgroups(Fig. 2A, B), while a higher level of IgA wasobservedin all 3 groups with no significant differences between groups. Mucosal IgM antibody levels in vaccinated lambs (Groups I & II) were significantly higher as compared to the challengecontrols (Fig. 2B) while bile IgM antibodies of Group I demonstrateda significantly higher level than either Group II or HI (Fig. 2A). The serumantibody isotypesreacting against the antigensusedfor immunization were determinedby ELISA and the results are presented in Fig. 3. Following vaccination, lambs of Group I showed significantly higher serumIgM activity aga&st larval Local and serum antibody responses against antigens surfaceantigensthan Group II or III but by the day used for vaccination of challengethe levelshad fallen and all vaccinated The abomasalmucosaand bile IgA, IgM and IgG sheepshowedsimilar levelsof this antibody isotype responses to 0. circumcincta infective larvae surface (Fig. 3a). A clear increasein levelsof IgA antibodies antigensused for immunization were evaluated by reacting with bile precipitated surface antigenswas ELISA three weeksafter challengewhen the animals observedin Group I after administrationof the first werekilled and the data am presentedin Fig. 2. The immunization and a significant anamnestic IgA level of mucosaland bile IgG to nematodesurface responsewas recorded in this group a week after

Surface antigens of 0. circumcinta

(a)

1115

(a)

0

I

I

I

I

I

I

I

7

14

21

28

35

42

49

,

0

56

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14

21

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42

49

56

0.35 0.30 0.25 0.20

a zs w I

I I

0

0.15 0.10 0.05

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0.40 r

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0.10 k

0.05t , 0

7

,

,

(

,

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14

21

28

35

42

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56

Day of experiment Fig. 3. Dynamics of serum IgM (a), IgA (b) and LgG (c) responses against infective larvae surface antigens precipitated with bile antibodies. Assayed by ELISA using the same systems as in Fig. 2. Each point represents mean of 4 (groups I & II) or 5 sheep (group III). Bars indicate standard errors, arrow indicates day of challenge with 5x 104 L3.

challenge(Fig. 3b). In contrast, the control group (Group III) only showedan increasepost-challenge while the Beryllium hydroxide/ovalbumin group (Group II) showed a steady rise in IgA over the course of immunization. IgG responsesto the bile IgA precipitated surface antigens,were lower than thoseobservedfor IgA or IgM (cf. Fig. 3a and b with c) although an increasein level was observedin the immunizedgroups.

Day of experiment Fig. 4. Dynamics of serum IgG (a), IgM (b) and IgA (b) responses againstovalbuminassessed by ELISA. Each point represents mean of 4 (groups I & II) or 5 sheep (group III). Bars indicate standard errors, arrows indicate day of challenge with 5 x 104 L3.

were observedin both the Group I lambs prior to challengeand the Group III animalspost-challenge. Challengecontrol IgG antibodiesshowedthe lowest titres (Fig. 4~). The local bile antibody response(post-challenge) of all 3 groupsof animalswasassessed usingELISA to ovalbumin. In all casesthe responseswere low (Fig. 5) and no significant differences between groups were observedwith IgA, IgG or IgM (Fig. 5a), however mucosallevels of IgM and IgG were Serumand local antibody responses againstegg high in Group II (immunizedwith ovalbumin) and albumin were significantly higher comparedto the other two Group II lambs,vaccinated with eggalbumin and groups (Fig. Sb). In the caseof IgA the Group II beryllium hydroxide, produced a significantlevel of responsewasalso high and, interestingly, so wasthe serum IgM antibodies reacting with this antigen response from Group I, both responsesbeing (Fig. 4a). The serumIgA levelswerelower, risingto a significantly different from the control group peak prior to challenge(Fig. 4b). Low levelsof IgG (Fig. 5b).

H. Wedrychowicz et nl.

lllb

(a) 0.20

R

Group1

lzil GroUPE 0 GroupBI

reactivity 7 days after challenge;this would suggest that an anamnesticresponseoccurred to surface epitopes cross-reactingwith ovalbumin. As found with mucosalIgA, the mucosalIgG antibodiesfrom Group I showedthe highestlevel of response. Polypeptides recognized infective larvae

by IgG in surface extracts cqf

Surface polypeptides,immunoprecipitatedby bile antibodies derived from lambs protected against infection (seeMaterials and Methods), wereanalysed by western blotting, with pooled sera or abomasal mucosaextracts of vaccinated and control lambs. Day 0 serum IgG of all groups, (Fig. 6. track 1) recognized polypeptides of molecular mass >200 kDa. The IgG of animals,after immunization (Day 35, track 2) with the IgA immunoprecipitated surfacepolypeptides(Group I) recognizedpolypeptidesof molecularmassrangingfrom 66 to 120kDa, the strongestreaction being to those of I IO and 97 kDa (Fig. 6, track 2) although the responseto these was reducedby the day of challenge(track 3). The serumIgG from animalsimmunizedwith ovalbumin and beryllium hydroxide recognizedsevenpolypepFig. 5. Local IgG, IgA and IgM antibodylevelsagainst tidesboth pre (track 4) and post (track 5) challenge; ovalbumindetectedbv ELISA on individualsaxnnles of the most prominent polypeptides being of 64 and sheepfrom groupsI, B & III. a. Bileantibodies from d56; 58 kDa together with a doublet of 11I!120 kDa. A b. Mucosalantibodiesfrom d56. Histogramsrepresent meansof 4 (groupsI & II) or 5 sheep(group3) andthe very similarpattern of polypeptidesis recognizedby serumfrom the challengecontrol group post infecbarsdepictstandarderrors. tion (track 6). Analysis of the polypeptides recognized by mucosal IgG from all three groups ot Immunojluorescence tests animals,post-challenge,showsthat this doublet of The serum,bile and mucosalIgA responses to the 11I,/120 kDa is also recognised(Fig. 6, tracks 7---Y) nematodesurfacewereexaminedby immunofluores- specificallyrelative to pooleduninfectedmucosalIgG cenceof exsheathedviable infective larvae and the (Fig. 6, track IO). resultsare presentedin Table 3. The strongestserum Theseresultsshow that the IgG response(in both IgA reaction to the infective larvae surface was serumand mucosa)recognizesa polypeptide doublet observedin Group I on the day of challengeand a of 111/120kDa when lambs are immunized with week later (Table 3A); similarly the bile and mucosal either surface polypeptidesor an irre1eva.mantigen IgA reactivity was the highest in this group. The (ovalbumin) and that this doublet is also recognized serum, bile and mucosal reactivities of IgM anti- on infection with the parasite. Serum IgG also bodies are presentedin Table 3B. Serum IgM of recognizes polypeptides of 64 and 58 kDa postGroup I (day of challenge) clearly recognized challenge and an immune responseto these, not epitopeson the surfaceof all exsheathedlarvae, but recognizedby control infected animals,is inducedby the level of theseantibodiesdropped after challenge the combination of beryllium and ovalbumin. As (Table 3B). A very strong IgM responseto the thesepolypeptidesare not clearly recognizedby prcinfective larval surface was observedin the control immunization serumIgG (track 1). it is presumed lambs(Group III) after challenge.Interestingly, the that this responseis specificand induced by crossmucosalIgM antibodies to the L3 surface did not reacting determinantson ovatbumin. These rest&s developin this group while in the protected Group I, may explain the level of protection obtained in the the responsewasvery strong (Table 3B). Serum,bile animals immunized with ovalbumin. The antigens and mucosalIgG reactivity to the surfaceof infective recognized by serumIgG from animalsimmunized larvae is shownin Table 3C. In Group I the clearest with the surfaceprotein fraction (Group I) recognize reaction was shownby serumIgG on day 56 of the surfacepolypeptidesof 97 and 94 kDa (track 2) which experiment while Group II demonstrateda similar are not recognizedby IgG from the other groups.

Surface antigens of 0. circumcinta Table 3-Immunofluorescence

reactivity of pooled sheep serum, bile and mucosal antibodies with the surface of exsheathed infective larvae of Ostertagia circumcinctu**

Day of experiment

A

SERA 0 35* 42 49 56 BILE 56 MUCOSA 56

25 (+++);

I

Groups II

(zk:) (f) (,I (k)

25+45 (+) 40 (k) 50 (k)

10(,I

75 100 75 50

50 (+), 50 (33 100 (+)

B SEBA 0 35* 42 49 56 BILE 56 MUCOSA 56

10(rt)

25 (k) 10(+I; 80 (k) 55 (+I; 30 (t)

III

10(k) 10(k) 10(k) 55 (k) 15 (k)

20(+) 20 (+); 50 (k)

w

10(+I 100(+)

50 (?I); 3OM (+) 100 (+) 10 (+++I, 90 (Tk)

0 10 (+++)

10 (k) loo (It)

0 20H (+) 0

80($-l

10(+I 50(k) 20H C+l 30 (+++j 10 (+++j 20(+), 50(+) 0

50 (+I; 50 (k)

C

SEBA 0 35: 42 49 56 BILE 56 MUCOSA 56

1117

W

0 10 (+), 20 IC loo (k), 30 IC (+) 100 (k) 100 (k)

0 10 (+I 100 (+) 100 (k) 100 (*)

0

80 (k)

0 0 lo(+) 15 (+I

25 (k) 20 (+I; 50 (*I

0 100 (+++) 50 (+I; 50 (k) **Percentage of larvae showing a positive fluorescence reaction (A) reactivity with IgA; (B) reactivity with IgM and (C) reactivity with IgG. *, day of challenge infection with 5 x lo4 L3; +/-, weak fluorescence of whole larval surface; +, clear fluorescence; +++, very strong fluorescence; H, fluorescence limited to head of the larvae; M, fluorescence only in the mouth (buccal cavity); IC, patches of immunocomplexes.

As the western blot analysispresentedin Fig. 6 could only detect denatured epitopes and only analysed the reactivity to surface polypeptides immunoprecipitated by IgA, immunoprecipitation experimentswere undertaken to examine the polypeptide profiles recognizedby the mucosal,bile and serumantibodiesof the different groupsof animals. Extracts of larvae, surface labelled with NHS-SSbiotin, were precipitatedwith seraof immunizedand control sheep and the immunocomplexeswere dissociated,electrophoresed,blotted on nitro-cellulose and then probed with streptavidin in order to speciIlcally detect the surface polypeptides. The results are presentedin Fig. 7 for pooled mucosal and bile antibodies (a) and for pooled sera from each group (b); theseresultsallow a comparisonto

be made betweenthe polypeptides recognized and the outcomeof challenge.The mucosalantibodiesof Group I (track l), group II (track 2) and group III (track 3) recognizea number of common polypeptides (Fig. 7a) with the profiles for the ovalbumin immunizedgroup being almost identical to that for the group immunizedwith surfaceantigens.A range of polypeptides (indicated by arrows, Fig. 7a) are specificallyrecognizedby the antibodiesfrom the 2 groupswhich showprotection againstchallengeand are not recognized by the antibodies from the challengecontrol animals.The sizesof the polypeptides recognized are listed in Table 4 with those specific to Groups I and II indicated in bold. In addition there is an enhanced responseto major polypeptides of 80 kDa and 27.5 kDa in the 2

1118

H. Wedrychowia

er al.

29

4 7 8 9 10 1 2 3 5 6 Fig. 6. Western blots of surface polypeptides extracted from infective larvae of 0. cirnuncbrctu recognized by IgG of control and vaccinated sheep. The surface polypeptide preparation is that used to immunize the group I lambs. Tracks l-3 = serum of Group I from day 0,35 and 56 respectively; tracks 4-5 = sera of sheep immtmimd with ovalbumin and collected on day 35 (4) or 56; (5 post-challenge); track 6 = d56 serum from challenge control animals (Group III); track 7 = mucosal IgG from Group I; track 8 = mucosal IgG from Group II; track 9 = mucosal IgG from Group III; track 10 = mucosai IgG from uninfected, non-vaccinated control. All mucosal samples were collected on day 56 of experiment ( = day 21 after challenge). The blots, after probing witb immune sera were developed using an alkaline phosphatase conjugated anti-ovine IgG antibody.

groups of protected animals relative to the response in the challenge controls (* in Table 4). At face value these results show a correlation between the specific recognition of certain surface polypeptides and the

Table 4-Molecular

weights of major polypeptides found in immunocomplexes formed by mucosal and bile antibodies and 0. circumcincta surface extracts

Source of antibodies Abomasal mucosa G-1

Bile GrollaI

outcome of challenge as well as the de&&ion of those polypeptides (37, 31 and 23.5 kDa) which must cross-react with ovalbumin. The polypepti&s recognized by bile antibodies in Groups I (track 9,

Molecular weights of precipitated polypeptides (kDa) 205; 122; 96; 80*; 60; 56; 54,5; 49; 45; 43; 39; 37; 35; 3; 27,5; 2435; 23.5 80*; 56; 54,5; 49; 45; 43; 39; 37; 35; 3l; 27.5*; 24 $72 5. 23 5 80; 54,5; 41,5 225; 122; 96; 80*; 60; 54,5; 51; 49; 47; 45; 43; 42; 39; 36; 33; 27.5; 21 14.5; 13.5 80; 56,5; 54,5; 51; 45; 43; 39; 33; 20

__ polypepkbs speck to either or both group I or group II. *, major polypeptides against which there is an enhanced response in groups I L II.

Surface antigens of 0. circumcinta

A

29-

I

B 2

3

45

6

I

2

3

1119 4

5

6

7

8

9

IO

II

12

* * -D

20-

Fig. 7. Biotinylated surface polypeptides of infective larvae precipitated by local (a) and serum (b) antibodies of vaccinated and control sheep and detected by alkaline-phosphatase. conjugated streptavidin. A. Tracks l-3 = polypeptides precipitated by mucosal antibodies of group I (track 1), group II (track 2) and group III (track 3). Tracks 46 = polypeptides precipitated by bile antibodies of group I (track 4), group II (track 5) and group III (track 5). B. Tracks l-6 = polypeptides precipitated by sera of group I collected on: day 0 (track 1); day 21 (track 2); day 28 (track 3); day 35 (track 4); day 49 (track 5) and day 56 (track 6). Lines 7-10 = polypeptides precipitated by sera of group II collected on: day 28 (track 7); day 35 (track 8); day 49 (track 9) and day 56 (track 10). Lines 11, 12 = polypeptides precipitated by sera of group III (challenge control) on day 49 (track 11) and day 56 (track 12)

Groups II (track 5) and Group III (track 6) are also shown in Fig. 7a and the sizesof the polypeptides recognized are listed in Table 4. The responsein Group II animalsis very low and only 2 polypeptides (14.5 & 13.5 kDa; arrowed in track 5) are weakly recognizedbut are specificto this group of animals.Comparisonof the polypeptidesrecognized by the antibodiesfrom Group I (track 4) with those recognizedby the challengecontrols (track 6) shows a seriesof polypeptideswhich are commonto both groups with an enhancedresponseto the polypeptides of 80 and 27.5 kDa from the protected group (* in Table 4). In addition a seriesof polypeptides recognized by sera from the protected group are observed(Table 4). Analysis of the surfacepolypeptidesrecognizedby serafrom the 3 groups of animalsare presentedin Fig. 7b. The serafrom Group I animalsrecognize10 polypeptides (arrows, Fig. 7b track 2) specifically (relative to a Day 0 control, track 1 during immunization (Fig. 7b tracks 2, 3, 4) and after challenge(Fig. 7b track 5 and 6), the most prominent polypeptide being of 44 kDa. Immunization with ovalbumin (tracks 7 & 8) alsoinduced a responseto this polypeptide as did challengeof either group II (tracks 9, 10)or the control group III (tracks 11, 12). In fact challengeof thesegroupsinduced antibodies recognizing, albeit weakly the main polypeptides recognizedspecificallyby Group I animals following immunization with surfacepolypeptides.

DISCUSSION

Lambsvaccinatedsubcutaneouslywith 2 low doses (25 pg/kg body weight) of partially purified infective larvae surface antigens and beryllium hydroxide showed 72% protection against challenge with a singledoseof 5 x lo4 infective larvae of 0. circumcincta, while lambsvaccinated with ovalbumin and beryllium hydroxide showedsome 54% protection relative to challengecontrols. The differencein worm burdensbetweenvaccinated groupsand the control group was statistically significant, but the difference betweenthe 2 groupsof vaccinatedanimalswasnot. Mild clinical changes(decreasein body weight and increasein percentageof blood eosinophils)were observed after challenge in all the experimental groups. Lambs vaccinated with infective larval surface antigens showed an anamnestic serum IgA responsea week after challengebut mucosal and bile IgA antibody levels against infective larval surfaceantigenswere rather low and no significant differenceswere observed between vaccinated and control lambs. The lambs vaccinated with surface antigensalsoshowedthe clearestserumIgA reaction in immunofluorescence tests.Westernblots using 0. circumcincta L3 surface antigens and pre-infection serafrom lambsvaccinated with ovalbumin showed that as many as 6 polypeptidesof molecular mass from 140 to 42 kDa possessed determinantscrossreactingwith ovalbumin.To our knowledgetherehas been no previous

report on parenteral,

non-specific

1120

H. Wedrychowin et al.

stimulation of protective immunity against 0. circumcincta infection. However, similar resultshave been obtained in rabbit-T. colubriformis system. Rabbits immunizedwith 3 dosesof 1 mg of normal rabbit serum proteins and beryllium hydroxide showednearly sterile immunity to subsequentchallengewith 1x lo4 infective larvae of the nematode. The mean worm burden found in intestines of vaccinated rabbits was 20 while that in challenge controls was2583(Wedrychowicz et al., 1992b).The mechanism of such non-specific stimulation of immunity is obscure. Hall (1988) suggestedthat Beryllium forms complexeswith plasma proteins which induce rapid lymphoproliferative responses in the regionallymph nodesand the appearanceof huge numbers of immunoblasts in the lymph. In the present experiment we monitored blood leukocyte composition during the experiment and found no significant changeswhich could be related to the lymphoproliferative action of beryllium. However, antibodiesinduced by infection appearedto crossreact with the unrelated protein ovalbumin. Challengecontrol lambsshowedincreasedlevelsof serum IgM, IgA and IgG antibodiesreacting with ovalbumin in ELISA tests. Ovalbumin has already been used as a T cell dependent antigen to study mechanismsof local antibody responses in sheepusingFreund’sadjuvant (Husband& Lascelles,1974;Beh & Lascelles,1981; 1984).It was found that the level of IgA responseto the ovalbumin dependedon the type of adjuvant (Beh & Lascelles,1984) and route of the antigen administration(Beh & Lascelles,1981).However, the cross-reactivity of antibodies induced against ovalbumin with 0. circumcincta antigenshas not been reported nor investigatedpreviously. Mucosal and bile IgA antibody responsesto infective larval surfaceantigenswere rather low and no significant differences were observed between vaccinated and control lambs. However, a clear increasein the concentrationof serumIgA antibodies reacting with 0. circumcincta surface antigenswas observedin Group I after the first immunizing dose of surfaceantigensand a significantanamnesticIgA response occurred in this group a week after challenge. Studies on local and systemic immune responsesagainst bacterial and viral antigenshave shownthat parenteralvaccination changedand often inhibited immune reactions on mucosal surfaces (Kenrick & Cooper, 1978; Pierce & Koster, 1980, Jackson& Cooper, 1981).The level of inhibition or modification of local immunemechanismsdepends on the nature of the antigen used for parenteral stimulation and also the vaccination procedure.The immunofluorescencetestscarried out in the present

study showedthe clearestserumIgA reaction with the infective larval surfacein group I on the day of challengeand a week later. Also bile and mueosal IgA reactivity was the hi t in this group. A very strong IgM responseto the in&&e larval surface wasobservedin ch&enge control lambsat the endof the experiment. Interest&&, mucosal IgM antibodies to the L3 surface did not develop in this group while in the protected Group I, the IgM responsefrom the mucosawas very strong. Unlike surface labelling methods, immunofluoreacencedetects surface-exposedepitopesrather than determinants on subsurfaceantigens. Moreover, immunofluorescenceanalysis will not be affected by the biochemicalnature of an epitope, which is a drawback of most surface radio-labeiling and biotinylation methods (Fraser & Kennedy, 1991). In agreementwith previous results (Wedrychowicz et al., 1992a)the present investigation only showeda positive immunof&oreseeneereaction with a proportion of exsheathedlarvae while the pattern and intensity of staining varied considerably. Evidence for heterogeneity in surface antigen expression among Ascaris larvae has been reported (Fraser & Kennedy. 1991)and, according to Kennedy (1991), such polymorphism will have fundamental implications not only for understandingof the dynamicsof parasite populations but also for the application of recombinantvaccines. In the presentexperimentIgM antibody responses both to immunizationand subsequentchallengewere much more marked than in our previous experiment (Wedrychowicz et al., 1992a).This was possibly a result of the different schedule of the present experiment as immunization doseswere given at 2week-intervalsand the challengeinfection at three weeks after the secondimmunization while in the previousexperiment4-week-intervalswereused.Our previous results (Wedrychowicz & Besubik, 1990; Wedrychowicz et al., 1992a)showedthat adjuvants can selectively and independentlyenhancediRerent aspectsof the immuneresponsessuchasprotection, isotype and epitope specificity and challengeprotected lambs demonstrated very strong bile IgA responseto surfacepolypeptides of infective larvae (Wedrychowicz et al., 1992a). The role of IgA responsesin immunity to 0. circumcincta infection hasyet to be resolved(Wedrychow&, 1995).Sincethe lambsimmunized with the fraction of infective larvae surfaceproteins precipitated by bile IgA from immune sheep showed a considerableimmunity againstchallenge,it may be assumed that some of the antigens possessed protective activity. However, although theseantigens weregiven in the sameprotein concentrationascrude

Surface antigens of 0. circumcinta

1121

intestinal sections. Parasite Immunology 3: 127-135. surface extracts in the previous experiment the degree Kennedy M. W. 199 1. The antibody repertoire in nematode of protection achieved was not better than that infections. In: Parasitic Nematodes-Antigens, Memobtained previously (Wedrychowicz et al., 1992a). branes and Genes (Edited by Kennedy M.F.), pp. 219PAGE and westernblot analysisof the antigensused 243. for immunization in the present experiment showed Laemmli U. K. 1970. Cleavage of structural proteins during that 2 polypeptides of 94 and 74 kDa predominated the assembly of the head of bacteriophage T4. Nature which could suggest that other less prominent 227: 680-685. polypeptides might be more protective. The present Lowry 0. H., Rosebrough N. J., Farr A. L. & Randall results indicate a need for further purification and R. J. 1951. Protein measurement with the Folin investigation of the surface polypeptides of the phenol reagent. Journal of Biological Chemistry 193:

infective larvae of 0. circumcincta in order to separateantigensinvolved in protection and pathogenesisof the nematodeinfection. Acknowledgements-The work reported here was supported by the Wellcome Trust through a Travelling Research Fellowship awarded to H. Wedrychowicz.

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