INFECTION AND IMMUNITY, Sept. 1990, p. 3101-3108 0019-9567/90/093101-08$02.00/0 Copyright X) 1990, Americn Society for Microbiology Vol. 58, No. 9 Protection of Sheep ginst Chlmydi psittci Infection with Subcellulr Vccine Contining the Mjor Outer Membrne Protein TIN -WEE TAN,t ALAN J. HERRING,* IAN E. ANDERSON, AND GARETH E. JONES Moredun Reserch Institute, 408 Gilmerton Rod, Edinburgh EH17 7JH, United Kingdom Received 22 Februry 1990/Accepted 29 June 1990 An outer membrne (OM) preprtion from elementry bodies (EBs) of Chlmydi psittci (ovine bortion strin) ws used to vccinte pregnnt ewes in single subcutneous dose nd ws found to chieve protection fter subcutneous chllenge with infectious orgnisms. Inctivted purified EBs used s single-dose vccine lso gve protection. The rtio of live to ded lmbs ws significntly higher in the vccinted groups (16:1 nd 15:1, respectively) thn in the plcebo group (8:9). Polycrylmide gel electrophoresis nd immunoblotting showed tht 40-kilodlton protein ws the min protein constituent of the OM preprtion, nd this ws positively identified s the mjor outer membrne protein by protein microsequencing. Electron microscopy reveled tht fine prticulte structures on the outermost surfce of the EB were lso present in the OM preprtion. The findings suggest tht the mjor outer membrne protein is n importnt immunoprotective determinnt in ovine bortion vccines. Ovine chlmydil bortion, lso known s ovine enzootic bortion (OEA) or enzootic bortion of ewes, is n economiclly importnt disese in mny countries (1). Infection of pregnnt ewes results in necrotizing plcentitis nd consequent bortion (40). Vccines prepred from egg-grown Chlmydi psittci inctivted with Formlin induced immunity in ewes ginst OEA (30) nd form the bsis of product tht hs been in use for severl decdes (20). Recently, the efficcy of this vccine hs been vrible nd there hve been outbreks in vccinted flocks (26). Heterologous chllenge experiments hve suggested the possibility of strin vrition in the field (2, 3). However, ttempts to distinguish between OEA isoltes hve not reveled ny obvious differences (4, 5, 22, 27). Confirmtion tht the OEA gent cn lso cuse bortion nd severe illness in pregnnt women (10, 22) hs dded impetus to efforts to understnd nd control the disese. An importnt objective hs been the identifiction of the immunoprotective ntigens tht cn ccount for the efficcy of OEA vccines. Evidence tht the mjor outer membrne protein (MOMP) of C. psittci my be useful for protection in sheep hs been reported (25, 44; I. E. Anderson, T. W. Tn, G. E. Jones, nd A. J. Herring, Vet. Microbiol., in press). However, it ws not possible to obtin pure MOMP in sufficient quntities for further vccine studies without using strongly denturing procedures. In this study, modified procedure for producing chlmydil outer membrne complexes (6, 12) hs been used to produce subcellulr vccine highly enriched for undentured MOMP. This preprtion, given s single dose, protected sheep ginst OEA, s did single dose of vccine prepred from purified elementry bodies. MATERIALS AND METHODS Chimydil culture nd purifiction. The ovine bortion isolte of C. psittci, S26/3, ws used in this study (4). An egg-grown orgnism ws used (4) for the complement fix- * Corresponding uthor. t Present ddress: Deprtment of Biochemistry, Ntionl University of Singpore, Kent Ridge, Republic of Singpore 0511. 3101 tion test. Chlmydil elementry bodies (EBs) for vccine production were purified from infected 5'-iodo-2'-deoxyuridine-treted BHK-21 cell monolyers s described previously (5, 27; Anderson et l, in press). Live orgnisms in plcentl smples were detected by culture (4). Preprtion of vccines nd plcebo. Purified orgnisms were divided into two liquots of 2 mg ech. One liquot ws inctivted nd formulted with djuvnt s described by Anderson et l. (in press) to produce the purified EB vccine contining pproximtely 160 pug of protein per dose. The other liquot ws subjected to two-step detergent extrction procedure bsed on procedures for prepring chlmydil outer membrne complexes (6, 12, 14). Elementry bodies were incubted in 100 mm phosphte buffer, ph 7.4, contining 10 mm EDTA nd 2% srcosyl (sodium N-luroylsrcosinte; Sigm Chemicl Co., St. Louis, Mo.) for 1 h t 37 C with occsionl mixing nd bth soniction (10-s bursts) to prevent ggregtion. The mixture ws then centrifuged t 100,000 x g for 45 min to pellet the insoluble mteril. The pellet ws resuspended nd further incubted in the sme solution contining 10 mm dithiothreitol, under the sme conditions. This mixture ws then centrifuged s before. The resultnt pellet, the outer membrne (OM) preprtion, ws suspended in phosphte-buffered sline, ph 7.4, inctivted nd formulted into vccine by the method used for the first vccine. Ech dose of this vccine contined bout 20,ug of protein. To produce the plcebo vccine, uninfected cell monolyers were hrvested in wy similr to tht used for chlmydil purifiction, concentrted by low-speed centrifugtion, nd formulted into vccine s for the other test vccines. Protein estimtion ws crried out by using dye-binding ssy (8). Sodium dodecyl sulfte-polycrylmide gel electrophoresis (SDS-PAGE) nd immunoblotting. The two procedures were crried out s described before (23, 25; Anderson et l., in press). Gels were stined with silver by the method of Morrisey (34). Protein microsequencing. The OM preprtion ws resolved by SDS-PAGE nd electroblotted onto glss fiber support (Glssybond; Biometr Ltd, Mnchester, United Kingdom) by semidry method (18). After the trnsfer, the
3102 TAN ET AL. INFECT. IMMUN. 1 2 3 4 5 6 7 b 1 2 3 4 Po p bumwb 116 93 78 66 45 _ - 36 "~ _0o 0-1.. t. 45 *jm 4m-MOMP-4 *40 'p 29-24 Pw 20 O 14 _* FIG. 1. Anlyses of the vccine preprtions by SDS-PAGE nd by immunoblotting. () SDS-PAGE profiles (silver stined) of the vccine preprtions used: BHK-21 cell preprtion used on group A ewes s plcebo vccine (lne 2); purified EBs used to vccinte group B ewes (lne 6); nd outer membrne preprtion used to vccinte group C ewes (lne 3, 3,ug; lne 7, 1,ug). Chlmydil components solubilized from the first detergent extrction (srcosyl) of EBs (lne 5) nd the second sequentil extrction (srcosyl-dithiothreitol) (lne 4) re lso shown. Moleculr mss stndrds re lbeled in kilodltons (lne 1). The stined mteril t the interfce of the stcking nd resolving gels is probbly nucleic cid which is not seen in gels stined with Coomssie blue. (b) Distribution of chlmydil ntigens during the sequentil extrction procedure s visulized by immunblotting with serum smple from postbortion ewe followed by utordiogrphy. Lne 1, Purified EBs; lne 2, srcosyl-soluble components from the first detergent extrction of EBs; lne 3, srcosyl-dithiothreitol-soluble components from the second sequentil extrction; lne 4, detergent-insoluble outer membrne preprtion constituting the subcellulr OM vccine. The position of MOMP is s mrked; the moleculr mss stndrds re lbeled in kilodltons. Glssybond filter ws rinsed with distilled wter nd stined with Coomssie blue. The dominnt 40-kilodlton (kd) protein ws identified nd excised for sequence nlysis. Amino cid sequencing ws performed on gs-phse microsequencer (model 477A; Applied Biosystems, Inc., Foster City, Clif.) (courtesy of Lind Fothergill-Gilmore, University of Edinburgh, Edinburgh, United Kingdom). Briefly, Polybrene (2 mg/30,ul) ws dded to the sintered glss fiber smple disk, which then underwent 3-h precycle of wshes before the protein smple ws loded nd subjected to 10 utomted sequencing cycles. Electron microscopy. Smples coted on copper grids were stined with either 1% phosphotungstic cid (ph 7.0) or 1% mmonium molybdte (ph 5.3) nd exmined under JEOL 1200EX trnsmission electron microscope operting t voltge of 80 kv. Serologicl test. The complement fixtion (CF) test ws crried out by the method of Stmp et l. (41) by using microtiter pltes. Animl procedures. The widespred occurrence of chlmydil infection in sheep flocks necessitted the stringent selection of experimentl nimls for this study. Ewes obtined from flock with no known history of OEA were serologiclly screened for chlmydi-specific ntibodies by the CF test nd by immunoblotting. Following synchroniztion of estrus, selected ewes were mted nd penned seprtely in three groups. Within month of mting, ewes from ech group were vccinted subcutneously with 1 ml of the plcebo (group A), purified EB (group B, 160,ug of protein), or subcellulr OM (group C, 20,ug of protein). Serum smples were tken t regulr intervls for nlysis by the CF test. After 70 dys of gesttion, ll pregnnt ewes were chllenged by subcutneous injection with 1 ml of live S26/3 30 orgnisms (105-5 chick embryo lethl doses). At prturition, plcentl tissues or vginl swbs were tken for isoltion of chlmydie (Anderson et l, in press). Sttisticl test. The Fisher's exct test (one-tiled) ws used to nlyze the dt for significnce in 2 by 2 contingency tble (19). RESULTS Production nd biochemicl nlyses of the vccines. The results of SDS-PAGE nd immunoblotting nlyses of the vccine preprtions nd the intermedite stges of preprtion re shown in Fig. 1. The first detergent extrction solubilized some chlmydil proteins; most ntigens, except 40-kD protein, were removed t this stge. The subsequent detergent-dithiothreitol extrction removed lmost ll low-moleculr-mss proteins, leving 40-kD protein in highly enriched stte (rrow). Some high-moleculr-mss polypeptides were still present (Fig. l, lne 7) nd prticulrly visible when the well ws hevily loded (lne 3). Densitometric scnning showed tht in the finl detergentinsoluble preprtion, the 40-kD polypeptide mde up in excess of 90% of the protein present. A bnd migrting t the dye front ws lso detected in both chlmydil vccine preprtions by silver stining, nd it possessed the mobility expected for chlmydil lipopolyscchride (LPS) (dt not shown). Immunoblotting of gel similr to tht in Fig. l with postbortion serum smple from n experimentlly infected convlescent ewe showed tht the 40-kD protein ws strongly ntigenic nd comigrted with the dominnt 40-kD bnd from whole orgnisms (Fig. lb). Protein microsequencing showed tht the 40-kD protein possessed the following mino-terminl sequence:
VOL. 58, 1990 CHLAMYDIAL MOMP VACCINE 3103 loqnm 0 -- + / FIG. 2. Electron microgrph of the purified EB vccine preprtion mgnified x 240,000. The rrows indicte obvious prticles on the EB surfce. (Br = 100 nm). 1 2 3 4 5 6 7 8 9 10 Leu-Pro-Vl-Gly-Asn-Pro-Al-Glu-Pro-Ser... This decpeptide sequence mtched the 23rd to 32nd residue of the mino cid sequence deduced from the S26/3 MOMP nucleotide sequence (24), indicting tht 22-residue leder peptide hs been cleved from the precursor S26/3 MOMP polypeptide. During the first two sequencing cycles, lterntive residues Asp-Gly were lso detected. Inspection of the deduced S26/3 MOMP sequence showed tht residues 14 nd 15 were Asp-Gly. This suggests tht n Asp-Gly terminus my hve risen from n lterntive processing site downstrem of Leu-1, but mild cid hydrolysis during the stining procedure or contmintion re lterntive explntions. Electron microscopy. Chlmydil EBs observed were typiclly coccoid nd bout 200 to 400 nm in dimeter (Fig. 2). A grnulrity of the surfce ws commonly observed in negtively stined EB preprtions. On the edges of the EB, the prticulte nture of the surfce ws clerly visible (rrows). After single detergent extrction, the mteril hd the ppernce of broken membrne frgments. Following the two-step srcosyl extrction, the preprtion hd the ppernce shown in Fig. 3. The most bundnt structures were fine, tightly pcked prticles very similr in size to those seen on the EB surfce. Where frgments of membrne were visible in n edge-on spect, knoblike prticles which ppered to project from continuous substrtum were seen (open rrow in Fig. 3). Another less bundnt structurl feture ws rosette consisting of nine subunits of bout 3 to 4 nm rrnged in ring bout 15 nm in dimeter with stin-filled centrl cvity (solid rrow in Fig. 3). Anlyses of the ntibody response. Within 3 weeks of the homologous chllenge, most control nimls injected with the plcebo vccine (group A) responded with log2 CF ntibody titers rnging from 4 to 6 (Fig. 4). Although three ewes hd responded slowly, ll control nimls eventully seroconverted. Vccintion with purified EBs (group B) or the subcellulr OM (group C) vccine induced primry response in most
3104 TAN ET AL. INFECT. IMMUN. 8A * -Z$ -L~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4.i IV*.; ik # JZ1. 0 #'.o.,,4. -40~ ~ ~ *~ ~~t :';t. *' ; '^ ' *~~~ ott*m_ ~~ b,. ;0 ',,"u-.-.!, j.,,0.. t;, %t4*~'~'&~-a I WOI~~~~~~~~~~~~~~~Iv FIG. 3. Electron microgrph of the subcellulr OM preprtion mgnified x240,000. The solid rrow indictes rosette visible s nine-membered ring. The open rrow indictes membrne frgment with protruding knoblike prticles. (Br = 100 nm). sheep within 42 dys. Titers generlly declined by the time of chllenge, whereupon secondry response ws induced in ll vccinted sheep (Fig. 4b nd c). One ewe from group B nd three from group C did not produce detectble CF titer before chllenge, but none of them becme infected or borted. As previously reported (11, 17, 20, 29), no significnt correltion ws found between the presence of prechllenge CF titers nd immunity. Figure 5 shows the immunoblots of prechllenge ser from ewes of ll three groups 4 weeks postvccintion. Lystes of whole EBs were used s ntigen. No chlmydi-specific ntibodies were detected in the ser of ewes injected with the plcebo. In contrst, ewes vccinted with the EB nd OM vccines produced chlmydi-specific ntibodies, primrily directed ginst MOMP. Lmbing nd isoltion results. Tble 1 shows the finl result of the study. All ewes in group C were protected ginst bortion; however, infectious orgnisms were isolted from one ewe. One of the live lmbs from the single infected ewe died. In group B, 6 of 7 ewes were protected nd similr low rtio of lmb mortlity ws recorded. In contrst, 7 of the 12 control nimls were infected, of which 5 subsequently borted. The lmb mortlity ws significntly higher compred with either of the vccine groups (P < 0.005). DISCUSSION In comprison with ttempts to vccinte ginst chlmydil infections in humns nd in niml models by using whole orgnisms or specific components s immunogens (16, 21, 45-47, 50), vccintion ginst bortion in ewes cused by C. psittci hs hd long nd successful history until recent yers (2, 20, 26, 29, 30). Vccines ginst OEA hve evolved from plcentl nd yolk sc preprtions of the 1950s (20, 29, 30) to cell-cultured vccines (48). Recently, purified chlmydil EBs from cell culture hve been successfully used in two-dose vccintion-chllenge experiment to estblish tht the immunoprotective components of these vccines resided in the chlmydil EB (Anderson et l., in press). This study hs extended tht finding by showing tht EBs re effective when given s single dose. As the next step in the logicl progression of vccine development, the subcellulr component(s) of the EB required for protection hs been shown to reside in the OM s described bove. SDS-PAGE nd immunoblotting nlyses showed tht the protective subcellulr preprtion consisted minly of 40-kD protein directly identified s MOMP by mino cid microsequencing. The sequence ws identicl to the first 9 residues of Chlmydi trchomtis L2 MOMP (35), confirming tht leder peptide clevge occurs t the sme site in C. psittci. A component with the mobility of Y
VOL. 58, 1990 CHLAMYDIAL MOMP VACCINE 3105 L Ai.-I Ai Ai 0 L U-, 13 L. 4* U- _L 01 0 8 6 4 2 e 6 4 2 e 8 e S K S 39 90 dows post-vccintion chlmydil LPS, chrcteristic component of the OM (37), ws lso present (dt not shown). The ppernce of the subcellulr preprtion in the electron microscope ws consistent with tht reported for, OM preprtions by Mtsumoto (32, 33). Fine prticles some / to 6 nm in dimeter pper to be the mjor structurl component of the outermost surfce of the EB. It must be ssumed tht these prticles re ggregtes of MOMP. This is consistent with the known properties of MOMP, nmely its predominnce in the OM (12), surfce exposure (12, 14, 51), porin function (6), nd close reltionship with LPS (7). The nine-membered rosettes hve lso been observed previously in both C. psittci (31) nd C. trchomtis (15). Since these structures re not numerous, they my be formed by the minor protein constituents detected in the preprtion. The nture nd significnce of the immune mechnisms elicited by chlmydil infections re not fully understood. XM> The most detiled studies hve been mde with nturl U/u 230C chlmydil infections of smll rodents, nd both cell-medited nd humorl immune responses hve been shown to possess role, s reviewed by Willims (49) nd by Rnk (39). A study of infection with OEA strin C. psittci in mouse model showed tht T-cell-medited immunity ws more efficiently trnsferred thn humorl immunity (11). Recently, studies on cellulr immune responses hve been reported for ovine bortion strins in sheep (17, 25; H.-S. Hung, M. Phil. thesis, University of Edinburgh, Edinburgh, United Kingdom, 1988), but the immunologicl mechnisms operting remin undefined. Whether the nti-momp ntibodies tht feture prominently in immunoblots of ser from vccinted sheep (44; Anderson et l., in press) re meditors of immunity or merely indictors of immunity needs to be resolved. Dt presented here suggest tht the immunoprotective element(s) of previous OEA vccines resides within the OM, nd the role of ech constituent of the OM preprtion now remins to be ssessed. Severl lines of evidence suggest tht MOMP is protective immunogen. Polyclonl nd monoclonl ntibodies to MOMP neutrlize C. trchomtis '~---~---~----' infectivity in vitro (13, 38) nd in vivo (51). Studies of 126 186 efferent lymph hve indicted tht MOMP is recognized erly in the immune response (25). Sheep protected by vccintion with purified EB preprtion expressed FIG. 4. Kinetics of the CF ntibody response of chllenged sheep strong ntibody response directed lmost exclusively to in groups A (, plcebo control; n = 12), B (b, purifiied EB vccine; MOMP (44; Anderson et l., in press). Functionlly, MOMP n = 7) nd C (c, subcellulr outer membrne vccinie; n = 11). The is involved in the developmentl cycle (6, 36) nd my hve men log2 CF titers (± stndrd devition) of ewes iri ech group re role in infectivity (43). A recent experiment testing SDSdy of homolo- extrcted MOMP s vccine by orl dminstrtion showed plotted ginst number of dys postvccintion. The gous chllenge with live C. psittci is indicted withi solid rrow. only slight effect ginst oculr C. trchomtis infection in monkeys (46). However, the use of SDS-dentured protein TABLE 1. Effect of vccinting ewes ginst chllenge with infectious C. psittci, OEA isolte S26/3 Totl no. ChIIgd No. infected: No. borted: Lmb rtio, Group Vccine vcci- Nonpregnnt ened uninfected unborted ded:live nted ewes' ewes (p <)b (p <)b (p <)b A Plcebo; uninfected 12 0 12 7:5 5:7 9:8 BHK cells B Purified EBs 12 5 7 1:6 (0.080) 1:6 (0.238) 1:10 (0.004) C Subcellulr OM 12 1 11 1:10 (0.019) 0:11 (0.024) 1:16 (0.003) preprtion Nonpregnnt ewes were not chllenged. b Probbility vlues quoted were clculted by using Fisher's exct test (one-tiled), testing ech vccine group ginst the plcebo group.
3106 TAN ET AL. INFECT. IMMUN. A B C 45 * 0 IMOMNA 4 + n npfnp np np + np + np FIG. 5. Immunoblots of prechllenge ser from ewes of ll three groups, 4 weeks postvccintion. (A) Immunoblots of four sets of pooled ser from three plcebo-injected nimls ech (group A); (B) immunoblots of ser from individul nimls vccinted with purified EBs (group B); (C) immunoblots of ser from individul nimls vccinted with the subcellulr OM preprtion (group C); (+), immunoblot of serum from postbortion ewe. Lystes of whole EBs from the S26/3 OEA isolte were used throughout s ntigen. Blots from nonpregnnt ewes which were subsequently not chllenged re indicted t the bottom s np; ech + represents ewe which ws subsequently found infected or borted. The position of MOMP is indicted; the moleculr mss stndrds re mrked in kilodltons. my hve destroyed epitopes necessry for protection (51). Other protein components present in the subcellulr vccine my lso be importnt in immunity, but it seems unlikely tht they were present in sufficient mounts to contribute significnt effect. Chlmydil LPS is generlly not thought to be importnt s protective ntigen in C. trchomtis infections (45, 51). Serum prepred ginst het-killed C. psittci orgnisms, which is likely to contin ntibodies ginst the thermostble genus-specific epitope(s) of LPS, did not confer protection in the pssive trnsfer experiments of Buzoni-Gtel et l. (11). Complement-fixing ntibodies, which do not correlte with protection ginst ovine bortion strins of C. psittci (11, 17, 20, 29), re lso thought to be directed ginst genusspecific epitope(s) of LPS. The immunoblotting technique used in this study nd in previous studies (28; Anderson et l., in press; M. McClenghn nd A. J. Herring, unpublished dt) did not detect consistent ntibody rection ginst LPS. Nevertheless, LPS my hve n djuvnt role in the OEA vccine. If MOMP is the mjor protective component in OEA vccines, it is possible, since serovr vrition in C. trchomtis hs been ttributed to sequence vrition in MOMP (14, 42), tht similr vrition in OEA strins cn ccount for the recent vccine brekdowns. We re currently investigting such vribility in OEA strins; nlysis is complete for five Scottish isoltes, but it hs not reveled ny sequence vrition (S. Bxter nd A. Herring, unpublished results). We re currently investigting further ruminnt bortion strins from continentl Europe nd Afric. To determine whether MOMP lone is sufficient for protection ginst OEA, recombinnt DNA pproch is being pursued. The MOMP gene of n OEA vccine strin (S26/3) hs been cloned, sequenced (24), nd expressed in Escherichi coli (T. W. Tn nd A. J. Herring, unpublished dt). Expression hs lso been chieved in Slmonell typhimurium roa mutnt (J. J. Oliver, A. J. Herring, nd G. D. Bird, unpublished dt), vector known to be cpble of eliciting both rms of the immune response (9). The recombinnt protein is being tested s vccine. It is our hope tht this nd other plnned studies of OEA will help to estblish the immune mechnisms involved in both ovine nd other chlmydil infections. ACKNOWLEDGMENTS T.W.T. is in receipt of Wooldridge Frm Livestock Fellowship from the Animl Helth Trust, United Kingdom. The uthors thnk E. Gry nd L. Inglis for electron microscopy, B. J. Ester nd A. Inglis for photogrphy, nd stff of the Deprtment of Clinicl Studies for the cre of the experimentl nimls. LITERATURE CITED 1. Aitken, I. D. 1983. Enzootic (chlmydil) bortion, p. 119-123. In W. B. Mrtin (ed.), Diseses of sheep. Blckwell Scientific Publishers, Ltd., Oxford. 2. Aitken, I. D., I. E. Anderson, nd G. W. Robinson. 1986. Ovine chlmydil bortion: limittions of inctivted vccine, p. 55-65. In I. D. Aitken (ed.), Chimydil diseses of ruminnts. Publiction EUR 10056 EN. Commission of the Europen Communities, Luxembourg, Luxembourg. 3. Aitken, I. D., G. W. Robinson, nd I. E. Anderson. 1981. Enzootic bortion: experimentl infection. Proc. Sheep Vet. Soc. 5:53-60. 4. Anderson, I. E. 1986. Comprison of the virulence in mice of 30
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