INFECTION AND IMMUNITY, Sept. 1990, p. 3101-3108 0019-9567/90/093101-08$02.00/0 Copyright X) 1990, American Society for Microbiology

Vol. 58, No. 9

Protection of Sheep against Chlamydia psittaci Infection with a Subcellular Vaccine Containing the Major Outer Membrane Protein TIN -WEE TAN,t ALAN J. HERRING,* IAN E. ANDERSON, AND GARETH E. JONES Moredun Research Institute, 408 Gilmerton Road, Edinburgh EH17 7JH, United Kingdom Received 22 February 1990/Accepted 29 June 1990

An outer membrane (OM) preparation from elementary bodies (EBs) of Chlamydia psittaci (ovine abortion strain) was used to vaccinate pregnant ewes in a single subcutaneous dose and was found to achieve protection after subcutaneous challenge with infectious organisms. Inactivated purified EBs used as a single-dose vaccine also gave protection. The ratio of live to dead lambs was significantly higher in the vaccinated groups (16:1 and 15:1, respectively) than in the placebo group (8:9). Polyacrylamide gel electrophoresis and immunoblotting showed that a 40-kilodalton protein was the main protein constituent of the OM preparation, and this was positively identified as the major outer membrane protein by protein microsequencing. Electron microscopy revealed that fine particulate structures on the outermost surface of the EB were also present in the OM preparation. The findings suggest that the major outer membrane protein is an important immunoprotective determinant in ovine abortion vaccines.

Ovine chlamydial abortion, also known as ovine enzootic abortion (OEA) or enzootic abortion of ewes, is an economically important disease in many countries (1). Infection of pregnant ewes results in a necrotizing placentitis and consequent abortion (40). Vaccines prepared from egg-grown Chlamydia psittaci inactivated with Formalin induced immunity in ewes against OEA (30) and form the basis of a product that has been in use for several decades (20). Recently, the efficacy of this vaccine has been variable and there have been outbreaks in vaccinated flocks (26). Heterologous challenge experiments have suggested the possibility of strain variation in the field (2, 3). However, attempts to distinguish between OEA isolates have not revealed any obvious differences (4, 5, 22, 27). Confirmation that the OEA agent can also cause abortion and severe illness in pregnant women (10, 22) has added impetus to efforts to understand and control the disease. An important objective has been the identification of the immunoprotective antigens that can account for the efficacy of OEA vaccines. Evidence that the major outer membrane protein (MOMP) of C. psittaci may be useful for protection in sheep has been reported (25, 44; I. E. Anderson, T. W. Tan, G. E. Jones, and A. J. Herring, Vet. Microbiol., in press). However, it was not possible to obtain pure MOMP in sufficient quantities for further vaccine studies without using strongly denaturing procedures. In this study, a modified procedure for producing chlamydial outer membrane complexes (6, 12) has been used to produce a subcellular vaccine highly enriched for undenatured MOMP. This preparation, given as a single dose, protected sheep against OEA, as did a single dose of a vaccine prepared from purified elementary bodies.

tion test. Chlamydial elementary bodies (EBs) for vaccine production were purified from infected 5'-iodo-2'-deoxyuridine-treated BHK-21 cell monolayers as described previously (5, 27; Anderson et al, in press). Live organisms in placental samples were detected by culture (4). Preparation of vaccines and placebo. Purified organisms were divided into two aliquots of 2 mg each. One aliquot was inactivated and formulated with adjuvant as described by Anderson et al. (in press) to produce the purified EB vaccine containing approximately 160 pug of protein per dose. The other aliquot was subjected to a two-step detergent extraction procedure based on a procedures for preparing chlamydial outer membrane complexes (6, 12, 14). Elementary bodies were incubated in 100 mM phosphate buffer, pH 7.4, containing 10 mM EDTA and 2% sarcosyl (sodium N-lauroylsarcosinate; Sigma Chemical Co., St. Louis, Mo.) for 1 h at 37°C with occasional mixing and bath sonication (10-s bursts) to prevent aggregation. The mixture was then centrifuged at 100,000 x g for 45 min to pellet the insoluble material. The pellet was resuspended and further incubated in the same solution containing 10 mM dithiothreitol, under the same conditions. This mixture was then centrifuged as before. The resultant pellet, the outer membrane (OM) preparation, was suspended in phosphate-buffered saline, pH 7.4, inactivated and formulated into vaccine by the method used for the first vaccine. Each dose of this vaccine contained about 20 ,ug of protein. To produce the placebo vaccine, uninfected cell monolayers were harvested in a way similar to that used for chlamydial purification, concentrated by low-speed centrifugation, and formulated into a vaccine as for the other test vaccines. Protein estimation was carried out by using a dye-binding assay (8). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The two procedures were carried out as described before (23, 25; Anderson et al., in press). Gels were stained with silver by the method of

MATERIALS AND METHODS Chiamydial culture and purification. The ovine abortion isolate of C. psittaci, S26/3, was used in this study (4). An egg-grown organism was used (4) for the complement fixa-

Morrisey (34). Protein microsequencing. The OM preparation was resolved by SDS-PAGE and electroblotted onto a glass fiber support (Glassybond; Biometra Ltd, Manchester, United Kingdom) by a semidry method (18). After the transfer, the

* Corresponding author. t Present address: Department of Biochemistry, National University of Singapore, Kent Ridge, Republic of Singapore 0511. 3101

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FIG. 1. Analyses of the vaccine preparations by SDS-PAGE and by immunoblotting. (a) SDS-PAGE profiles (silver stained) of the vaccine preparations used: BHK-21 cell preparation used on group A ewes as a placebo vaccine (lane 2); purified EBs used to vaccinate group B ewes (lane 6); and outer membrane preparation used to vaccinate group C ewes (lane 3, 3 ,ug; lane 7, 1 ,ug). Chlamydial components solubilized from the first detergent extraction (sarcosyl) of EBs (lane 5) and the second sequential extraction (sarcosyl-dithiothreitol) (lane 4) are also shown. Molecular mass standards are labeled in kilodaltons (lane 1). The stained material at the interface of the stacking and resolving gels is probably nucleic acid which is not seen in gels stained with Coomassie blue. (b) Distribution of chlamydial antigens during the sequential extraction procedure as visualized by immunblotting with a serum sample from a postabortion ewe followed by autoradiography. Lane 1, Purified EBs; lane 2, sarcosyl-soluble components from the first detergent extraction of EBs; lane 3, sarcosyl-dithiothreitol-soluble components from the second sequential extraction; lane 4, detergent-insoluble outer membrane preparation constituting the subcellular OM vaccine. The position of MOMP is as marked; the molecular mass standards are labeled in kilodaltons.

Glassybond filter was rinsed with distilled water and stained with Coomassie blue. The dominant 40-kilodalton (kDa) protein was identified and excised for sequence analysis. Amino acid sequencing was performed on a gas-phase microsequencer (model 477A; Applied Biosystems, Inc., Foster City, Calif.) (courtesy of Linda Fothergill-Gilmore, University of Edinburgh, Edinburgh, United Kingdom). Briefly, Polybrene (2 mg/30 ,ul) was added to the sintered glass fiber sample disk, which then underwent a 3-h precycle of washes before the protein sample was loaded and subjected to 10 automated sequencing cycles. Electron microscopy. Samples coated on copper grids were stained with either 1% phosphotungstic acid (pH 7.0) or 1% ammonium molybdate (pH 5.3) and examined under a JEOL 1200EX transmission electron microscope operating at a voltage of 80 kV. Serological test. The complement fixation (CF) test was carried out by the method of Stamp et al. (41) by using microtiter plates. Animal procedures. The widespread occurrence of chlamydial infection in sheep flocks necessitated the stringent selection of experimental animals for this study. Ewes obtained from a flock with no known history of OEA were serologically screened for chlamydia-specific antibodies by the CF test and by immunoblotting. Following synchronization of estrus, selected ewes were mated and penned separately in three groups. Within a month of mating, ewes from each group were vaccinated subcutaneously with 1 ml of the placebo (group A), purified EB (group B, 160 ,ug of protein), or subcellular OM (group C, 20 ,ug of protein). Serum samples were taken at regular intervals for analysis by the CF test. After 70 days of gestation, all pregnant ewes were challenged by subcutaneous injection with 1 ml of live S26/3

organisms (105-5 chick embryo lethal doses). At parturition, placental tissues or vaginal swabs were taken for isolation of chlamydiae (Anderson et al, in press). Statistical test. The Fisher's exact test (one-tailed) was used to analyze the data for significance in a 2 by 2 contingency table (19). RESULTS Production and biochemical analyses of the vaccines. The results of SDS-PAGE and immunoblotting analyses of the vaccine preparations and the intermediate stages of preparation are shown in Fig. 1. The first detergent extraction solubilized some chlamydial proteins; most antigens, except a 40-kDa protein, were removed at this stage. The subsequent detergent-dithiothreitol extraction removed almost all low-molecular-mass proteins, leaving a 40-kDa protein in a highly enriched state (arrow). Some high-molecular-mass polypeptides were still present (Fig. la, lane 7) and particularly visible when the well was heavily loaded (lane 3). Densitometric scanning showed that in the final detergentinsoluble preparation, the 40-kDa polypeptide made up in excess of 90% of the protein present. A band migrating at the dye front was also detected in both chlamydial vaccine preparations by silver staining, and it possessed the mobility expected for chlamydial lipopolysaccharide (LPS) (data not shown). Immunoblotting of a gel similar to that in Fig. la with a postabortion serum sample from an experimentally infected convalescent ewe showed that the 40-kDa protein was strongly antigenic and comigrated with the dominant 40-kDa band from whole organisms (Fig. lb). Protein microsequencing showed that the 40-kDa protein possessed the following amino-terminal sequence:

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FIG. 2. Electron micrograph of the purified EB vaccine preparation magnified x 240,000. The arrows indicate obvious particles on the EB surface. (Bar = 100 nm).

9 10 7 8 6 4 5 3 2 Leu-Pro-Val-Gly-Asn-Pro-Ala-Glu-Pro-Ser ... This decapeptide sequence matched the 23rd to 32nd residue of the amino acid sequence deduced from the S26/3 MOMP nucleotide sequence (24), indicating that a 22-residue leader peptide has been cleaved from the precursor S26/3 MOMP polypeptide. During the first two sequencing cycles, alternative residues Asp-Gly were also detected. Inspection of the deduced S26/3 MOMP sequence showed that residues 14 and 15 were Asp-Gly. This suggests that an Asp-Gly terminus may have arisen from an alternative processing site downstream of Leu-1, but mild acid hydrolysis during the staining procedure or contamination are alternative explana1

tions. Electron microscopy. Chlamydial EBs observed were typically coccoid and about 200 to 400 nm in diameter (Fig. 2). A granularity of the surface was commonly observed in negatively stained EB preparations. On the edges of the EB, the particulate nature of the surface was clearly visible (arrows).

After a single detergent extraction, the material had the appearance of broken membrane fragments. Following the two-step sarcosyl extraction, the preparation had the appearance shown in Fig. 3. The most abundant structures were fine, tightly packed particles very similar in size to those seen on the EB surface. Where fragments of membrane were visible in an edge-on aspect, knoblike particles which appeared to project from a continuous substratum were seen (open arrow in Fig. 3). Another less abundant structural feature was a rosette consisting of nine subunits of about 3 to 4 nm arranged in a ring about 15 nm in diameter with a stain-filled central cavity (solid arrow in Fig. 3). Analyses of the antibody response. Within 3 weeks of the homologous challenge, most control animals injected with the placebo vaccine (group A) responded with log2 CF antibody titers ranging from 4 to 6 (Fig. 4). Although three ewes had responded slowly, all control animals eventually

seroconverted. Vaccination with purified EBs (group B) or the subcellular OM (group C) vaccine induced a primary response in most

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sheep within 42 days. Titers generally declined by the time of challenge, whereupon a secondary response was induced in all vaccinated sheep (Fig. 4b and c). One ewe from group B and three from group C did not produce a detectable CF titer before challenge, but none of them became infected or aborted. As previously reported (11, 17, 20, 29), no significant correlation was found between the presence of prechallenge CF titers and immunity. Figure 5 shows the immunoblots of prechallenge sera from ewes of all three groups 4 weeks postvaccination. Lysates of whole EBs were used as antigen. No chlamydia-specific antibodies were detected in the sera of ewes injected with the placebo. In contrast, ewes vaccinated with the EB and OM vaccines produced chlamydia-specific antibodies, primarily directed against MOMP. Lambing and isolation results. Table 1 shows the final result of the study. All ewes in group C were protected against abortion; however, infectious organisms were isolated from one ewe. One of the live lambs from the single infected ewe died. In group B, 6 of 7 ewes were protected and a similar low ratio of lamb mortality was recorded. In contrast, 7 of the 12 control animals were infected, of which 5 subsequently aborted. The lamb mortality was significantly higher compared with either of the vaccine groups (P < 0.005).

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DISCUSSION In comparison with attempts to vaccinate against chlamydial infections in humans and in animal models by using whole organisms or specific components as immunogens (16, 21, 45-47, 50), vaccination against abortion in ewes caused by C. psittaci has had a long and successful history until recent years (2, 20, 26, 29, 30). Vaccines against OEA have evolved from placental and yolk sac preparations of the 1950s (20, 29, 30) to cell-cultured vaccines (48). Recently, purified chlamydial EBs from cell culture have been successfully used in a two-dose vaccination-challenge experiment to establish that the immunoprotective components of these vaccines resided in the chlamydial EB (Anderson et al., in press). This study has extended that finding by showing that EBs are effective when given as a single dose. As the next step in the logical progression of vaccine development, the subcellular component(s) of the EB required for protection has been shown to reside in the OM as described above. SDS-PAGE and immunoblotting analyses showed that the protective subcellular preparation consisted mainly of a 40-kDa protein directly identified as MOMP by amino acid microsequencing. The sequence was identical to the first 9 residues of Chlamydia trachomatis L2 MOMP (35), confirming that leader peptide cleavage occurs at the same site in C. psittaci. A component with the mobility of

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chlamydial LPS, a characteristic component of the OM (37), also present (data not shown). The appearance of the subcellular preparation in the

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electron microscope was consistent with that reported for ,a OM preparations by Matsumoto (32, 33). Fine particles some / to 6 nm in diameter appear to be the major structural component of the outermost surface of the EB. It must be assumed that these particles are aggregates of MOMP. This is consistent with the known properties of MOMP, namely its predominance in the OM (12), surface exposure (12, 14, 51), porin function (6), and close relationship with LPS (7). The nine-membered rosettes have also been observed previously in both C. psittaci (31) and C. trachomatis (15). Since these structures are not numerous, they may be formed by the minor protein constituents detected in the

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FIG. 4. Kinetics of the CF antibody response of c hallenged sheep in groups A (a, placebo control; n = 12), B (b, purifiied EB vaccine; n = 7) and C (c, subcellular outer membrane vaccinie; n = 11). The mean log2 CF titers (± standard deviation) of ewes ir each group are plotted against number of days postvaccination. The day of homologous challenge with live C. psittaci is indicated with ia solid arrow. i

The most detailed studies have been made with natural

chlamydial infections of small rodents, and both cell-mediated and humoral immune responses have been shown to possess a role, as reviewed by Williams (49) and by Rank (39). A study of infection with OEA strain C. psittaci in a mouse model showed that T-cell-mediated immunity was more efficiently transferred than humoral immunity (11). Recently, studies on cellular immune responses have been reported for ovine abortion strains in sheep (17, 25; H.-S. Huang, M. Phil. thesis, University of Edinburgh, Edinburgh, United Kingdom, 1988), but the immunological mechanisms operating remain undefined. Whether the anti-MOMP antibodies that feature prominently in immunoblots of sera from vaccinated sheep (44; Anderson et al., in press) are mediators of immunity or merely indicators of immunity needs to be resolved. Data presented here suggest that the immunoprotective element(s) of previous OEA vaccines resides within the OM, and the role of each constituent of the OM preparation now remains to be assessed. Several lines of evidence suggest that MOMP is a protective immunogen. Polyclonal and monoclonal antibodies to MOMP neutralize C. trachomatis infectivity in vitro (13, 38) and in vivo (51). Studies of efferent lymph have indicated that MOMP is recognized early in the immune response (25). Sheep protected by vaccination with a purified EB preparation expressed a strong antibody response directed almost exclusively to MOMP (44; Anderson et al., in press). Functionally, MOMP is involved in the developmental cycle (6, 36) and may have a role in infectivity (43). A recent experiment testing SDSextracted MOMP as a vaccine by oral adminstration showed only

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TABLE 1. Effect of vaccinating ewes against challenge with infectious C. psittaci, OEA isolate S26/3 Total no.

Group A

Vaccine

Placebo; uninfected

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Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein.

An outer membrane (OM) preparation from elementary bodies (EBs) of Chlamydia psittaci (ovine abortion strain) was used to vaccinate pregnant ewes in a...
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