INFECTION AND IMMUNITY, Mar. 1984, p. 986-993 Vol. 43, No. 3 0019-9567/84/030986-08$02.00/0 Copyright C) 1984, American Society for Microbiology Campylobacter jejuni Outer Membrane Proteins Are Antigenic for Humans MARTIN J. BLASER,1,2* JANET A. HOPKINS,' AND MICHAEL L. VASIL3 Medical Service, Veterans Administration Medical Center,1 and Division of Infectious Diseases, Department of Medicine,2 and Department of Microbiology and Immunology,3 University of Colorado School of Medicine, Denver, Colorado 80220 Received 7 November 1983/Accepted 16 December 1983 All Campylobacter jejuni strains have a major outer membrane protein (OMP) that migrates between a molecular weight of 41,000 (41K) and 45K and represents more than 50% of protein present, plus several more minor bands. Using 125I-radiolabeled C. jejuni cells in a radioimmunoprecipitation procedure to assess whether the OMPs were antigenic, we studied serum from rabbits immunized with C. jejuni cells, from humans convalescent after C. jejuni infection, and from appropriate controls. In this assay, the major OMP was the major antigen for both homologously and heterologously immunized rabbits and infected humnans but not for controls. Minor bands at 29K and 50K were also antigenic. We tested human and animal sera in a Western blot procedure using anti-immunoglobulin A (IgA), anti-IgG, or anti-IgM conjugates. Homologous and heterologous immune rabbit serum, but not control serum, recognized a large number of membrane proteins between 15K and 91K, including the major OMP. Both Campylobacter spp.-infected and healthy humans showed IgA, IgG, and IgM responses to the major OMP, although the response was more pronounced in the former group. Sera from infected humans recognized several minor bands to a significantly greater extent than control sera did. Our data suggest that there is antigenic similarity between the OMPs of different C. jejuni strains and that some of these OMPs recognized by infected animals and humans have vaccinogenic potential. For Campylobacterjejuni, an important cause of diarrheal illness in humans (3, 6), the pathogenesis of infection is not clearly understood. Most evidence suggests that the orga- nism invades the gastrointestinal mucosa of the host (1, 6), and biologically significant toxins (20) may or may not exist. As with other invasive organisms, immunological interven- tions should be based on components of the bacterial cell itself. For other gram-negative invasive organisms, three cellular constituents, polysaccharide capsules, lipopolysaccharide, and outer membrane proteins (OMPs), or combinations of these (23), have been studied to determine their usefulness as vaccines. C. jejuni does not have a polysaccharide capsule, although a protein microcapsule has been reported (19). Because there is considerable heterogeneity of the antigenic specificity of the lipopolysaccharide (17), it is unlikely that a vaccine based on components from one or more strains would have the broad specificity needed for protection, and endotoxin-based vaccines often have unac- ceptable toxicity. The OMPs of other gram-negative pathogens have been found to be antigenic (7, 15, 21, 23), and several are being considered as vaccine candidates (7, 23). Recently, we (2) and others (10) have developed methods for identifying and characterizing the OMPs of C. jejuni. As a result of this work, we believe that it is useful to determine whether any of the C. jejuni OMPs identified are antigenic to naturally or experimentally infected hosts. We have adapted the staphylococcal protein A-dependent radioimmunoprecipitation (RIP) method (8), which has been used successfully for characterizing the antigenic proteins of other gram-negative pathogens, to identify those proteins in the C. jejuni outer membrane that are antigenic to infected mammalian hosts. We have also employed a second method, * Corresponding author. 986 immunoblotting (5, 22), to detect antigenic proteins in the C. jejuni outer membrane. The data presented in this paper demonstrate that humans convalescing from Campylobacter enteritis have higher levels of serum immunoglobulin A (IgA), IgG, and IgM antibodies to several of the C. jejuni OMPs than do healthy controls. Data obtained in a similar fashion from infected animal hosts corroborate these find- ings. MATERIALS AND METHODS Bacterial strains and media. The isolate used in most studies was C. jejuni PEN1, the type strain of Penner serotype 1 based on somatic heat-stable antigens (17). Other isolates used were the PEN2 and PEN3 strains from the same system; all three were originally isolated from the feces of patients with diarrhea. The isolate of Campylobacter fetus subsp. fetus (80-109) used was originally isolated from the bloodstream of a patient with acute onset of abdominal pain. Plate cultures of C. jejuni were grown at 42°C in a microaero- bic atmosphere on Mueller-Hinton Agar plates as previously described (2). The C. fetus strain was grown under similar conditions but was incubated at 37°C. Radioiodination of intact C. jejuni cells. C. jejuni cells were extrinsically radiolabeled as previously described (2). In brief, cells from 24-h cultures grown confluently on Mueller- Hinton plates were added to Dulbecco phosphate-buffered saline to make a suspension of standard optical density. A 1.5-ml sample of this suspension was transferred to a 1.5-ml polypropylene centrifuge tube and was centrifuged at 12,800 x g to pellet the cells. According to the method of Swanson (21), 40 p.J of Dulbecco phosphate-buffered saline and 5 p.l of 10-5 M potassium iodide were added to the pellet, and the suspension was transferred to an Iodogen-coated (Pierce Chemical Co., Rockford, Ill.) tube to which 500 ,uCi of 125I was added. The suspension was agitated for 10 min at 25°C and then was added to 1.0 ml of Dulbecco phosphate- on January 28, 2018 by guest http://iai.asm.org/ Downloaded from
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INFECTION AND IMMUNITY, Mar. 1984, p. 986-993 Vol. 43, No. 30019-9567/84/030986-08$02.00/0Copyright C) 1984, American Society for Microbiology
Campylobacter jejuni Outer Membrane Proteins Are Antigenic forHumans
MARTIN J. BLASER,1,2* JANET A. HOPKINS,' AND MICHAEL L. VASIL3
Medical Service, Veterans Administration Medical Center,1 and Division of Infectious Diseases, Department of Medicine,2and Department of Microbiology and Immunology,3 University of Colorado School of Medicine, Denver, Colorado 80220
Received 7 November 1983/Accepted 16 December 1983
All Campylobacter jejuni strains have a major outer membrane protein (OMP) that migrates between amolecular weight of 41,000 (41K) and 45K and represents more than 50% of protein present, plus severalmore minor bands. Using 125I-radiolabeled C. jejuni cells in a radioimmunoprecipitation procedure to assesswhether the OMPs were antigenic, we studied serum from rabbits immunized with C. jejuni cells, fromhumans convalescent after C. jejuni infection, and from appropriate controls. In this assay, the major OMPwas the major antigen for both homologously and heterologously immunized rabbits and infected humnansbut not for controls. Minor bands at 29K and 50K were also antigenic. We tested human and animal sera in aWestern blot procedure using anti-immunoglobulin A (IgA), anti-IgG, or anti-IgM conjugates. Homologousand heterologous immune rabbit serum, but not control serum, recognized a large number of membraneproteins between 15K and 91K, including the major OMP. Both Campylobacter spp.-infected and healthyhumans showed IgA, IgG, and IgM responses to the major OMP, although the response was morepronounced in the former group. Sera from infected humans recognized several minor bands to asignificantly greater extent than control sera did. Our data suggest that there is antigenic similarity betweenthe OMPs of different C. jejuni strains and that some of these OMPs recognized by infected animals andhumans have vaccinogenic potential.
For Campylobacterjejuni, an important cause of diarrhealillness in humans (3, 6), the pathogenesis of infection is notclearly understood. Most evidence suggests that the orga-nism invades the gastrointestinal mucosa of the host (1, 6),and biologically significant toxins (20) may or may not exist.As with other invasive organisms, immunological interven-tions should be based on components of the bacterial cellitself.For other gram-negative invasive organisms, three cellular
constituents, polysaccharide capsules, lipopolysaccharide,and outer membrane proteins (OMPs), or combinations ofthese (23), have been studied to determine their usefulnessas vaccines. C. jejuni does not have a polysaccharidecapsule, although a protein microcapsule has been reported(19). Because there is considerable heterogeneity of theantigenic specificity of the lipopolysaccharide (17), it isunlikely that a vaccine based on components from one ormore strains would have the broad specificity needed forprotection, and endotoxin-based vaccines often have unac-ceptable toxicity.The OMPs of other gram-negative pathogens have been
found to be antigenic (7, 15, 21, 23), and several are beingconsidered as vaccine candidates (7, 23). Recently, we (2)and others (10) have developed methods for identifying andcharacterizing the OMPs of C. jejuni. As a result of thiswork, we believe that it is useful to determine whether any ofthe C. jejuni OMPs identified are antigenic to naturally orexperimentally infected hosts.We have adapted the staphylococcal protein A-dependent
radioimmunoprecipitation (RIP) method (8), which has beenused successfully for characterizing the antigenic proteins ofother gram-negative pathogens, to identify those proteins inthe C. jejuni outer membrane that are antigenic to infectedmammalian hosts. We have also employed a second method,
* Corresponding author.
986
immunoblotting (5, 22), to detect antigenic proteins in the C.jejuni outer membrane. The data presented in this paperdemonstrate that humans convalescing from Campylobacterenteritis have higher levels of serum immunoglobulin A(IgA), IgG, and IgM antibodies to several of the C. jejuniOMPs than do healthy controls. Data obtained in a similarfashion from infected animal hosts corroborate these find-ings.
MATERIALS AND METHODSBacterial strains and media. The isolate used in most
studies was C. jejuni PEN1, the type strain of Pennerserotype 1 based on somatic heat-stable antigens (17). Otherisolates used were the PEN2 and PEN3 strains from thesame system; all three were originally isolated from the fecesof patients with diarrhea. The isolate of Campylobacterfetussubsp. fetus (80-109) used was originally isolated from thebloodstream of a patient with acute onset of abdominal pain.Plate cultures of C. jejuni were grown at 42°C in a microaero-bic atmosphere on Mueller-Hinton Agar plates as previouslydescribed (2). The C. fetus strain was grown under similarconditions but was incubated at 37°C.
Radioiodination of intact C. jejuni cells. C. jejuni cells wereextrinsically radiolabeled as previously described (2). Inbrief, cells from 24-h cultures grown confluently on Mueller-Hinton plates were added to Dulbecco phosphate-bufferedsaline to make a suspension of standard optical density. A1.5-ml sample of this suspension was transferred to a 1.5-mlpolypropylene centrifuge tube and was centrifuged at 12,800x g to pellet the cells. According to the method of Swanson(21), 40 p.J of Dulbecco phosphate-buffered saline and 5 p.l of10-5 M potassium iodide were added to the pellet, and thesuspension was transferred to an Iodogen-coated (PierceChemical Co., Rockford, Ill.) tube to which 500 ,uCi of 125Iwas added. The suspension was agitated for 10 min at 25°Cand then was added to 1.0 ml of Dulbecco phosphate-
buffered saline at 4°C, centrifuged at 12,800 x g, andsuspended in 30 ml of 0.01 M Tris (pH 7.4).
Preparation of Campylobacter outer membranes. C. jejunicells were fractionated to prepare outer membranes aspreviously described (2). A pellet of unlabeled cells was usedfor the immunoblot procedures, and the 125I-labeled cellswere used for the RIP procedure. Cells were sonicated on icefour times with a Branson Sonifier (model S-75; BransonInstruments Co., Danbury, Conn.) for 30 s with 30-s rests.The preparation was then centrifuged two times at 5,000 x gfor 20 min to remove whole cells, and the supernatant wascentrifuged for 1 h at 100,000 x g at 40C (L8-70 Ultracentri-fuge, Beckman Instruments, Inc., Fullerton, Calif.). Thepellet was suspended in 3 ml of sterile distilled water andadded to 20 ml of 1% sodium lauryl sarcosinate (Sarcosyl) in7 mM EDTA for a 20-min incubation at 37°C. This suspen-sion was centrifuged at 100,000 x g for 2 h at 4°C. The pelletwas suspended in Tris buffer and recentrifuged at 100,000 x
g for 2 h, and the resultant Sarcosyl-insoluble pellet was
suspended in 1.0 ml of sterile distilled water and stored at40C.
Rabbit and mouse antisera. After samples of preimmuneserum were obtained, adult New Zealand white rabbits were
intravenously immunized with the PEN1, PEN2, or PEN3C. jejuni strain as described elsewhere (2a). Immune serum
was defined as having an indirect fluorescent antibodyreciprocal titer of at least 640. We obtained serial serum
samples from uninfected HA-ICR adult mice or from micethat had been orally, intraperitoneally, or intravenouslyinfected with C. jejuni in an experimental model (1). Allserum samples were stored at -20°C until used for theseassays.Human sera. From our serum collection we used for this
study serum samples from persons in the following groups:(i) random healthy adults and children; (ii) healthy adults andchildren with reciprocal serum IgG titers by C. jejuni en-
zyme-linked immunosorbent assay (ELISA) of less than 50(M. J. Blaser and D. J. Duncan, Clin. Res., in press); (iii)random patients 2 to 3 weeks after the onset of C. jejunienteritis (convalescent); (iv) convalescent patients with re-
ciprocal ELISA titers greater than 200; (v) convalescentpatients after C. fetus infections. Serum samples had beenstored at -20 or -70°C for 1 to 5 years before use in thisstudy.RIP procedure. The RIP method used to identify antigenic
C. jejuni OMPs was adapted from that of Hansen andcolleagues (7). Outer membranes were prepared from ra-
dioiodinated C. jejuni cells, and 9 x 105 cpm of 125I-labeledOMPs was mixed with 100 ,ul of human serum that was
previously heated at 56°C for 30 min to destroy complementactivity. This suspension was incubated at 4°C for 18 h, andthen the membrane particles, together with any adherentantibodies, were collected by centrifugation at 80,000 x g for1 h at 40C. The resultant pellet was suspended in 1.0 ml of a
solubilization buffer composed of 10 mM Tris-hydrochloride(pH 7.8) containing 1% (vol/vol) Triton X-100, 150 mMNaCl, and 10 mM EDTA and was incubated at 370C for 1 h.Use of 0.5 or 1.0% sodium dodecyl sulfate (SDS), 2% Brij, or
2% Triton showed no greater solubilization than the 79.5%produced by 1% Triton. The suspension was then subjectedto centrifugation at 80,000 x g for 1 h, and the uppermost 900,ul of supernatant liquid containing solubilized antibodieswas carefully drawn off. At least 40% of the initial radioac-tivity added to the system was recovered in this supernatant,to which was then added 400 ,ul of a 10% (wt/vol) formalde-hyde-treated suspension of the Cowan I strain of Staphylo-
coccus aureus prepared by the method of Kessler (8). Thissuspension was incubated at 4°C for 60 min, and the result-ant S. aureus-antibody-membrane complexes were spundown at 12,000 x g and were washed five times with 10 mMTris-hydrochloride (pH 7.8) containing 1% (vol/vol) TritonX-100, 150 mM NaCl, 10 mM EDTA, 0.2% (wt/vol) sodiumdeoxycholate, and 0.1% (wt/vol) SDS (W buffer). Approxi-mately 1 to 2% of the solubilized radiolabeled proteinoriginally present in the supernatant preparation was specifi-cally bound by antibodies to S. aureus. The S. aureus-
adsorbed antibody-membrane complexes were recovered bysuspending the washed S. aureus antibody-membrane pelletin 200 ,ulof 0.0625 M Tris-hydrochloride, pH 6.8, containing2% (wt/vol) SDS and 10% (vol/vol) glycerol (D buffer),heating this suspension at 100°C for 4 min, and removing theS. aureus from the suspension by centrifugation at 12,000 x
g for 2 min. In all cases, over 90% of the radioactivityoriginally bound to the S. aureus was recovered in thesupernatant fluid. The supernatant material was then heatedat 100°C for 3 min in the presence of 5% 2-mercaptoethanoland 4% SDS in sample buffer, and the radioiodinated anti-gens present in the supernatant were identified by SDS-polyacrylamide gel electrophoresis (PAGE).SDS-PAGE. Preparations of total and outer membrane C.
jejuni proteins for SDS-PAGE were made as previouslydescribed (2). Stacking and separating gels consisted of 4.5and 7.0 or 10% acrylamide (Bio-Rad Laboratories, Rich-mond, Calif.) and protein concentrations of preparationsstandardized (14). Electrophoresis was carried out with a
Protean Dual 16-cm slab cell apparatus (Bio-Rad) with a
constant current of 35 mA per gel. Gels were stained with themodified silver stain of Oakley, dried with a gel dryer (Bio-Rad), and processed for autoradiography as previously de-scribed (2).Immunoblot procedure. The methods of Towbin et al. (22)
as modified by Burnette (5) formed the basis for the Westernblot procedure we employed. The procedure was done as
follows. After SDS-PAGE, the gels were covered withnitrocellulose paper (NCP) that had been soaked in electrodebuffer (192 mM glycine, 25 mM Tris base, 20% methanol).The electroblotting sponges were rinsed in deionized waterand then saturated with the electrode buffer. After the gel (orgel slices) was placed on the sponge, the NCP was laid overthe gel, and then the second sponge was overlaid. Thissandwich was placed in an electroblotting apparatus (Bio-Rad), and the proteins were electrophoresed at 100 mA for18 h. Upon completion of the electroblotting, the gels were
removed and stained with Coomassie blue to determinewhether transfer was complete. NCP strips containing themolecular weight markers and the Campylobacter antigenswere stained with amido black for molecular weight calcula-tions. The NCP strips were rinsed in borate buffer (pH 8.0)with 0.5% Tween 80 and then were incubated for 1 h at 42°Cwith 1% bovine serum albumin (Sigma Chemical Co., St.Louis, Mo.) in borate buffer. After a rinsing in borate buffer,the NCP was incubated at 250C for 4 h in a 1:100 dilution ofthe test serum samples in 1% bovine serum albumin-boratebuffer. After being washed in borate buffer, the NCP was
incubated at 250C for 1 h with optimal horseradish peroxi-dase (HRPO)-conjugated antiserum dilutions as determinedexperimentally. Antisera used as capture antibodies were
HRPO conjugates of S. aureus protein A (Amersham Corp.,Arlington Heights, Ill.) or goat anti-human IgA (Tago, Bur-
lingame, Calif.) or rabbit anti-human IgG or IgM (AccurateChemical, Westbury, N.Y.) diluted in bovine serum albu-min-borate buffer with 2% OCT compound (Lab-Tek Prod-
ucts, Div. of Miles Laboratories, Inc., Naperville, Ill.). Aftera washing in borate buffer, the NCP was placed in DABsolution (50 mM Tris with 0.025% diaminobenzidine with 2drops of H202) for 5 to 10 min until reaction products wereoptimally developed. After a washing in tap water, the NCPwas photographed then air dried in the dark.
RESULTS
Radiolabeling of C. jejuni cells. Radioiodination of intact C.jejuni PEN1 cells with subsequent fractionation of the outermembrane-enriched preparation showed that the major sur-face-exposed peptide migrates at a molecular weight of44,000 (44K) (Fig. 1, lane a). Minor bands also were resolvedat 30K and 50K. A wide band of radiolabel was seen at thebottom of the gel, corresponding to unresolved low-molecu-lar-weight protein or degraded peptides.RIP. According to the RIP method, sera from homolo-
gously and heterologously immunized rabbits recognizedseveral of the surface-exposed OMPs (Fig. 1). The mostradiolabel was detected at 44K or at the bottom of the gelrepresenting degraded peptides. No radiolabeled proteinbands were detected when normal rabbit serum or phos-phate-buffered saline was incubated with the outer mem-brane preparations.By means of the RIP method, serum samples from humans
convalescent from Campylobacter enteritis and from healthycontrols were compared (Fig. 2). Although not as clear-cutas in the rabbit studies, a difference in the amount ofradiolabel visualized in these two groups was easily dis-cerned. Again, most activity occurred in the protein bandmigrating at about 44K.Western blots with serum from experimentally infected
animals. Using the Western blot method with a swine anti-
b c d e f g h
U
4-N4U- 'd -0p 1 11 -
m n 0 p
b C d e
.450K
44K
30K _
FIG. 1. RIP of 125"-labeled C. jejuni OMPs with convalescent-phase and control rabbit serum. Serum samples were mixed withradioiodinated outer membranes, and then the antigen-antibodycomplexes were isolated by adsorption to S. aureus and processedby SDS-PAGE followed by autoradiography to detect radiolabeledprotein antigens, as described in the text. Lane a, '25I-labeled OMPspresent in C. jejuni outer membranes used as antigen for thisexperiment; lane b, 125I-labeled OMPs precipitated by 100 ,ul ofPBS; lane c, 125I-labeled OMPs precipitated by 100 ,ul of convales-cent-phase serum from an isoimmunized rabbit; lane d, 125I-labeledOMPs precipitated by 100 ,ul of convalescent-phase serum from a
heteroimmunized rabbit; lane e, 125I-labeled OMPs precipitated by100 ,ul of preimmune serum from the isoimmunized rabbit. Values toleft show molecular weights.
MO
FIG. 2. Autoradiograph of SDS-PAGE after RIP of C. jejuniOMPs by human serum diluted 1:100. Shown are preparations fromouter membrane fractions of extrinsically 125I-labeled C. jejuniPEN1 whole cells (lane a), outer membrane fraction of extrinsically1251-labeled C. jejuni with serum from patients convalescent fromCampylobacter enteritis (lanes b through k), and healthy controls(lanes 1 through p).
rabbit immunoglobulin conjugate, we found that immunerabbit serum recognized a wide variety of C. jejuni OMPs,whereas normal serum did not (Fig. 3). Immune serumdiluted 1:1,000 still produced strong reactions (data notshown). The range of proteins recognized was from 15K to92K. As in the RIP procedure, serum samples from bothhomologously and heterologously immunized rabbits con-tained antibodies to these proteins. In contrast, amongserum samples from mice that had been experimentallyinfected with C. jejuni, only serum raised after intraperitone-al challenge had significant antibody and only at a low (1:20)detected serum dilution (data not shown).Western blots with serum from humans. We then used the
same rabbit sera shown in Fig. 1 to establish a Western blotsystem. The S. aureus protein A conjugate captured specificantibody to C. jejuni OMPs that was in the serum of theimmune rabbit but not in the normal rabbit serum. Based onthese findings, the use dilution for S. aureus protein A infuture assays was 1:1,000. Similarly, using serum from ahealthy human and a human convalescent from Campylo-bacter enteritis, we determined that the appropriate dilutionof a rabbit anti-human IgG was 1:800. Using these samehuman sera, we determined the use dilutions of a goat anti-
FIG. 5. Western blot of human serum IgG against C.jejuni PEN1outer membrane preparations. Sera were the same as in Fig. 4.Second antibody was rabbit anti-human IgG-HRPO conjugate(1:800).
FIG. 3. Western blot of rabbit serum against C. jejuni PEN1outer membrane preparations. Sera were from rabbits immunizedwith PEN1 (lane a), with PEN2 (lane b), with PEN3 (lane c), andunimmunized (lane d). All sera were diluted 1:100. Second antibodyis swine anti-rabbit immunoglobulin-HRPO conjugate (1:200).
human IgA conjugate (1:1,000) and a rabbit anti-human IgMconjugate (1:800) (data not shown).We then studied serum from humans. As shown in Fig. 4,
both persons convalescent from Campylobacter enteritisand healthy persons had IgA antibodies to the C. jejuniOMPs that resolved in the Western blot procedure. Themajor band was the most antigenic band for both ill andhealthy persons, but the serum from convalescent ill personshad a stronger response. Several minor bands were recog-nized by the serum from ill persons much more than by theserum from healthy ones, especially a band migrating at62K.IgG antibody to the major band was just as pronounced in
a b r d e h
the serum from healthy people as in the serum from ill ones(Fig. 5). Again, differences in serum responses of convales-cent ill persons and of healthy persons to the minor proteinbands were seen. Most prominent were proteins migrating at62K, 54K, and 30K.One explanation for the apparent recognition of the major
OMP by normal serum is that the second antibody source,the rabbit anti-human IgG, has activity against the OMP. Totest this hypothesis, we did several experiments using thesecond antibody alone (without preincubation of the NCPwith the first antibody). In the absence of human serum,there was no recognition of the major OMP by the rabbitserum.For IgM, the picture was similar to that for IgA and IgG
(Fig. 6). There was differentiation between the amount ofrecognition of the OMPs by serum from ill and healthypersons, but there was no all-or-none phenomenon. Severalminor bands appeared to provide better differentiation.As illustrated in Fig. 7, serum samples obtained from
persons convalescent from C. jejuni infections who had high
d e f 9 h
66K-
45K - _ -A _ - _0 66K-
4 5K-31K -
31K--
FIG. 4. Western blot of human serum IgA against C.jejuni PEN1outer membrane preparations. Sera, all diluted 1:100, were from fivepatients convalescent after C. jejuni infection (sera 82-439, 82-442,82-445, 82-32, and 82-37; lanes a through e) and from healthycontrols (sera 82-422, 82-424, 82-427, 82-364, and 82-367; lanes fthrough j). Second antibody was goat anti-human IgA-HRPO conju-gate (1:1,000).
FIG. 6. Western blot of human serum IgM against C. jejuniPEN1 outer membrane preparations. Sera were the same as in Fig.4. Second antibody was rabbit anti-human IgM-HRPO conjugate(1:800).
FIG. 7. Western blot of human serum immunoglobulin against C.jejuni PEN1 outer membrane preparations. The first antibody wasfrom serum from a patient convalescent with Campylobacter enteri-tis with an IgG ELISA reciprocal titer greater than 200 (82-454; lanesa) or from a healthy control with a reciprocal titer less than 50 (82-67; lanes b). Second antibody conjugates were as specified in thelegends to Fig. 4 (IgA), 5 (IgG), and 6 (IgM).
titers in the C. jejuni ELISA showed strong responses to theprotein bands migrating at 65K, 62K, 58K, 44K, 42K, 31K,and 23K. In contrast, persons with known low ELISA titersshowed little reactivity to any OMPs in the IgA or IgMassays; however, there was strong recognition of the 44Kband in the IgG assay.Western blots of other body fluids from humans. Since
normal human serum appears to have IgG to the major OMP,we were interested in whether these specific antibodies weretransferred to the fetus. Not surprisingly, cord blood con-tained IgG but not IgA or IgM antibodies that reactedwith the OMP preparations (Fig. 8). Similarly, we examinedbreast milk from women from Bangladesh, Mexico, and theUnited States. Up to a 1:100 dilution, all three women had
a b c d e f q
66K
45K-
IgA antibody to several OMPs, and the antibody was espe-cially reactive with the major band (Fig. 9). By inspection ofthe NCP strips, concentration of antibody was highest in themilk from the Bangladeshi woman and lowest in the milkfrom the American woman.
Bile specimens from three healthy persons in the UnitedStates were studied for IgA antibody to the OMPs (Fig. 10).Two of the three persons had antibody demonstrated at 1:10dilutions, but tests at further 10-fold dilutions were negative.Western blots with other Campylobacter strains as antigen
sources. We then assessed serum antibody response toOMPs from two other C. jejuni isolates and one C. fetusisolate. Convalescent-phase serum from a patient with Cam-pylobacter enteritis showed a strong IgA response to allthree C. jejuni preparations and a weaker response to the C.fetus strain (Fig. 11). Convalescent-phase serum from apatient with C. fetus systemic infection (bacteremia andmeningitis) had antibody to C. fetus OMPs, but the majorantigen appeared to be lipopolysaccharide. The level ofreactivity of this serum to the C. jejuni OMPs was similar tothat of the serum from a healthy control. Serum from thehealthy control had little antibody to the C. fetus majorbands. For serum IgG (Fig. 12) and IgM (Fig. 13) from thesesame persons, similar phenomena were observed.
DISCUSSIONThe purpose of our study was to determine which, if any,
OMPs of C. jejuni are antigenic for infected animal andhuman hosts. In our previous study (2), we used 1251extrinsic labeling of intact C. jejuni cells to determine whichOMPs were surface exposed. When this technique was usedin the present study, the major surface-exposed peptidemigrated at 44K, and minor bands were seen at 50K and30K. To determine which of these were antigenic, we usedthe RIP technique and found that for both immunized rabbitsand humans convalescent after natural infection, the major44K band was antigenic. Sera from uninfected humansrecognized this protein but to a much lesser extent. Thesedata suggest that the antibodies present in sera had been
h i j k I m n o
a a**
31K -
FIG. 8. Western blot of human cord blood against C. jejuni PEN1 outer membrane preparations. The first antibody was from cord bloodfrom five healthy control mothers and infants as follows: 82-134 (lanes a, f, and k); 82-135 (lanes b, g, and 1); 82-136 (lanes c, h, and m); 82-137(lanes d, i, and n); and 82-138 (lanes e, j, and o). The second antibody was goat anti-human IgA-HRPO conjugate (lanes a through e), rabbitanti-human IgG-HRPO conjugate (lanes f through j), and rabbit anti-human IgM-HRPO conjugate (lanes k through o).
FIG. 9. Western blot of human breast milk IgA against C. jejuniPEN1 outer membrane preparations. The first antibody was frombreast milk from an American woman (lanes a, d, and g), a Mexicanwoman (lanes b, e, and h), or a Bangladeshi woman (lanes c, f, andi). Breast milk was diluted 1:10 (lanes a through c), 1:100 (lanes dthrough f), or 1:1,000 (lanes g through i). Second antibody was goatanti-human IgA-HRPO conjugate.
acquired as a result of the specific infection, although somebackground cross-reacting antibodies are present in normaladult human serum.To confirm these findings, we used an alternative tech-
nique for specific antigen detection, the Western blot proce-dure. In this method,electrophoresed in an SINCP, and test antibodiThere are advantages atRIP or the Western btechnique permits recog
a b
116K -
92.5K -
o b c d e f g h i j k
66K- *4_
45K
31K-
FIG. 11. Western blot of human serum IgA against C. fetus andC. jejuni outer membrane preparations. Antigens were C. fetus(lanes a, e, and i) and C. jejuni PEN3 (lanes b, f, and j), PEN2 (lanesc, g, and k) and PEN1 (lanes d, h, and 1). Sera were from a patientconvalescent after Campylobacter enteritis (82-442, lanes a throughd), a patient convalescent after C. fetus meningitis (81-167, lanes ethrough h), and a healthy person (82-424, lanes i through 1). Secondantibody was goat anti-human IgA-HRPO conjugate.
outer membrane preparations are sarily surface exposed, and because of procedures necessaryDS-PAGE system and transferred to to solubilize the membrane proteins for electrophoresis, theies are permitted to adhere (5, 22). proteins are often denatured, with resulting changes in theirnd disadvantages to using either the tertiary structure and thus in their antigenicity.)lot techniques. The Western blot Other C. jejuni surface components such as lipopolysac-,nition of proteins that are not neces- charide are probably precipitated in the RIP procedure.
Since only proteins are radioiodinated, analysis is limited tothese surface-radiolabeled proteins. Not all proteins are
C d e f g h necessarily radiolabeled, nor is radiolabeling of OMPs uni-form in the RIP procedure. Furthermore, not all IgG sub-classes bind the staphylococcal protein A used (9). In
a b c d e f g h j k I
66K -
66K -
45K- -o45K -
31K -
31K -
FIG. 10. Western blot of human bile IgA against C. jejuni PEN1outer membrane preparations. The first antibody was from bileobtained from three healthy persons at the time of cholecystectomy(patient 1, lane a through c; patient 2, lanes d through f; patient 3,lanes g through i). Bile was diluted 1:10 (lanes a, d, and g), 1:100(lanes b, e, and h), or 1:1,000 lanes c, f, and i). Second antibody was
goat anti-human IgA-HRPO conjugate.
I
FIG. 12. Western blot of human serum IgG against C. fetus andC. jejuni outer membrane preparations. Antigens and first antibodywere as described in the legend to Fig. 11. Second antibody wasrabbit anti-human IgG-HRPO conjugate.
C. jejuni outer membrane prewere as described in the legrabbit anti-human IgM-HRP(
contrast, the Western bloto any outer membraneNCP. In addition, the s(means of the protein A maof several OMPs that dboiling SDS. As such, antaggregate may result inproteins.To summarize our find
additional minor OMPs Mhuman serum than by notization of the antigenicitynondenaturing conditionstheir value as vaccine can(ed persons show antibodibut possibly the C. jejudeterminants with those owhich normal persons artbility is that because Cam,lent, the healthy persons xwith C. jejuni or related c
This study and previouthat the OMPs of C. jejurwhat has been reported fThis evidence includes iantigenic cross-reactionconvalescent-phase humathere is little cross-reacti4OMP antigens. The levelantigens present in seruiinfection was similar to tluals. C. fetus lipopolysamajor antigens recognize4with C. jejuni immune rabsion showed recognition 4jejuni strains but not fromH. A. Cody, unpublishedBoth the RIP and W
marked differences in the oof immune rabbits and a cence in antibody response
e f g h j k mal human serum was not nearly as pronounced. Oneexplanation for this diversity may be the sensitivity of theassays, the difference between hyperimmune and normalrabbit serum being greater than that between convalescent-phase and normal human serum. A similar problem withmeningococcal cell wall antigens was resolved by using a
more sensitive antibody-detection assay (18).The results obtained from the RIP and Western blot assays
were different in several areas. Whereas a protein migratingat about 62K was strongly recognized by immune humanserum samples in the Western blot assay, it was not antigen-ic at all in the RIP procedure. One explanation may be thatthe amino acid moieties necessary for the iodination reactionto occur (13) were not surface exposed in the tertiary proteinstructure. Logan and Trust (11) have presented evidence
A that a major OMP migrating in the 62K to 63K regionrepresents flagellar protein. Flagellar proteins, which areusually very hydrophobic, may not have been sufficientlysolubilized to permit recognition in the RIP procedure.
human serum IgM against C. fetus and In the RIP procedure, immune rabbit and human serum
-parations. Antigens and first antibody showed major recognition to a protein migrating at aboutrend to Fig. 11. Second antibody was 44K, in the region of the major OMP, and control sera were) conjugate. nonreactive. However, in the Western blot procedure, a
wide variety of peptide bands were recognized by immuneserum, and some were recognized by control human serum.
it procedure will detect antibodies There are several possible explanations for this discrepancy.constituent transferred onto the Because the Western blot procedure requires processes thatAlubilized protein precipitated by denature proteins, protein structures may be altered toty consist of multimeric aggregates resemble other antigenic proteins. Recently, core lipopoly-issociate into monomers only in saccharide structures have been identified which are presentibody binding to one protein in the in a wide variety of gram-negative organisms (4); our find-coprecipitation of nonrecognized ings suggest that common core OMP antigens could be
present.[ings, both the major and several Logan and Trust (10) have shown that the 44K major bandvere better recognized by immune probably represents the C. jejuni porin; porins are universal-nimmune serum. Better character- ly found in the outer membranes of gram-negative orga-of the peptides, especially under nisms. Mackie and colleagues have noted that conservation
I will permit further evaluation of of antigens that closely resemble one another in structuiedidates. The reasons why uninfect- and function with their subsequent physiological exposure toes to the major band are not clear, animals explains the existence of natural antibodies in manymni major OMP shares antigenic species (12). In mice, immunization with P. aeruginosa,f other gram-negative organisms to which shares antigenic determinants with a wide variety ofe usually exposed. Another possi- flora, resulted in more of a polyclonal response than did,pylobacter infections are so preva- immunization with Neisseria meningitidis, a strain virtuallymay have been previously infected unknown to mice.)rganisms. We have previously shown that mice challenged with C.Is studies (2, 10) provide evidence jejuni develop a rapid specific serum IgG, but not an IgM orni are highly conserved, similar to IgA, response (1). The level of recognition of the OMPs byor Pseudomonas aeruginosa (16). immune mouse sera may in part be artifically lowered by thehighly similar OMP profiles and technique used. Staphylococcal protein A binds mouse IgGof specific major OMPs. Using poorly (9) and does not bind IgM and IgA. Use of specificin serum samples, we found that anti-mouse immunoglobulin conjugates should determineon between C. fetus and C. jejuni whether OMP-specific antibodies are present.of specific antibody to C. jejuni The breast milk and bile studies show that IgA antibodiesm from the patient post-C. fetus to C. jejuni OMPs are naturally present in two body fluidshat in serum from normal individ- that are of physiological significance in relation to enteric.ccharide appears to contain the infections. Immunization with specific antigens could en-d in this system. Recent studies hance antibody levels in these fluids. This observation may)bit serum in double immunodiffu- be important in suggesting immunization strategies for chil-of soluble antigens from three C. dren in developing countries. Immunization of mothers alsoC. fetus strains (M. J. Blaser and may protect their infants for their first several monthsdata). through passive transplacental IgG antibody transfer.lestern blot procedures showed Mechanisms by which antibodies to specific C. jejuniC. jejuni-OMP antibody responses OMPs may play a protective role include complement-control rabbit, whereas the differ- dependent bactericidal reactions, opsonization for phagocy-is in convalescent-phase and nor- tosis, antibody-dependent cellular cytotoxicity, and inhibi-
tion of adherence to mucosal epithelium. Whether thesemechanisms occur in vivo is currently being studied in ourlaboratory.
ACKNOWLEDGMENTS
This work was supported by the U.S. Army Research andDevelopment Command under contract DAMD 17-83-C-2227 and bythe Medical Research Service of the Veterans Administration.
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