Distribution of Immunogenic Epitopes on the Two Major ... · Universite´ de la Mediterrane´e, Marseille, France Received 30 June 1997/Accepted 29 August 1997 Forty-four monoclonal

CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/97/$04.0010

Nov. 1997, p. 753–763 Vol. 4, No. 6

Copyright © 1997, American Society for Microbiology

Distribution of Immunogenic Epitopes on the Two MajorImmunodominant Proteins (rOmpA and rOmpB) of

Rickettsia conorii among the Other Rickettsiaeof the Spotted Fever Group

WENBIN XU AND DIDIER RAOULT*

Unite des Rickettsies, CNRS UPRES-A 6020, Faculte de Medecine,Universite de la Mediterranee, Marseille, France

Received 30 June 1997/Accepted 29 August 1997

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Ofthese 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein(rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiproteinmonoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the twomajor immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes werelocated on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein.R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes,whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with theexception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additionalanalyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae werelocated on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on theR. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some werefound on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing thecommon epitopes were found to have different sizes in the SFG rickettsial species. The different distributionsof common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demon-strated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and wasgenerally consistent with previously proposed taxonomies.

Rickettsia conorii, one species of the spotted fever group(SFG) rickettsiae, is the causative agent of Mediterraneanspotted fever (17). Unlike other pathogenic SFG rickettsiae,R. conorii is widely distributed geographically, as it is found insouthern Europe, Africa, the Middle East, and the Indiansubcontinent (9, 57). Phylogenetic assessment of R. conoriistrains from different geographical sources has demonstratedtheir diversity, which is reflected in antigenic variation, and thespecies is often considered the R. conorii complex (24, 56).

With the isolation and identification of an increasing num-ber of new SFG rickettsiae worldwide (7, 8, 10, 21, 22, 28, 61),recognition of taxonomic and evolutionary relationships be-tween newly and previously described SFG rickettsiae appearsto be more important. Perhaps the different geographical re-gions and vectors explain the ecological differences in the SFGrickettsiae (9, 24). However, their antigenic and evolutionaryrelationships have usually been revealed from phenotypic andgenotypic studies (11, 20–22, 24, 33, 37, 44, 47, 48, 55, 56, 61).Following these investigations, the SFG rickettsiae have beenphylogenetically divided into several subgroups, i.e., one clus-ter including R. africae, R. parkeri, and strain S, and anothercluster including R. massiliae, Bar29, R. aeschlimannii, andR. rhipicephali. In addition, two different SFG rickettsiae, As-trakhan fever rickettsia (isolated from the Astrakhan region,Russia) and Israeli tick typhus rickettsia, have been shown to

be close relatives of R. conorii and then included in anothercluster with R. conorii. These two rickettsiae are now consid-ered members of the R. conorii complex (20, 24, 37, 55).

In previous studies, a monoclonal antibody technique whichcan be used to accurately analyze the particular immunogenicepitopes located on the complicated cellular antigens playedan important role in investigating the antigenicity and predom-inant immunogenic proteins of the SFG rickettsial species (1,2, 23, 36, 59, 60). Moreover, by analyzing the cross-reactivitiesof monoclonal antibodies with different SFG species, whichrevealed different distributions of the epitopes among theother rickettsiae, this technique has shown potential as a toolto study taxonomic relationships between the SFG rickettsialspecies (59). Therefore, in order to study (i) the major immu-nogenic epitopes of R. conorii, the most complex species of theSFG rickettsiae, (ii) the distribution of the R. conorii epitopesamong the SFG rickettsiae, and (iii) the antigenic taxonomicrelatedness between the R. conorii strains and the other SFGrickettsial species, including the Astrakhan fever rickettsia andthe Israeli tick typhus rickettsia, we produced a large mono-clonal antibody panel to R. conorii Seven (Malish), the pro-posed type strain of R. conorii species (46). All previouslydescribed rickettsial species—and some unrecognized strainsof the SFG; R. prowazekii, R. typhi, and R. canada of the typhusgroup; R. bellii; Orientia tsutsugamushi; and Coxiella burnetii—were included in this study for evaluating reactivity of mono-clonal antibodies. The epitopes located on the two majorimmunodominant surface proteins of R. conorii Seven, i.e.,the rickettsia outer membrane A (rOmpA) protein and therOmpB protein, were studied, and their distributions among

* Corresponding author. Mailing address: Unite des Rickettsies,Faculte de Medecine, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 5,France. Phone: (33) 4 91 32 43 75. Fax: (33) 4 91 83 03 90. E-mail:[email protected].

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Reactivities

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Monoclonalantibody

IsotypeSpecificity

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22

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22

22

22

22

22

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22

22

22

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RC

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22

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22

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22

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11

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22

22

22

22

22

22

22

22

22

22

22

22

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RC

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12

22

22

22

22

22

22

22

22

22

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22

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22

21

22

22

22

22

22

22

22

22

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22

22

22

22

22

22

22

22

22

22

22

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22

22

22

22

22

22

22

22

22

22

22

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22

22

22

22

22

22

22

22

22

22

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11

11

11

22

22

22

22

22

22

22

22

22

22

22

22

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11

11

21

22

22

22

22

22

22

22

22

22

22

22

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11

11

11

11

21

22

22

22

22

22

22

22

22

22

22

22

RC

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11

12

11

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22

22

22

22

22

22

22

22

22

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11

11

21

11

22

22

22

22

22

22

22

22

22

22

RC

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11

11

11

11

12

21

22

22

22

22

22

22

22

22

22

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mpB

11

11

11

21

11

11

11

22

22

22

22

22

22

22

22

22

RC

10-B1

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rOm

pB1

11

11

11

11

11

21

12

22

22

22

22

22

22

22

22

2R

C8-F

12IgG

1rO

mpB

11

11

11

11

11

12

11

12

22

22

22

22

22

22

22

22

RC

8-D1

IgG2a

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pB1

11

11

11

11

11

11

11

22

22

22

22

22

22

22

22

2R

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2IgG

1rO

mpB

11

11

11

11

12

11

22

21

22

22

22

22

22

22

22

22

RC

5-E10

IgG2b

rOm

pA1

11

12

21

21

11

21

11

12

22

22

22

22

22

22

22

2R

C4-C

5IgG

2brO

mpB

11

11

11

21

12

22

11

12

12

22

22

22

22

22

22

22

RC

9-B12

IgG3

rOm

pB1

11

11

12

11

22

21

11

21

22

22

22

22

22

22

22

2R

C2-F

2IgG

1rO

mpB

11

11

11

11

21

11

11

11

12

22

22

22

22

22

22

22

RC

9-B8

IgG2a

rOm

pB1

11

11

11

12

11

11

11

11

22

22

22

22

22

22

22

2R

C3-G

4IgG

2brO

mpA

11

11

22

11

21

11

11

21

21

12

22

22

22

22

22

22

RC

5-E3

IgG1

rOm

pA1

11

11

12

21

11

11

12

21

11

22

22

22

22

22

22

2R

C2-D

9IgG

3rO

mpB

11

11

11

21

12

22

11

22

12

21

22

22

22

22

22

22

RC

1-B7

IgG2a

rOm

pB1

11

11

11

11

11

11

22

22

22

11

22

22

22

22

22

2R

C8-F

8IgG

2brO

mpB

11

11

11

11

12

11

11

11

21

21

12

22

22

22

22

22

RC

7-A2

IgG3

rOm

pB1

11

11

11

11

11

11

11

12

11

11

22

22

22

22

22

2R

C1-H

10IgG

1rO

mpB

11

11

11

12

11

11

11

11

21

11

11

12

22

22

22

22

RC

3-E2

IgG2b

rOm

pA1

11

11

11

11

11

11

11

11

11

11

11

22

22

22

22

2R

C10-A

3IgG

1rO

mpB

11

11

11

11

11

11

11

11

11

11

11

12

22

22

22

22

RC

5-A5

IgG3

LPS-like

11

11

11

11

11

11

11

11

11

11

11

11

11

22

22

22

RC

4-F6

IgML

PS-like1

11

11

11

11

11

11

11

11

11

11

11

11

12

22

22

2R

C7-A

12IgM

LPS-like

11

11

11

11

11

11

11

11

11

11

11

11

11

22

22

22

RC

10-D6

IgML

PS-like1

11

11

11

11

11

11

11

11

11

11

11

11

12

22

22

2

Total d

4444

4444

4444

3936

2727

2223

2220

2321

1614

1211

1011

107

74

44

00

00

00

aT

heantigen

againstw

hichthe

monoclonalantibody

isdirected;rO

mpA

,therickettsialouter

mem

braneprotein

A(116

kDa);rO

mpB

,therickettsialouter

mem

braneprotein

B(124

kDa);L

PS-like,theL

PS-likeantigen.

bR

eactivity(1

)or

lackof

reactivity(2

)of

hybridoma

culturesupernatants

diluted1:4

inthe

micro-IF

assay.cT

herickettsialspecies

testedw

ereR

.conoriiSeven(Sev),R

.conoriiIndiantick

typhusrickettsia

(Ind),R.conoriiK

enyatick

typhusrickettsia

(Ken),R

.conoriiManuel(M

an),R.conoriiM

-1(M

-1),R.conoriiM

oroccan(M

or),Astrakhan

feverrickettsia

(AF

),Israeliticktyphus

rickettsia(Itt),“R

.slovaca”(Slo),strain

S(S),R

.africae(A

fr),R.parkeri(Par),“R

.mongolotim

onae”(H

A),R

.sibirica(Sib),R

.rhipicephali(Rhi),R

.rickettsii(R

ic),R.japonica

(Jpn),R.honei(H

on),Thaitick

typhusrickettsia

(Ttt),B

ar29(B

ar),R.m

assiliae(M

as),R.aeschlim

annii(Aes),R

.montana

(Mon),R

.helvetica(H

el),R.australis

(Aus),R

.akari(Aka),R

.prowazeki

(Pro),R.typhi(T

yp),R.canada

(Can),R

.bellii(Bel),O

.tsutsugamushi(T

su),andC

.burnetii(Cox).

dT

henum

berof

monoclonalantibodies

with

which

differentrickettsialspecies

cross-reacted.

VOL. 4, 1997 IMMUNOGENIC EPITOPES ON RICKETTSIA CONORII PROTEINS 755


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the other R. conorii strains and other SFG rickettsiae werefurther demonstrated. The taxonomic relationships betweenthe R. conorii strains and the other members of the SFGrickettsia were also established based on the distribution ofR. conorii epitopes among the SFG rickettsiae revealed by theircross-reactivities with this monoclonal antibody panel.

MATERIALS AND METHODS

Rickettsial strains. Thirty-two strains of rickettsiae, including 26 different SFGrickettsiae, three typhus group rickettsiae, R. bellii, O. tsutsugamushi, and C.burnetii, were used in this study (Table 1).

All the strains were obtained from the American Type Culture Collection(Rockville, Md.), with the following exceptions. R. australis, Israeli tick typhusrickettsia, R. rhipicephali, “R. slovaca,” and Thai tick typhus rickettsia were kindlyprovided by G. A. Dasch (Naval Medical Research Institute, Bethesda, Md.).R. bellii, R. honei, R. japonica, R. montana, and R. parkeri were kindly providedby D. H. Walker (University of Texas, Galveston). R. helvetica was kindly sup-plied by W. Burgdorfer (Rocky Mountain Laboratory, Hamilton, Mont.).R. conorii M-1 and Indian tick typhus rickettsia, R. sibirica, strain S, and R. can-ada were obtained from the Gamaleya Research Institute of Epidemiology andMicrobiology (Moscow, Russia). Astrakhan fever rickettsia, R. africae, Bar29,R. conorii Manuel, “R. mongolotimonae,” R. massiliae, and R. aeschlimannii wereisolated in our laboratory.

Rickettsial culture and purification. SFG rickettsiae, typhus group rickettsiae,and C. burnetii were propagated as described previously (59). O. tsutsugamushiwas cultivated in L929 cell monolayers overlaid with Dulbecco modified Eagle’sminimal essential medium (Eurobio, Les Ulis, France) in the presence of 1%(wt/vol) glucose (Gibco BRL, Life Technologies Ltd., Paisley, Scotland), 10%fetal bovine serum (Eurobio), and 2 mM L-glutamine (Gibco BRL) at 35°C.Heavily infected cells were harvested and stored at 280°C. These unpurifiedinfected cells were used as antigens in the microimmunofluorescence (micro-IF)assay.

Rickettsial organisms were further purified from infected cells for immuniza-tion and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis(PAGE) as follows. Ten to 20 150-cm2 flasks (Nunc, Roskilde, Denmark) ofheavily infected cells were harvested by glass beads and then sedimented bycentrifugation at 13,500 3 g for 12 min. The pellets were resuspended in 12-mlportions of K36 solution (16.5 mM KH2PO4, 33.5 mM K2HPO4, 100 mM KCl,15.5 mM NaCl), intact cells were sheared by sonication, and cell debris wasremoved by two centrifugations at 135 3 g for 12 min each time. The supernatantcontaining rickettsial organisms was layered onto an equal volume of 25% (wt/vol) sucrose and then centrifuged at 5,000 3 g for 30 min. The supernatant wasthen carefully removed, and the pellet was resuspended with 1.7 ml of K36solution. This suspension was layered onto a 28 to 45% (vol/vol in K36 solution)Renografin (Radioselectan; Schering, Lys-Lez-Lannoy, France) continuous den-sity gradient (58). Rickettsiae were separated from cell debris by ultracentrifu-gation at 120,000 3 g for 1 h. The bands containing purified rickettsiae werecollected, washed twice with 20 ml of K36 solution, and centrifuged at 13,500 3g for 15 min each time. The purified organisms were resuspended in distilledwater and stored at 280°C. The protein contents of the purified suspensionswere quantitated by the Lowry method (41).

Production and characterization of monoclonal antibodies. The 6- to 8-week-old BALB/c mice were immunized intraperitoneally three times with 0.5 ml of

purified viable rickettsial suspension containing ;2 3 104 organisms at 1-weekintervals. One week after the last injection, mice were given booster doses byinjection of 0.1 ml of purified viable rickettsial suspension containing ;4 3 103

organisms into the tail vein. Three days later, the animals were sacrificed andspleen cells were fused with SP2/0-Ag14 myeloma cells by using 50% (wt/vol)polyethylene glycol (molecular weight, 1,300 to 1,600; Sigma Chemical Co., St.Louis, Mo.) (30). The fused hybridoma cells were cultured in the hybridomamedium (Seromed, Berlin, Germany) supplemented with 13 hypoxanthine, am-inopterin, and thymidine selection medium (Sigma) as previously described (30,59).

Antibodies in hybridoma culture supernatant were detected by using the mi-cro-IF assay as described elsewhere (59), incorporating rickettsia-infected L929

FIG. 1. Western immunoblotting of mouse polyclonal antisera and representative monoclonal antibodies with R. conorii Seven proteins subjected to differenttreatments. Native proteins (N), proteins digested with proteinase K (Boehringer GmbH, Mannheim, Germany) at 28 U/ml at 37°C for 1.5 h (D), and proteins heatedat 100°C for 5 min (H) were used and are shown in three lanes for each protein. Lanes 1, mouse polyclonal antisera; lanes 2, monoclonal antibody RC1-C7; lanes 3,RC2-E3; lanes 4, RC8-C9; lanes 5, RC3-G4; lanes 6, RC5-E3; lanes 7, RC8-D1; lanes 8, RC1-H10; lanes 9, RC10-A3; lanes 10, RC4-F6; lanes 11, RC5-A5; lanes 12,RC7-A12. The positions of molecular mass standards (in kilodaltons) are indicated.

TABLE 3. Distribution of common epitopes located on the rOmpAand rOmpB proteins of R. conorii Seven in the SFG rickettsiae

SFG rickettsia

No. (%) of epitopeslocated on proteina: Total no.

(%)rOmpA rOmpB

R. conorii Sevenb 11 (100) 18 (100) 29 (100)R. conorii Indian tick typhus rickettsia 11 (100) 18 (100) 29 (100)R. conorii Kenya tick typhus rickettsia 11 (100) 18 (100) 29 (100)R. conorii Manuel 11 (100) 18 (100) 29 (100)R. conorii M-1 7 (63.6) 18 (100) 25 (86.2)R. conorii Moroccan 5 (45.5) 18 (100) 23 (79.3)Astrakhan fever rickettsia 7 (63.6) 12 (66.7) 19 (65.5)Israeli tick typhus rickettsia 4 (36.4) 15 (83.3) 19 (65.5)“R. slovaca” 5 (45.5) 12 (66.7) 17 (58.6)Strain S 6 (54.5) 11 (61.1) 17 (58.6)R. africae 4 (36.4) 12 (66.7) 16 (55.2)R. parkeri 4 (36.4) 10 (55.6) 14 (48.3)“R. mongolotimonae” 5 (45.5) 12 (66.7) 17 (58.6)R. sibirica 4 (36.4) 11 (61.1) 15 (51.7)R. rhipicephali 2 (18.2) 8 (44.4) 10 (34.5)R. rickettsii 2 (18.2) 7 (38.9) 9 (31.0)R. japonica 2 (18.2) 4 (22.2) 6 (20.7)R. honei 3 (27.3) 4 (22.2) 7 (24.1)Thai tick typhus rickettsia 3 (27.3) 3 (16.7) 6 (20.7)Bar29 1 (9.1) 6 (33.3) 7 (24.1)R. massiliae 1 (9.1) 5 (27.8) 6 (20.7)R. aeschlimannii 1 (9.1) 2 (11.1) 3 (10.3)R. montana 1 (9.1) 2 (11.1) 3 (10.3)R. helvetica 0 0 0R. australis 0 0 0R. akari 0 0 0

a Shared epitopes distributed in the different SFG rickettsiae.b Eleven and 18 distinct epitopes were considered to be located on the rOmpA

and rOmpB proteins of the R. conorii Seven strain, respectively (see text).



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cells as antigens. Dichlorotriazinyl amino fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG) and IgM (heavy and light chains) (AffiniPure;Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) antibodies wereused to detect bound antibodies. Pooled sera from the immunized mice used forhybridoma formation and uninfected mice were used as positive and negativecontrols, respectively, in the micro-IF assay.

Hybridomas which secreted monoclonal antibodies with different reactivitiesamong the SFG rickettsiae were selected and subcloned twice by limiting dilu-tions by standard methods (30). The immunoglobulin class and subclass of thesemonoclonal antibodies were determined with an immunotype mouse monoclonalantibody isotyping kit (stock no. ISO-1; Sigma) following the manufacturer’sinstructions.

SDS-PAGE and Western immunoblotting. SDS-PAGE was performed as de-scribed elsewhere (34, 59). Ten to 30 ml of a purified rickettsial suspensioncontaining 20 to 50 mg of protein was dissolved in an equal volume of Laemmlibuffer, and polypeptides were electrophoretically separated and then visualizedby staining with Coomassie brilliant blue or transferred onto a nitrocellulosemembrane (0.45-mm pore size; Bio-Rad, Richmond, Calif.) (50). SDS-PAGEmolecular weight standards (low- and high-range; Bio-Rad) were included ineach run to estimate protein band size.

Western immunoblotting was performed as described previously (59). Thehybridoma supernatants diluted 1:8 and the peroxidase-conjugated F(ab9)2 frag-ment goat anti-mouse IgG (heavy and light chains) (AffiniPure; Jackson Immu-noResearch) diluted at 1:200 were used in epitope detection. The controls wereapplied as described above for the micro-IF assay.

Numerical taxonomic analysis. The reactivity of each monoclonal antibodywith each rickettsial strain was assessed by the micro-IF assay incorporatinghybridoma cultural supernatant at a 1:4 dilution. The reactivity of each rickettsiawith each monoclonal antibody was scored as either 1 (positive) or 0 (negative)(59). Based on these scores, Jaccard coefficients (SJ) for each pair of rickettsiaewith each monoclonal antibody were obtained as follows: SJ 5 a/(a 1 b), wherea was the number of positive matches and b was the number of negative matches(49). Cluster analysis was carried out on the coefficients obtained by the un-weighted pair group method with arithmetic means (UPGMA) available in thePC-TAXAN software package (Sea Grant College, University of Maryland,College Park).

RESULTS

Production and characterization of monoclonal antibodies.Ten days after fusion, 543 wells containing viable hybridomaswere observed, and 233 of these wells (42.9%) were found tosecrete monoclonal antibodies against R. conorii Seven.Among these 233 hybridomas, 44 hybridomas were selected forfurther study and their reactivities against each antigen arepresented in Table 2.

The antigens against which each monoclonal antibody wasdirected are also presented in Table 2. The two major immu-nodominant proteins, rOmpA and rOmpB proteins, exhibitedapparent molecular masses of 116 and 124 kDa based on theirelectrophoretic mobilities and reacted with 13 and 27 mono-

clonal antibodies, respectively (Fig. 1 and Table 2). All mono-clonal antibodies directed against proteinic epitopes belongedto the IgG class, including IgG1, IgG2a, IgG2b, and IgG3subclasses. The monoclonal antibodies directed against thelipopolysaccharide (LPS)-like antigen belonged to the IgM andIgG3 classes (Table 2).

Epitopes of R. conorii Seven and their distribution amongthe SFG rickettsiae. None of the typhus group rickettsiae,R. bellii, R. tsutsugamushi, and C. burnetii reacted with any ofthese monoclonal antibodies. All members of the SFG rickett-siae reacted with the monoclonal antibodies which recognizedepitopes on LPS. The monoclonal antibodies directed againstepitopes on the rOmpA and rOmpB proteins, however,showed different reactivities with the SFG rickettsiae, exceptfor R. helvetica, R. akari, and R. australis, which failed to reactwith any of them (Table 2).

Several of the monoclonal antibodies directed against pro-teins shared the same reactivity patterns as one or more othermonoclonal antibodies. In such cases, all were considered to bedirected against the same epitope. Accordingly, the 40 mono-clonal antibodies were directed against 29 distinct epitopeslocated on these two proteins (11 on the rOmpA protein and18 on the rOmpB protein) of R. conorii Seven.

All 29 epitopes were expressed on the Manuel, Indian ticktyphus rickettsia, and Kenya tick typhus rickettsia strains ofR. conorii. All other SFG rickettsiae, except R. helvetica,R. akari, and R. australis, possessed between 3 and 25 of theepitopes. The epitopes located on the rOmpB protein wereusually expressed more often than those located on therOmpA protein in the SFG rickettsiae (Table 3).

Further analyses found that all strains of R. conorii possessedall the detected epitopes on rOmpB. Israeli tick typhus rick-ettsia also possessed more of these rOmpB epitopes (83.3%)than the other SFG rickettsiae. However, the distribution ofthe 11 rOmpA proteinic epitopes was generally less wide-spread in the SFG rickettsiae. Even with R. conorii, the M-1and Moroccan strains possessed only some of these epitopes,and among the SFG rickettsiae it was the Astrakhan feverrickettsia and not the Israeli tick typhus rickettsia that pos-sessed most of these rOmpA proteinic epitopes (63.6%) (Ta-ble 3).

Protein polymorphism of the SFG rickettsiae. The electro-phoretic mobilities of the proteins of the R. conorii strains and

TABLE 4. Protein polymorphism of the SFG rickettsiae exhibiting common epitopes with the rOmpA and rOmpB proteinsof R. conorii Seven demonstrated by Western immunoblotting

Monoclonalantibody

Shared epitope on proteins of SFG rickettsiaa:

Sev Ind Ken Man M-1 Mor AF Itt Sib HA Afr Par Slo Rhi Ric Jpn Mas Bar Aes

RC1-C7 116 136 136 116 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2RC2-E3 116 136 136 116 ND 2 2 2 2 2 2 2 2 2 2 2 2 2 2RC1-C2 116 136 136 116 2 2 120 138 2 2 2 2 2 2 2 2 2 2 2RC4-C1 116 136 136 116 ND ND 120 2 2 2 2 2 2 2 2 2 2 2 2RC5-E10 116 136 136 116 2 2 120 2 132 140 ND 2 142 ND ND 2 2 2 2RC3-G4 116 136 136 116 2 2 120 138 132 140 124 126 2 2 ND 2 2 2 2RC5-E3 116 136 136 116 ND ND 2 2 132 140 124 126 142 2 2 140 2 2 2RC8-C9 124 124 124 124 124 124 2 2 2 2 2 2 2 2 2 2 2 2 2RC7-G11 124 124 124 124 124 124 120 116 2 2 2 2 2 2 2 2 2 2 2RC8-C10 124 124 124 124 124 124 2 116 114 112 116 116 115 2 2 2 2 2 2RC8-D1 124 124 124 124 124 124 120 116 114 112 116 116 115 ND 2 2 2 2 2RC9-B8 124 124 124 124 124 124 120 116 114 112 116 116 2 ND ND 122 2 2 2RC1-H10 124 124 124 124 124 124 120 2 114 112 116 116 115 148 130 2 102 102 2RC10-A3 124 124 124 124 124 124 120 116 114 112 116 116 115 148 130 122 102 102 148

a The numbers indicate the molecular masses of the protein (in kilodaltons) on which the common epitope was located. 2, no reactivity; ND, not detected. Theabbreviations of rickettsial species are defined in footnote c of Table 2.



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other SFG rickettsiae on which the shared epitopes withR. conorii Seven lay were further analyzed by Western immu-noblotting (Fig. 2 and 3). The electrophoretic mobilities of theproteins analogous to the R. conorii Seven immunogenic pro-teins varied among the different species and strains (Table 4).

FIG. 2. Polymorphism of proteins of the SFG rickettsiae containing theshared rOmpA protein epitopes of R. conorii Seven detected by Western immu-noblotting incorporating the monoclonal antibodies. Lanes 1, R. conorii Seven;lanes 2, R. conorii Indian tick typhus rickettsia; lanes 3, R. conorii Kenya ticktyphus rickettsia; lanes 4, R. conorii Manuel; lanes 5, R. conorii M-1; lanes 6,R. conorii Moroccan; lanes 7, Astrakhan fever rickettsia; lanes 8, Israeli ticktyphus rickettsia; lanes 9, R. sibirica; lanes 10, “R. mongolotimonae”; lanes 11,R. africae; lanes 12, R. parkeri; lanes 13, “R. slovaca”; lanes 14, R. rickettsii; lanes15, R. rhipicephali; lanes 16, R. japonica. The positions of molecular mass stan-dards (in kilodaltons) are indicated.

FIG. 3. Polymorphism of proteins of the SFG rickettsiae containing theshared rOmpB protein epitopes of R. conorii Seven detected by Western immu-noblotting incorporating the monoclonal antibodies. Lanes 1, R. conorii Seven;lanes 2, R. conorii Indian tick typhus rickettsia; lanes 3, R. conorii Kenya ticktyphus rickettsia; lanes 4, R. conorii Manuel; lanes 5, R. conorii M-1; lanes 6,R. conorii Moroccan; lanes 7, Astrakhan fever rickettsia; lanes 8, Israeli ticktyphus rickettsia; lanes 9, R. sibirica; lanes 10, “R. mongolotimonae”; lanes 11,R. africae; lanes 12, R. parkeri; lanes 13, “R. slovaca”; lanes 14, R. rickettsii; lanes15, R. rhipicephali; lanes 16, R. japonica; lanes 17, R. massiliae; lanes 18, Bar29;lane 19, R. aeschlimannii. The positions of molecular mass standards (in kilo-daltons) are indicated.



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Although all the R. conorii Seven rOmpA protein epitopeswere expressed on a protein of the same size on the Manuelstrain (116 kDa), they existed on markedly larger proteins (136kDa) on the Indian tick typhus rickettsia and Kenya tick typhusrickettsia strains (Fig. 2). Among other SFG rickettsiae, pro-teins of different molecular masses were found to express theR. conorii Seven rOmpA epitopes, ranging from 120 kDa forAstrakhan fever rickettsia to 142 kDa for “R. slovaca” (Fig. 2and Table 4).

For the rOmpB epitopes, protein of indistinguishable size(124 kDa) was demonstrated to contain the epitopes in allR. conorii strains (Fig. 3). These shared epitopes, however,were observed on different-molecular-mass proteins amongother SFG rickettsiae, ranging from 102 kDa for R. massiliaeand Bar29 to 148 kDa for R. rhipicephali and R. aeschlimannii(Fig. 3 and Table 4).

With the exception of the Astrakhan fever rickettsia, all SFGrickettsiae analyzed were demonstrated to possess two differ-ent high-molecular-mass proteins on which shared epitopeswere distributed in the same way as those on R. conorii Seven(Fig. 2 and 3 and Table 4). These two major immunodominantproteins, in particular rOmpB, were also observed on proteinprofiles by SDS-PAGE (Fig. 4). Furthermore, the most prom-inent protein bands correlated with the rOmpB bands onWestern immunoblots performed with the anti-R. conoriiSeven rOmpB protein epitope monoclonal antibodies (Fig. 3and 4B).

Taxonomic relationships of SFG rickettsiae revealed by thismonoclonal antibody panel. The dissimilarities of each SFGrickettsia pair as assessed with this monoclonal antibody paneland the SJ values calculated for each SFG rickettsia pair arepresented in Table 5. The dendrogram constructed from thismatrix by UPGMA analysis is presented in Fig. 5 and demon-strates that the SFG rickettsiae are divided into two deeplydivergent clusters. The species R. aeschlimannii and R. mon-tana and the species R. helvetica, R. akari, and R. australis,which showed identical reactivities with this monoclonal anti-body panel, therefore, could not be distinguished between inthis analysis.

When a cutoff value of 70.0% for SJ was used, severalsmaller clusters were apparent (Fig. 5). Six strains of R. conoriiformed an independent cluster at an SJ of 85.2%. R. sibiricaand “R. mongolotimonae,” Thai tick typhus rickettsia andR. honei, and R. massiliae and Bar29, which had SJs of 91.3,90.1, and 91.7%, respectively, also demonstrated very closeantigenic relationships. Astrakhan fever rickettsia and Israelitick typhus rickettsia clustered at an SJ of 74.2%, and R. africae,R. parkeri, and strain S exhibited high antigenic similarity,forming a cluster at an SJ of 72.7% (Fig. 5). R. akari, R. aust-ralis, and R. helvetica, which reacted with only four anti-LPSmonoclonal antibodies, showed marked antigenic diversityfrom other SFG rickettsiae (Fig. 5 and Tables 2 and 5).

DISCUSSION

Among the SFG rickettsiae, two high-molecular-mass pro-teins, rOmpA and rOmpB (25–27), have been demonstrated tobe the major immunodominant surface proteins and have beenshown to play an important role in protective antigenicity (1, 2,36). These two proteins, particularly rOmpB, also show strongimmunogenicity in the humoral response of BALB/c mice (6),and most monoclonal antibodies raised from the SFG rickett-siae have been directed against their rOmpB proteins (1, 36,59, 60). Monoclonal antibodies against R. conorii, the mostwidely distributed SFG rickettsial species, have been producedon three occasions. In 1987, Feng et al. (23) produced six

monoclonal antibodies to R. conorii Seven to analyze the T-cell-dependent and -independent antigens of this organism(23); one year later, Li et al. (36) produced 23 monoclonalantibodies to R. conorii during study of the protective antigensof SFG rickettsiae. Twenty-two monoclonal antibodies havealso been raised by Vitale et al. (54) in 1989 against R. conoriiMAVI, an isolate from a Sicilian human patient with Mediter-ranean spotted fever, to compare antigenic phenotypes (54). Adecade later, we used monoclonal antibodies to study the epi-topes located on the rOmpA and rOmpB proteins of R. conoriiand the distribution of these epitopes among the other SFGrickettsiae.

In this study, the rOmpA and rOmpB proteins of R. conoriiSeven displayed molecular masses of 116 and 124 kDa, respec-tively. These proteins were demonstrated to possess a numberof specific epitopes which stimulated the production of mono-clonal antibodies with different reactivities among other SFGrickettsial strains. The rOmpB protein possessed more immu-nogenic epitopes than the rOmpA protein, although it is not anunexpected observation, as the rOmpB protein is strongly ex-pressed among the SFG rickettsiae and is recognized as beingthe predominant immunogenic SFG rickettsial surface antigen(3, 36, 59, 60). The gene encoding the rOmpA protein, inaddition to being smaller and less strongly immunogenic thanthat encoding the rOmpB protein, contains multiple (8 to 15)copies of a large, highly conserved repeating unit (18, 25, 27).The high level of primary structure conservation introduced

FIG. 4. Profiles of whole native protein from rickettsial strains. Coomassiebrilliant blue-stained proteins from rickettsial strains were separated electro-phoretically with 11% resolving gel (A) and with 7.5% resolving gel (B). Lanes1, R. conorii Seven; lanes 2, R. conorii Indian tick typhus rickettsia; lanes 3,R. conorii Kenya tick typhus rickettsia; lanes 4, R. conorii Manuel; lanes 5,R. conorii M-1; lanes 6, R. conorii Moroccan; lanes 7, Astrankhan fever rickettsia;lanes 8, Israeli tick typhus rickettsia; lanes 9, R. sibirica; lanes 10, “R. mongolo-timonae”; lanes 11, R. africae; lanes 12, R. parkeri; lanes 13, “R. slovaca”; lanes 14,R. rickettsii; lanes 15, R. rhipicephali; lanes 16, R. japonica; lanes 17, R. massiliae;lanes 18, Bar29; lanes 19, R. aeschlimannii; lanes 20, R. helvetica; lanes 21,R. akari; lanes 22, R. australis. The positions of molecular mass standards (inkilodaltons) are indicated.



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into the gene by the presence of these units may reduce sec-ondary and tertiary structure heterogeneity.

The monoclonal antibodies directed against the epitopes onthe rOmpA protein of R. conorii Seven exhibited differentpatterns of reactivities among the SFG rickettsiae from thoseof the epitopes on the rOmpB protein, indicating that nocommon epitope existed on these two proteins. Further anal-ysis found that the R. conorii strain-specific epitopes were ex-pressed on the rOmpA protein, whereas the species- and sub-group-specific epitopes were expressed on the rOmpB protein.The epitopes on the R. conorii rOmpB protein were generallyshared more widely among the SFG rickettsiae than those onthe rOmpA protein. Among R. conorii strains, not all epitopeson the Seven strain rOmpA protein were expressed by the M-1and Moroccan strains, although several were expressed byother SFG rickettsiae. Sequence analysis of the rOmpA pro-tein-encoding gene has demonstrated significant deletions inthose of the M-1 and Moroccan strains, and it is feasible thatthese losses prevent expression of epitopes on the encodedprotein (46).

The vast majority of epitopes on the rOmpA and rOmpBproteins of R. conorii Seven were expressed on the correspond-ing proteins of the other SFG rickettsiae, despite the differentmolecular weights of each protein in the different species.However, for R. aeschlimannii, R. rhipicephali, and R. rickettsii,the epitopes on the rOmpB protein of R. conorii were notexpressed on their rOmpB protein but on their rOmpA pro-

teins. The basis for this unexpected observation is unclear, butit may be that the rOmpA and rOmpB proteins have a similarfunction and possess regions of homologous structure. Evolu-tionary conservation of rOmpA and rOmpB suggests that theymay be derived from a common ancestor, but similar functionsmay also have resulted from convergent evolution (24, 26).

Interestingly, reactivity between some monoclonal antibod-ies and the R. conorii M-1 and Moroccan strains, R. rhipi-cephali, and R. rickettsii could not be demonstrated by Westernimmunoblotting, although reactivity was observed by the mi-cro-IF assay. These epitopes were perhaps disrupted by con-formational changes in the molecules induced during the treat-ment of samples with SDS and b-mercaptoethanol prior toPAGE. Alternatively, the low-level expression of the commonepitopes may go undetected by the Western immunoblotting ifit were less sensitive than the micro-IF assay.

The antigenic relationships between R. conorii and otherSFG rickettsiae were revealed by assessing the differences inepitope expression among species using numerical taxonomy.Within the R. conorii complex, marked antigenic differenceswere observed between the Astrakhan fever rickettsia and theIsraeli tick typhus rickettsia. The Astrakhan fever rickettsiaexpressed more of the R. conorii Seven rOmpA protein epi-topes than the Israeli tick typhus rickettsia, whereas the Israelitick typhus rickettsia possessed a markedly higher number ofthe R. conorii Seven rOmpB protein epitopes. After clusteranalysis, several small clusters determined by using a cutoff of70.0% for SJ were observed to generally correlate with theresults of recent phylogenetic studies (24, 47, 48). Two pairs ofthe SFG rickettsiae, the Thai tick typhus rickettsia and R. honeipair and the R. massiliae and Bar29 pair, exhibited an almostidentical number of shared epitopes with only one difference ineach pair. The close relationship of these strains has beendemonstrated previously (11, 24, 47, 48, 60). Also, R. africae,R. parkeri, and strain S were similar antigenically, as noted inprevious studies (24, 47, 48, 59). However, as it appears thatthe distribution of common rOmpA protein epitopes amongthe SFG rickettsiae is different from that of common rOmpBprotein epitopes, taxonomic analysis based on either individualmolecule may result in markedly different dendrograms, andthe dendrogram presented in this study may merely representa compromise between the two. Furthermore, since all rela-tionship assessments in this analysis were made relative toR. conorii, conclusions regarding SFG taxonomy would bestronger if more species were used. To this end, we are pres-ently reassessing taxonomic relationships among the SFG rick-ettsiae using a far larger panel of monoclonal antibodies raisedagainst a number of different species.

When we compared the reactivities of our monoclonal an-tibodies with those previously raised against R. africae andR. massiliae (59, 60), no identical patterns were found, indicat-ing that all antibodies were directed against different epitopes.This finding suggests that although a number of protein epi-topes are expressed by several different SFG rickettsiae, thelevel at which they are expressed on each species is different.For example, of the 23 R. africae monoclonal antibodiesscreened against all SFG rickettsiae, two pairs yielded reactiv-ity patterns identical to each other, indicating that they recog-nize the same epitopes, which were, therefore, likely to bestrongly expressed and highly immunogenic (unpublisheddata). Although these two epitopes were also shown to bepresent on R. conorii, they were not recognized by any of themonoclonal antibodies raised against R. conorii. The levels ofexpression of these two epitopes are therefore likely to bemarkedly lower in R. conorii than in R. africae. Indeed, al-though results of micro-IF assay screening of the R. africae

FIG. 5. Dendrogram showing the antigenic relationships of 28 SFG rickett-siae based on the distribution of common epitopes revealed by the cross-reac-tivities of anti-R. conorii monoclonal antibodies.



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monoclonal antibodies were only qualitatively presented (59),unpublished quantitative results demonstrated the monoclonalantibodies discussed above to react to a titer of 1/1,024 toR. africae but only 1/64 to R. conorii Seven.

The complex distribution of epitopes we observed here high-lights shortfalls in previous assessments of monoclonal anti-body specificity which used only a few SFG rickettsiae forevaluating reactivities of the anti-SFG rickettsia monoclonalantibodies and defining the strain- and species-specific epi-topes (1, 3, 35, 36, 39, 52). For example, had we screened themonoclonal antibodies herein using R. rickettsii, R. sibirica,R. conorii, R. akari, and R. australis, a previously employedantigen panel, 22 monoclonal antibodies would have been con-sidered species specific. However, with our much larger panelof antigens, only six monoclonal antibodies were clearly di-rected against species-specific epitopes, with 12 showing dif-ferent cross-reactivities with Astrakhan fever rickettsia, Israelitick typhus rickettsia, strain S, R. africae, R. parkeri, R. slovacaand/or “R. mongolotimonae” but not with R. rickettsii, R. sibiri-ca, R. akari, and R. australis (Table 2). Therefore, when estab-lishing the strain- or species-specific monoclonal antibody, allrecognized rickettsiae should be included.

This study clearly demonstrates the complex nature in whichepitopes are distributed on the rOmpA and rOmpB proteinsand reaffirms the diversity of these proteins among the SFGrickettsiae. Understanding the genetic basis of the variabilitymay be the best approach to assessing its true scale, and thisapproach is now being pursued in our laboratory.

ACKNOWLEDGMENTS

We are indebted to Herve Tissot-Dupont for help in the clusteranalysis. We are also grateful to Gregory A. Dasch for helpful discus-sions and Richard J. Birtles for critically reviewing the manuscript.

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Distribution of Immunogenic Epitopes on the Two Major ... · Universite´ de la Mediterrane´e, Marseille, France Received 30 June 1997/Accepted 29 August 1997 Forty-four monoclonal