Med MicrobiolImmunol(1992) 181:191-207

9 Springer-Verlag 1992

Molecular analysis of the outer surface protein A (OspA) of Borrelia burgdorferi for conserved and variable antibody binding domains Bettina Wilske 1, Benjamin Luft 2, William H. Schubach 2, Gitta Zumstein 1, Sigrid Jauris 1, Vera Preac-Mnrsic 1, and Michael D. Kramer 3 1 Max yon PettenkoferInstitut ftir Hygieneund MedizinischeMikrobiologie, Universit~itMtinchen,Pettenkoferstrasse9a, W-8000Mtinchen2, Federal Republicof Germany 2 State Universityat Stony Brook, Stony Brook, New York 3 Institut for Immunologieund Serologie,Universit~itHeidelberg,Im NeuenheimerFeld 305, W-6900 Heidelberg,Federal Republic of Germany Received May 18, 1992

Abstract. The outer surface protein A (OspA) of Borrelia burgdorferi is a major candidate for development of a borrelia vaccine. However, vaccine development may be aggravated by the immunological heterogeneity of OspA. In this respect the knowledge about conserved and variable epitopes is of major interest. In this study truncated proteins derived from two different OspA serotypes of B. burgdorferi were mapped for conserved and specific antibody-binding domains. The OspA fragments were reacted in the Western blot with eight different OspAspecific monoclonal antibodies recognizing between one and seven of the seven OspA serotypes previously described. The two broadly reacting antibodies (recognizing all serotypes) react with N-terminal fragments of 93 and 214 amino acids, respectively, whereas antibodies recognizing only one and two to four of the seven serotypes are reactive with C-terminal fragments of amino acid 143-273 and 109-273, respectively. Thus, conserved antibody-binding domains are located nearer to the N terminus than serotype-specific ones. Comparison of the results from western blot mapping with OspA sequence data suggested certain conserved or variable regions as probable candidates for antigenic sites involved in linear or conformationally dependent epitopes. This, however, needs to be confirmed by epitope mapping using the respective synthetic peptides.

Introduction Lyme borreliosis, the most common tick-borne disease in Europe and in North America, is caused by the spirochete Borrelia (B.) burgdorferi [9]. Lyme borreliosis is a multisystem disorder. The initial skin lesion, erythema migrans, may be followed by systemic complications as multiple skin lesions, meningitis or meningoradiculitis, carditis and arthralgias or arthritis. In some patients, especially if not treated with antibiotics, B. burgdorferi may persist in the nervous system, the joints, and/or the skin. The infection may recur as chronic

Correspondence to: B. Wilske

192 encephalomyelitis, chronic Lyme arthritis or acrodermatitis, a chronic skin disease [29]. In chronic disease, inflammatory processes resulting in organ damage may be initiated and sustained by immune reactions to certain borrelial proteins. Among the proteins that may trigger immunopathological processes those associated with the surface of the spirochete are of special interest. A large family of surface-related proteins has been demonstrated using two-dimentional (2D) gel analysis [21]. Among these, the outer surface protein A (OspA) [4], which is an abundant protein of B. burgdorferi, seems of major importance in the development of protective immunity. The genetics of the OspA have been intensively studied. The OspA-encoding gene is located on a linear plasmid of B. burgdorferi [1], and this gene forms one transcriptional unit with the gene encoding a second major outer surface protein, i.e., OspB. The osp genes of the American-type strain ofB. burgdorferi have been cloned and sequenced by Bergstr6m et al. [6]. The Osp proteins are integral lipoproteins of the borrelial outer membrane [7]. Despite their surface exposure and high concentration in the borrelial cell, the Osp proteins are of low immunogenicity in patients (humans and dogs) with early Lyme borreliosis; in contrast artificially infected or immunized animals respond early to the Osp proteins [3, 16, 32, 33]. In patients with chronic disease, antibodies to OspA are observed with higher frequency. However, even in late disease, many patients remain still negative for antibodies against OspA. In some patients with chronic Lyme disease a new IgM immune response has been described to emerge in association with exacerbation of clinical symptoms [10]. Thus, the appearance of IgM antibodies to OspA seems to be a marker for a relapse. In OspA antibodynegative patients, the OspA-specific antibodies may be bound in immunocomplexes [25] or the immune response may be restricted to T cells [11]. Especially in European patients a negative reaction to the OspA may be due to infection with an OspA serotype different from that of the infecting strain [33]. Passive immunization with OspA-specific monoclonal antibodies and active immunization with recombinant OspA have been effective in protecting mice from infection with B. burgdorferi [13, 23]. In contrast to North American isolates, OspA of European isolates has been observed to be immunologically heterogeneous [2, 5, 31, 32, 34]. This may be important for the development of a Borrelia vaccine and may account for the observations of Johnson et al. [17] in which immune serum against a North American isolate did not protect hamsters against the challenge with an European isolate and vice versa. In this respect it seems important to note that different monoclonal antibodies vary in their neutralizing ability. Such variations may be due to differences in the isotype as assumed by Simon et al. [28] but may also due to differences in the OspA epitope which is recognized by the antibody [26]. In two previously published reports, truncated OspA fusion proteins of two North American isolates (B31 and N40) were investigated for B cell epitopes [24, 26]. Several antibody-binding domains were recognized, which are distributed throughout the sequence except for the N-terminal end. Shanafelt et al. [27] reported that especially the C terminus is involved in B and T cell recognition sites. The antibodies used in these studies, however, have not been defined in respect to their reactivity with different OspA serotypes. To localize specific and conserved antigenic sites of the OspA protein we investigated truncated OspA proteins derived from two different OspA serotypes (strain B31 serotype 1 and strain PKo serotype2) with a spectrum of monoclonal antibodies that recognize

193 serotype-specific a n d c o n s e r v e d e p i t o p e s [35]. O u r studies s h o w e d t h a t c o n s e r v e d antigenic sites are l o c a t e d n e a r e r to the N t e r m i n u s t h a n serotype-specific ones. C o m p a r i s o n o f the results o b t a i n e d b y m o l e c u l a r m a p p i n g with O s p A sequence d a t a f r o m different s e r o t y p e s [37] revealed t h a t c o n s e r v e d a n d v a r i a b l e O s p A regions m a y be c o r r e l a t e d with c o n s e r v e d a n d v a r i a b l e antigenic sites.

Materials and methods

Borrelia burgdorferi isolates The American type strain B31 was obtained from Dr. Willy Burgdorfer (Hamilton, Montana). The European strains PKo, PBi and PHei were isolated from human skin (PKo) and CSF (PBi and PHei) by V. Preac-Mursic [22].

Truncated OspA proteins Truncated OspA proteins used in this study were: (a) 11 fusion proteins derived from OspA strain B31 described by Schubach et al. [24], and (b) three non fusion proteins derived from strain PKo described by Zumstein et al. [37].

Monoclonal and polyclonal antibodies Monoclonal antibodies (mAb) against OspA used in this study were L32 1F 11, L32 2E7, L32 1C8, H5332, LA 26, LA 5, LA 2 and H3TS. H5332 and H3TS were provided by A. G. Barbour. The mABs LA 2, LA 5 and LA 26 were produced as previously described [ 19]. Monoclonal antibodies L32 1F11, L32 2E7 and L32 1C8 were obtained from BALB/c mice immunized intraperitoneally with whole washed B. burgdorfericells containing about 100 lag protein/dose. A mixture of strains T25 and B31 was used for production of L32 IF11 and L32 1C8; a mixture of strains PKo and PGau was used for production of L32 2E7. The first immunization was performed with complete Freund's adjuvant, the second (day 21) and third (day 42) immunizations were with incomplete Freund's adjuvant. Finally, the mice were boosted three times on days 56, 59 and 60 without adjuvant but increasing the protein content per injection to 300 lag. Hybridomas were produced according to standard protocols [ 15]. Fusion was carried out 1 day after the last boost using HAT (hypoxanthine-aminopterin-thymidine)-sensitiveAg8-653 mouse myeloma cells. Identification and cloning of antibody-producing hybridomas were accomplished as described previously [ 14]. Serotype specificity and isotype of the antibodies are given in Table 1. The isotypes of the antibodies were determined using the mouse monoclonal antibody isotyping kit (Amersham Buchler GmbH, W-3300 Braunschweig, Germany). The reactivity of the mAb used in this study with the different OspA serotypes has been described previously [34, 35]. Polyclonal rabbit antibodies (immune serum against OspA from strain B31 eluted from SDSgel described in Wilske et al. [34] and immune serum against recombinant OspA from strain PKo) and a serum from a patient with chronic Lyme borreliosis containing OspA-reactive IgM antibodies were included in the study. The immune serum against the recombinant OspA (strain PKo) was obtained from a rabbit immunized subcutaneously with 200 lag purified OspA (first injection with complete Freund's adjuvant, second and third injection with incomplete Freund's adjuvant).

Strain

B31 PKo PBr PBi PHei TN T25

Biological origin

I dammini Skin CSF CSF CSF I. ricinus I. ricinus

1 2 3 4 5 6 7

OspA serotype

.

LA2 (IgG2b)

H3TS (IgG1)

+ § + + + § §

§ + § + + § §

§

§

§

+

+

§

--

+

§

+

+

.

--

+

§

. __

--

§

+

. __

--

§

_

_

_

--

§

+

+

+

-r

§

+

+

§

+

+

§

§

+

+

Rabbit Ig Human IgM anti-OspA OspA positive

LA5 (IgG2a)

L32 1Fll L322E7 (IgG2a) (IgG2b)

L32 1C8 H5332 (IgG2a) (IgG2a)

Polyclonal antibodies

OspA-specific monoclonal antibodies LA26 (IgG2b)

Table 1. Reactivity of Borrelia burgdorferi strains of distinct outer surface protein A (OspA) serotypes with different monoclonal and polyclonal antibodies 4~

195

Western blot SDS-PAGE and Western blots were performed as described previously [8]. For analysis of fusion proteins we used a 17.5% gel; for analysis of non-fusion proteins a 20 % gel. Immunocomplexes were detected with horseradish peroxidase conjugates (Dakopatts, Copenhagen, Denmark) specific for the respective antibodies.

Results and discussion All m A b and polyclonal antibodies used in this study were reactive with the OspA of strain B31 using Western blot analysis (Table 1). The antibodies used for analysis of proteins derived from strain PKo were reactive on Western blot analysis of this strain.

Location of anti OspA antibody-binding domains in OspA of strain PKo The full-length OspA was reactive with all four m A b tested (Figs. 1,2). Deletion of the leader sequence abrogated or markedly decreased reactivity o f m A b H5332 and m A b LA 26, respectively. Reactivity was also abrogated by deletions at the C terminus. Thus, the epitopes for H5332 and L A 2 6 may be conformational. A similar phenomenon was noted by Schubach et al. [24] in the identification of the epitope of m A b 105.5 for the B31 OspA. The epitope for m A b L32 2E7 is located on the aa 1-93 fragment, whereas antigenic sites involved in reactivity with L32 1F11 are probably located closer to the C terminus but not between aa 228 and 273. The most conserved epitopes, namely those of m A b L32 2E7 and L32 1F11 (which are reactive with all seven serotypes) seem to be located closer to the N

OspA-spectficmonoclonalantibody

poiydonal

antibodies

rabbit Ig

truncated non-fusionproteinsof OspA / strain PKo aa1-93

J

aa94-228

[

L32 1Fll L32 2E7

H5332

LA26

anti rec.OspA/PKo

aa229-273

Fig. 1. Analysis of the outer surface protein A (OspA) of strain PKo for antibody-binding domains. Full-length (aa 1-273) and truncated versions [aa 1-93, aa 1-228 and aa 17-273 (version without leaderpeptide)] of recombinant OspA were probed with monoclonal antibodies L32 1F 11, L32 2E7, H5332 and LA 26 and with rabbit immune serum against recombinant OspA of strain PKo

196

+

0

0

~

~

L32 1F11

w

+

0

~

~

~

~

~

0

0

~

.

L32

2E7

w

+

0

~

~

~

~

~

0

0

~

~

0

w

H5332

Fig. 2. Western blots of full-length (aa 1-273 - OspA/+) and truncated (aa 1-93, aa 1-228 and aa 17-273 OspA/-) versions of recombinant OspA of strain PKo probed with monoclonal antibodies L32 1F11, L32 2E7 and H5332. OspA/+ contains the leaderpeptide, O s p A / - has the leader sequence deleted

t e r m i n u s t h a n the m o r e specific e p i t o p e s o f H5332 (reactive with f o u r out o f seven serotypes) a n d L A 26 (reactive with only two out o f seven serotypes).

Location of anti-OspA antibody-binding domains in OspA of strain B31 Except for a n t i b o d y L 3 2 2 E 7 , all a n t i b o d i e s are reactive with r e c o m b i n a n t fulllength O s p A (Figs. 3-5). All a n t i b o d i e s r e c o g n i z e d at least one e p i t o p e on at least

r

Fig. 3. Analysis of the OspA of strain B31 for conserved and variable antibody-binding domains. Eleven truncated versions of OspA fusion proteins and the full-length fusion protein were probed with eight monoclonal antibodies and two polyclonal antibodies; the spectrum of reactivity of the different antibodies with the seven OspA serotypes is given just below the designation of the antibodies

1 - 273

[

d 181 -23" I

d 100- 21~ I

d 108-143 I

I

I

109-214

217-273:

[

[

62 - 214

I

109-273

I

I

1 -214

I

I

I

I. 1~,-23,V--1

]

11217-273

~10~-2f7 V-----1

,00_2,

truncatedfusion proteinsof OspA / strain 1331

62-100

I

1 - 108

Eta - ea

I,-8, 162o,08 I 1.61 [~1

fragment

L322E7 L321C8 H5332

LA26

LA5

LA2

rabbit Ig humanIgM

( +I

7/7

~;~#:: ~ ~!~i~!~:.:.~

6/7

~:.~:~ ~ ~ ~~~ ~ i ~ . ~ ' ~ :;::~$.$ ~i:::::::::::::::::::::::::::::::::::::::::::::::::::: ~,:~:::::~:~::..

~ii~iiiiii .............................

7/7

4/7

2/7

2/7

-

1/7

::::::::::::::::::::::::::::::::::::::::::

1/7

(+I

(+)

(-I- 1

~:;i~~ ~;:~;:~!~

~ r



7//7

~!~ii:~;i~:i!~i:':~':~:~:~:~: :~;:~!i!~i~i:~i~i~-~:,~:

~ ~-~ ! ~ ! .,...~ ~ .~.......

~#. ! .............. :::::=:~::::~ ..........

7/7

H3TS antlOspA antiOspA

number of serotypes reactive with the respective monoclonal and po~yclonalantibodies

L321F11

OspA-specificmonoclonalantibody

198

a

L32 ~F~ ~

d

LA S

199

Fig. 4 a-g. Western blots of full-length and truncated versions of recombinant OspA of strain B31 probed with monoclonal antibodies (a) L321FI1, (b) L32 1C8, (c) LA26, (d) LA 5, (e) LA 2, (f) H3TS and (g) rabbit immune serum against OspA g

rabbit immuneserum

one of the truncated proteins except for the aa 1-61 fragment. This fragment was also non-reactive with the two polyclonal antibodies, suggesting that it does not bear an immunodominant linear epitope. The epitope of L32 2E7 is located on the aa 1-108 fragment. Considering that fragments aa 1-61 and aa 62-108 are not reactive, it seems reasonable that the cleavage site between aa 61 and 62 is part of the epitope. L32 2E7 mAb is reactive with the same OspA fragments as mAb 184.1 described by Schubach et al. [24] (see Table 2). L32 2E7 mAb has a considerably lower reactivity with OspA from strain B31 compared to strain OspA from strain PKo. L32 1F 11 mAb is reactive with the aa 62-214 fragment and with the aa 181-232deleted clone, thus localizing its antigenic site between aa 62 and aa 181. Fragmentation between aa 108 and 109 destroys antibody binding, indicating that this region is part of the epitope. The L32 1C8-specific antigenic sites are probably located between aa 143 and aa 181. This is suggested by the fact that the aa 108-143 and the aa 181-232 deletion clones are reactive, whereas the aa 108-232 deletion clone is non-reactive. The finding that all three deletion clones comprising deletions from aa 108-232 are nonreactive with H5232, whereas the aa 109-273 fragment is reactive, argues for the fact that the H5332-epitope is conformation dependent. This has also been previously discussed by Sears et al. [26]. The similar reactivity pattern to that seen with H5332 was observed using mAb LA 5. Results were also similar with LA 26, for which the only difference was that the aa 108-143-deletion clone had some weak reactivity. These findings indicate that LA 26 and LA 5 also recognize conformation-dependent epitopes. The reactivity of the aa 109-273 fragment and that of the aa 108-143 deletion clone localizes the antigenic sites for mAb LA 2 and H3TS between aa 143 and aa 273. Fragmentation at aa 214 and aa 217 abrogates reactivity, indicating that the aa 214/217 cleavage site is part of the epitopes for both antibodies.

200 O3

I~

T03

o

a

r-

~

r

'~"

0

o

~

,-

~

,

L32

0,1 C~

0~1

04

0

0

,

,

,-

,-

,-

2E7

Fig. 5 a-c. Western blots of full-length and truncated versions of recombinant OspA of strain B31 probed with monoclonal antibodies (a) L322E7, (h) H5332 and (c) human serum with IgM antibodies against OspA Regarding the reactivity of the polyclonal antibodies, it is of interest that the human IgM antibodies recognize a relatively small fragment (aa 62 - aa 108) which is not reactive with any of the mAb. The reactivity of this fragment with the rabbit immune serum was only weak. The human sera from the study of Schubach et al. [24] did not recognize this fragment. Overlapping fragments containing the aa 62108 sequence have also been negative with sera from patients with Lyme arthritis [26]. The small fragment possibly has a better reactivity with the IgM isotype. In our study as well as in other studies C-terminal fragments of about 50-60 aa are only weakly recognized by polyclonal immune sera, and sera from patients with Lyme borreliosis have been negative [24, 26, 27]. However, binding of human IgG antibodies has been abrogated by deletions at the C-terminal region, indicating that the epitopes involved are dependent on this portion of OspA and are of the conformational type. T cells have been shown to recognize linear epitopes on the C-terminal end [27]. In contrast the C-terminal end was not reactive with antibodies.

Comparison of epitope analysis with OspA sequence The early sequence data on the OspA of three different strains provided by Bergstr6m et al. [6], Wallich et al. [30] and Fikrig et al. [13] suggested that the

(Q

~o

3

r-

::T

o

- 217 - 232

de1108 de1181

del 108 - 143

217 - 273

de1181 - 232

de1108 - 217

de1108 - 143

217 - 273

109 - 273

109-214

62- 214

62 - 108

1 -214

1 - 108

1 -61

1 - 273

E coli + vector

B31

1 0 9 -~2 7 3

r PO

01

-1-

O"

109 - 214

62- 214

62 - 108

1 -214

1 - 108

1 - 61

1 - 273

E. coil + vector

B31

F.)

5, 6, 7 7 5, 6, 7 6, 7

17- 93 1-108 61-181 143-181 109-273 109-273 109-273 143-273 143-273 143-273

Location on aa - aa

Conformationally dependent Conformationally dependent Conformationally dependent Fragmentation at aa 214/217 Fragmentation at aa 214/217 Fragmentation at aa 214/217

214/217 214/217 214/217

a

epitope epitope epitope destroys reactivity destroys reactivity destroys reactivity

Fragmentation at aa 61 destroys reactivity Fragmentation at aa 61 destroys reactivity Fragmentation at aa 108 destroys reactivity

Comments

61 61 108

Antigenic sites probably at aa

Degree of reactivity different among serotypes for L32 2E7, strong reactivity with all serotypes for L32 1F11 b Data from [24]

105.5 b

4, 6, 4, 5, 6,

1, 2, 1, 4, 1, 2, 1, 3, 1, 2, 1, 2 1, 3 1 1 1

L322E7 a 184.1 b L32 1Fll a L32 1C8 H5332 LA 26 LA5 H3TS LA 2

3, 5, 3, 4, 3,

Epitope conserved in Ospa serotypes

Monoclonal antibody

Table 2. Conserved and variable antibody-binding domains of OspA

b~

t~

203 10 20 30 40 50 MKKYLLGI GLI LALIACKQNVS S LDEKNSVSVDLPGEMKVLVSKEKNKDGKY

OA-PKO

MKKYLI~ IGLI LALIACKQNVS S LDEKNSASVDLPGEMKVLVS KEKDKDGKYS LKATVDK

2

OA-PBI

MKKYLLG IGLI LALIACKQNVS S LDEKNSVSVDLPGEMKVLVS KEKDKDGKYS LMATVDK

4

I OA-PHEI

20

30

OA-PKO

9

OA-PBI

n

i

l

i

n

9

50

OA-PHEI

i

I

i

i

I

9

I

i

~

i

KDKT

i

~

i

OA-PKO

I i n

I

STDEMFNEKGELSAKTMTRENGTKLEYTEMKS

I nimBI OA-PBI

I m e

~

n

~

i

OA-PHEI

I i n

i

I

KVTLE

i

180 190 200 210 220 230 VKEGTVTLS KNISKSGEVSVELNDTDSSAATKKTAAWNSGTSTLTITVNSKKTKDLVFTK

OA-PKO

i

ill

9

I i I

In

OA-PBI

I

Ill

9

i I I

VTEGTVVLSKHI PNSGEITVELNDSNSTQATKKTGKWDSNTSTLTI

l i m B OA-PHEI

i

ill

9

B i n

iI

SVNSKKTKNIVFTK

in

I

I

i

I

i

i

l

i

I

KTLDELKNALK

i

l

l

i

conserved

4

i 5

1 2 4

BiB

OA-PHEI EDTITVQNYDSAGTNLEGKAVEITTLKELKNALK 240 250 260 270

aminoacids

2

IlI

EDT ITVQKYDSAGTNLEGNAVE IKTLDELKNALK

I

1

IlI

QDTITVQKYDSAGTNLEGTAVEI

I

270 IKNALK

5

i

VTEGTVTLSKNI SKSGE ITVALDDTDSS -GNKKSGTWDSGTSTLTI SKNRXKTKQLVFTK 190 200 210 220 230

240 250 260 ENTITVQQYDSNGTKLEGSAVEITKLDE

4

I

V K E G T V T L S KE I A K S G E V T V A L N D T N T T Q A T K K T G A W D S K T S T LT I S V N S K K T T Q L V F T K

i l i I

2

II

S T E E K F N E K G E I S E K T I V R A N G T R L E Y T D I KS D K T G K A K E V L K D F T L E G T L A A D G K T T L K 130 140 150 160 170 180

B R i m

1

In

S IEEKFNAKGELSEKTI LRANGTRLEYTEIKSDGTGKAKEVLKDFALEGTLAAD-KTTLK

i iIiIi

5

II

DGTGKAKEVLKNFTLEGKVAND-

I

4

I

LELKGTS DKNNGSGTLEGEKTDKSKVKLTIAEDLSKTTFE IFKEDGKTLVSKKVTLKDKS 70 80 90 i00 ii0 120

i IIiiI

2

i

130 140 150 160 170 STEEKFNEKGEVSEKI ITRADGTRLEYTGIKS DGSGKAKEVLKGYVLEGTLTAE -KTTLV

OA-B31

1

mB

LELKGTS DKSNGSGTLEGEKSDKSKAKLTI SEDLSKTTFEI FKEDGKTLVSKKVNSKDKS

~

5

60

mu i

1

|

IELKGTS DKDNGSGVLEGTKDDKSKAKLTIADDLSKTTFELFKEDGKTLVSRKVSS

~

OA-PBI

n

40

iilnn

OA-PKO

m

70 80 90 i00 ii0 120 LE L K G T S D K N N G S G V L E G V K A D K S K V K L T I S D D L G Q T T L E V F K E D G K T L V S K K V T S K D K S

OA-B31

OA-B31

i

M K K Y L L G I G LI L A L I A C K Q N V S S L D E K N S V S V D L P G G M K V L V S K E K D K D G K Y S L M A T V E K I0

OA-B31

~

60 DLIATVDK

type

OA-B31

5

among all 4 strains

Fig. 6. Comparison of the OspA sequences of different OspA serotypes of Borreliaburgdorferi (OA-B31 - O s p A of strain B31, O A - P K o - OspA of strain PKo, OA-PBi- OspA of strain PBi, OA-PHei = OspA of strain PHei). B31 (type 1), PKo (type 2), PBi (type 4) and PHei (type 5)

OspA sequence is highly conserved. This assumption, however, was in disagreement with the finding that the OspA protein is immunologically heterogeneous [5, 32]. With more OspA sequence data from immunologically defined strains now being available, it appears that the OspA is heterogeneous at the molecular level [12, 18, 37 and W. Jiang, unpublished work]. Figure 6 shows a comparison of the

204 aa sequences of the strains PKo (serotype 2) and PBi (serotype4) analyzed by Zumstein et al. [37] compared to strains B31 (serotype 1) and PHei (serotype 5). For this study the PHei ospA gene has been amplified by polymerase chain reaction from genomic DNA of strain PHei, cloned and sequenced as previously described [37]. The DNA sequence data will be published elsewhere. Comparison of the OspA sequences from representative strains of serotypes 1, 2, 4 and 5 revealed a highly conserved region at the N terminus comprising about 50 aa. The rest of the sequence is partially conserved but the conserved regions which are scattered over the rest of the sequence are shorter. The more conserved aa 1-61 fragment was not reactive with any of the antibodies tested including also the rabbit immune serum against OspA and the anti-OspA IgM-positive human serum. Non-reactivity of N-terminal fragments has been observed also by Schubach et al. [24] for the aa 1-61 fragment using two mAb and seven human OspA-positive sera, and also by Sears et al. [26] for the aa 1-76 fragment using four mAb and seven human OspA-positive sera. The latter authors found some weak reactivity with several immune sera against recombinant OspA raised in mice; a smaller fragment of aa 1-36, however, was non-reactive. It has been discussed by Schubach et al. [24] that the low reactivity of the N-terminal fragment may be due to the fact that the N-terminal region is anchored in the borrelial membrane and, thus, is not exposed to the surface. Conserved epitopes are recognized by mAb L32 1Fll and mAb L322ET. Whereas the latter antibody shows differences in the intensity of reactivity among strains mAb L32 1F 11 is uniformly strongly reactive independent of the serotype. This antibody is specific for B. burgdorferi and not reactive with relapsing fever borreliae [35]. It has been reactive with all strains expressing the OspA (more than 100) that have been tested to date (unpublished results). Western blot analysis suggested that the epitope of L32 1F11 may be located around aa 108. At this location we find a region from aa 102 to aa 111 conserved among the four sequenced OspA proteins. However, immunological analysis of overlapping peptides would be necessary to exactly localize the epitope. For vaccine development, it would be of interest to know whether mAb L32 1Fll can protect experimentally infected animals. At the probable location of the mAb L32 2ETand the mAb 184.1-specific antigenic sites we also see a conserved region ranging from aa 59 to aa 69 with the exception of strain PKo which has an aa exchange at position 60. This finding is quite interesting and is in accordance with the observations that mAb L32 2E7 has the strongest reactivity with PKo and that mAb 184.1 is not reactive with this strain but reactive with the three other strains. For the three antibodies recognizing between two and four of the seven serotypes (LA 26, LA 5 and H5532) we cannot give a more exact location for aa residues involved in the respective antigenic domains than aa 109-273. No further information can be drawn from comparing the sequences of the different strains to each other. mAb L32 IC8 is reactive with strains B31, PBi and PHei but not with strain PKo. Within the aa 143-181 partial sequence only one region is more conserved among B31, PBi and PHei than between any of the strains compared to PKo. This is the N-terminal end of the aa 143-181 fragment ranging from 143 to 153. Among these 11 amino acids B31, PBi and PHei have 10 aa in common, whereas only 8 aa of strain PKo are conserved between PKo and the other strains. This is in agreement with the observation that L32 1C8 is reactive with B31, PBi and PHei but not reactive with PKo.

205 The serotype 1-specific mAb (Table 2) recognize an epitope on the aa 143-273 fragment which is destroyed by cleavage at aa 214 and 217. The region (aa 214-219) around this cleavage sites varies considerably among the different strains, It has been discussed by Sears et al. [26] that the neutralizing antibody CIII.78 is conformationally dependent. This antibody reacted with an aa 133-273 fragment. Further deletions at either the N-terminal or C-terminal end abrogate antibody binding. Laver et al. [20] postulate that antibody-binding determinants of native proteins are always of the conformational type and that such epitopes are discontinuous, comprising between 2 and 7 aa residues. They further stress that such an epitope must be composed of at least 15-22 aa. Regarding the aa 143-273 fragment which definitely bears the LA 2 and the H3TS epitope, we can define two other highly variable regions that might be involved in a conformationally dependent epitope: aa 164-175 and aa 197-208. Especially for LA2, which is a neutralizing antibody [23], we must assume that this antibody binds to the native protein and should recognize a conformationally dependent and, therefore, discontinuous epitope [20]. In addition we subjected the sequence data of the OspA proteins of strains B31 and PKo to computer analysis for prediction of antigenic determinants according to Wolf et al. [36]. This analysis program is based on probable secondary structure features, regional backbone flexibility as well as parameters relating to surface accessibility. Even when considering the limitations of this approach it seems of interest that, despite the fact that the homology of the aa sequences of strains B31 and PKo is only 77 %, we obtained a nearly identical pattern for the antigenic index values. We did not obtain further information by this method for a better localization for OspA-specific epitopes reacting with the mAbs used in this study. High index values are scattered all over the sequence except at the very N-terminal end. In conclusion, we localized the more-conserved antigenic sites closer to the N terminus and the more variable ones closer to the C terminus. We identified conserved and variable regions on the OspA at a molecular level that probably are part of antibody-binding determinants. More conclusive data might be obtained from analysis of synthetic peptides in respect to mapping linear epitopes. Such epitopes may be of special interest for diagnostic and taxonomic questions. However, for identification of epitopes recognized by antibodies with biological function binding to the native protein may be a prerequisite. Therefore, further research should comprise analysis of fragments designed according to the information obtained here, i.e., analysis of smaller fragments containing the prospective epitopes. Also it would be interesting to investigate the OspA proteins of other strains, for example PBi and PHei, for conserved and variable epitopes to determine whether a similar distribution of specific and conserved antibodybinding domains can be seen in these strains. It is worthwhile to mention that PHei has more similarity to PBi than to the other strains. This means that strains with different OspA serotypes may have separated at different times during evolution.

Acknowledgements. We thank Dr. W. Burgdorfer for providing us with strain B31, Dr. A. G. Barbour for mAb H5332 and mAb H3TS, and Dr. T. Krech for the human serum containing IgM antibodies against OspA. We also thank Ingrid Pradel for excellent technical assistance, Helga Schmidt for photographic work and Dr. E. Soutschek for valuable discussion. This work was supported by grants from the Max von Pettenkofer Institute. We would like to express our thanks to Prof. Dr. F. Deinhardt for the generous support.

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Molecular analysis of the outer surface protein A (OspA) of Borrelia burgdorferi for conserved and variable antibody binding domains.

The outer surface protein A (OspA) of Borrelia burgdorferi is a major candidate for development of a borrelia vaccine. However, vaccine development ma...
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