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Vaccination against Lyme Disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses
Vaccine 18 (2000) 540±548
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Vaccination against Lyme Disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses Yung-Fu Chang a,*, Vesna Novosol a, Sean P. McDonough b, Chao-Fu Chang a, Richard H. Jacobson a, Thomas Divers c, Fred W. Quimby b, Sang Shin a, Donald H. Lein a a
Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA b Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA c Department of Clinical sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received 11 March 1999; received in revised form 21 April 1999; accepted 22 April 1999
Abstract Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the ®rst vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis ) collected from Westchester County, New York (tick infection rate r60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kineticELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA ®rst appeared in the sera of vaccinated ponies three weeks after the ®rst vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Immunogenicity; OspA; Aluminium hydroxide; Borrelia burgdorferi; Lyme borreliosis; Equine
1. Introduction Lyme Disease (LD) is the most important arthropod-borne bacterial infection in the United States. Aecting people, dogs, horses, cattle and cats, LD is caused by the spirochete Borrelia burgdorferi transmitted primarily by Ixodes ticks [1±7]. The incidence of equine Borrelia infection appears to be increasing in the northeastern United States, the Midwest, Texas and California [7]. The clinical features of Lyme dis* Corresponding author. Tel.: +1-607-253-3675; fax: +1-607-2532943. E-mail address: [email protected] (Y.F. Chang)
ease in horses including sporadic lameness, swollen joints, facial paralysis and encephalitis [7]. Results from a previous study in our laboratory indicated that ponies can be infected by exposure to B. burgdorferi-infected ticks [8]. In that study, 7 ponies were successfully infected after B. burgdorferi-tick exposure. Infection with B. burgdorferi was detected from skin biopsies and various tissues at post-mortem by culture and PCR. Also, these animals seroconverted. This indicated that we could use this equine Lyme model to evaluate the ecacy and safety of an equine Lyme vaccine. Because of the increasing risk of equine Lyme disease, the development of a safe and eective vaccine
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Table 1 B. burgdorferi isolation from skin biopsies, blood and tissues and histopathology from ponies immunized with 100 mg of rOspA plus adjuvanta Pony number
Vaccination
Histopathologyd
B. burgdorferi isolation skin biopsies months postchallenge
2 4 5 8 9 10 11 1 3 6 7
OspA+A OspA+A OspA+A OspA+A OspA+A OspA+A OspA+A A A A A
1
2
3
± ± ± ± ± ± ± + + ± ±
± ± ± ± ± ± ± ± + ± +
± ± ± ± ± ± ± + + ± +
bloodb
tissuesc
± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± L,M,T J,L, M L L, M
NL NL NL NL NL NL NL NS NL NL NS, NSPA, NSP, N
a
Vaccinated horses were inoculated with 100 mg of rOpsA in 1% aluminium hydroxide at day 0, 20 and 82 (A) and were challenged with B. burgdorferi-infected ticks at 30 days after the last vaccination (day 112 of the experiment). b Blood cultures were performed monthly and were all negative for B. burgdorferi. c Tissues positive for isolation of B. burgdorferi: J=joint capsules; L=lymph node; M=muscle; T = thyroid gland. d NL=no signi®cant lesion; NS=nonsuppurative deep dermatitis at tick attachment site; NSPA=nonsuppurative polyarthritis; NSP= nonsuppurative perineutritis; N=neuritis.
against this disease is urgently needed. In the past several years, it has been demonstrated that vaccination with outer surface protein A (OspA) from the organism could prevent B. burgdorferi infection in animal and human studies [9±17]. In this study, we demonstrate that a recombinant OspA vaccine can also protect ponies against infection.
2. Materials and methods 2.1. Animals Twelve SPF ponies, 1-year-old, (Table 1) from Cornell University, College of Veterinary Medicine were kept in P2 isolation units, fed a commercial ration and provided water ad libitum. The protocol of this study was approved by IACUC (Institutional Animal Care and Use Committee) at Cornell University to comply with Federal Law (PL99-198). All work was conducted in compliance with regulations, policies and principles of the Animal Welfare Act, the Public Health Service for Policy on Humane Care and Use of Laboratory Animals used in Testing, Research and Training, the NIH Guide for the Care and Use of Laboratory Animals and the New York State Department of Public Health regulations. All ponies were observed for clinical signs and their body temperatures were recorded daily. Body weights were measured weekly. One pony (12) was terminated
because of serious diarrhea at the beginning of this study. 2.2. Overexpression of recombinant OspA rOspA derived from B. burgdorferi strain B31 was overexpressed by a T7 promoter and puri®ed by immobilized metal ion anity chromatography as previously described [18]. Puri®ed rOspA (100 mg/ml) was kept at ÿ208C until used. 2.3. Vaccination of ponies Ponies were randomly allotted to either a vaccination group (eight ponies) or a non-vaccinated control group (four ponies). Each pony in the vaccinated group was injected intramuscularly three times at day 0, 20 and 82 with 100 mg of rOspA in adjuvant (1% v/ v aluminium hydroxide). The non-vaccinated control ponies were injected intramuscularly with adjuvant only. All ponies were challenged with B. burgdorferiinfected ticks 112 days after the ®rst vaccination (Table 1). 2.4. Ticks Adult ticks (Ixodes scapularis ) infected with B. burgdorferi were collected by ¯agging in a forested area of Westchester County, New York. Ticks were maintained at the Cornell Entomology Laboratory at 94% relative humidity and 108C for two months. To deter-
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mine the percentage of ticks infected with B. burgdorferi, 20 male or female ticks were ground and cultured individually in BSK-2 medium with 8 mg/ml kanamycin and 50 mg/ml rifampicin as previously described [9,19,20]. The cultures, examined weekly over a 6-week period for B. burgdorferi by dark®eld microscopy and IFA, showed a 55 to 60% infection rate. 2.5. Exposure of ponies to ticks One hundred and twelve days after the ®rst vaccination, ponies were exposed to 20 female and 10 male ®eld collected adult ticks (I. scapularis ) by placing the ticks onto the clipped side of each pony as previously reported [9]. Dexamethasone (0.2 mg/lb per day, Schering±Plough Animal Health, Kenilworth, NJ) was given intramuscularly for 5 consecutive days starting on the ®rst day of adult tick exposure. Ticks were allowed to feed and engorge for 7 days, when at least 50% of the female ticks were fully engorged; at this time all ticks were manually removed from the ponies. 2.6. Serum and tissue samples A serum sample was obtained from each pony before vaccination and then every two weeks for six months. Sera were tested by Western blotting, kineticELISA (KELA) and growth-inhibition test. After tick exposure (attachment), skin biopsies were taken at monthly intervals for isolation of spirochetes. Four months after challenge, all ponies were euthanized and tissues were harvested aseptically for culture of B. burgdorferi and for histopathology. 2.7. Isolation of B. burgdorferi To test for infection, attempts were made monthly to isolate B. burgdorferi from skin biopsies at the site of tick attachment and from various tissues at postmortem. Samples from skin punch biopsies (4 mm) and blood collected at monthly intervals after tick exposure and pieces of tissue (approx. 0.2±1 g) obtained aseptically at necropsy (Table 1) were homogenized in 5 ml BSKII medium in a tissue homogenizer (Stomacher: Tekmar, Cincinnati) and then transferred to 25 ml of prewarmed BSKII medium. For blood culture, 100 ml of blood was transferred to 6 ml of prewarmed BSKII medium. The cultures were checked weekly for up to six weeks for the presence of B. burgdorferi by dark ®eld examination and IFA. 2.8. Serology: KELA, immunoblots and growth inhibition tests KELA for measuring levels of serum antibody to B. burgdorferi was described previously [9,21]. Each unit
of slope was designated as a KELA unit. Correlation with western blotting analysis indicated that the cuto separating negative from positive sera was 100 KELA units [9,21]. Brie¯y, diluted serum was added to duplicate wells in microtiter plates containing antigens of French-pressed B. burgdorferi (B31) lysate. Bound antibody was detected with horseradish peroxidase conjugated goat anti-horse IgG (HRP; Cappel Research Products, Durham, NC). Color development using the chromogen tetramethylbenzidine with H2O2 as a substrate was measured kinetically and expressed as the slope of the reaction rate between enzyme and substrate solution. Western blot analysis was performed as previously described [9,21]. French-pressed B. burgdorferi lysate was used as an antigen and subjected to SDS-PAGE [9,21]. Western blot analysis was performed in a miniblotter [9,21]. Test sera from experimental animals were used as the primary antibody, followed by goat antihorse IgG conjugated to HRP as a second antibody. The growth inhibition assay was done as described elsewhere [9,22]. Brie¯y, serial dilutions of serum in microtitration plates were incubated with 106/ml of live B. burgdorferi in BSKII medium for 30 min and guinea pig complement was then added. Microtitration plates were sealed and incubated at 348C for four to six days. Bacterial growth was measured as a function of the pH indicated by a color change from red to yellow, which was determined in a microplate reader at 570/630 nm. 2.9. Gross pathology and histopathology All vaccinated and non-vaccinated ponies were euthanized approx. 4 months after tick exposure and examined for gross and histopathologic lesions. The following tissues were ®xed in 10% neutral buered formalin: joint capsules and synovial membranes (right and left elbow, shoulder, sti¯e, carpus, tarsus, fetlock), cerebellum, cerebrum, meninges, spinal cord, myocardium, urinary bladder, thyroid, liver, spleen, kidney, lung, stomach, intestine, skeletal muscles, aorta, eyes, nerves (left and right brachial plexus, trigeminal ganglion, cervical and thoracic nerve roots, median, ulnar, radial, sciatic, tibial, ®bular, facial) and lymph nodes (axillary, prescapular and popliteal). Tissues were embedded in paran wax, sectioned and stained with Hematoxylin & Eosin by conventional methods for histopathologic evaluation. 3. Results 3.1. Clinical signs No ponies showed any clinical signs (lameness, anor-
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Fig. 1. KELA antibody levels from vaccinated and unvaccinated ponies. The line at 100 KELA units represents the cuto between positive and negative sera. Solid symbols indicated that ponies were vaccinated. Open symbols indicated that ponies were nonvaccinated. Vaccinated ponies were inoculated three times with 100 mg rOspA plus 1% aluminium hydroxide intramuscularly at day 0, 20 and 82 and were challenged with B. burgdorferi-infected ticks on day 112 after the ®rst vaccination.
exia or depression) or elevated body temperature either following vaccination or tick challenge except for one of the vaccinates. Two weeks after tick exposure, pony 4 had a high fever (1058F) and was given Flunixin Meglumine intramuscularly (1 g daily) for two days. Examination of peripheral blood revealed that this
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Fig. 3. Growth inhibition antibody titers from the vaccinated and unvaccinated ponies. Solid symbols indicated that ponies were vaccinated. Open symbols indicated that ponies were nonvaccinated. Sera were from the same group of ponies shown in Fig. 1. Data points oset to reveal superimposed lines.
pony was infected with Human Granulocytic Ehrlichiosis agent (HGE) with morulae clearly visible in neutrophils. 3.2. Isolation of B. burgdorferi B. burgdorferi was not isolated from any of the skin biopsies taken from the vaccinated ponies at monthly intervals after challenge or from any of the other tissues examined at necropsy (skeletal muscles, joint cap-
Fig. 2. Representative western blot analysis of antibody response in pony 2 vaccinated with 100 mg rOspA in adjuvant (A: lanes 1±14; Table 1) and in unvaccinated pony 1 (B: lanes 1±14; Table 1). Lane 1, preimmune serum; lane 2 to 14, two-week intervals after ®rst vaccination (A) or adjuvant control (B). Biotinylated SDS-PAGE standard-broad range molecular markers were used (Bio-Rad Laboratories, Richimond, CA). The numbers at the right indicate molecular weights.
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Fig. 4. Skin, pony 7. Lymphocytic accumulation around a small muscular arteriole in the deep dermis.
sules, pericardium, peritoneum, lymph nodes and meninges). All 4 non-vaccinated, challenged control ponies were culture positive (Table 1). B. burgdorferi were isolated from lymph nodes (left prescapular and right popliteal), muscles (left and right triceps brachii, left and right vastus lateralis) and thyroid gland in pony 1; lymph nodes (right axillary and prescapular), muscle (right triceps brachii) and joint (right shoulder) in pony 3; left prescapular lymph node in pony 6 and left prescapular lymph node and muscles (left triceps brachii and left vastus lateralis) in pony 7. 3.3. Serology At the time of challenge, all vaccinated ponies had KELA titers to B. burgdorferi between 500 to 590 KELA units. These titers gradually declined after challenge (Fig. 1). KELA antibodies in the non-vaccinated, challenged control ponies were detectable by eight to ten weeks after challenge (Fig. 1). Western blot analysis showed OspA antibody at about 32 kDa three
Fig. 6. Nonsuppurative perineuritis, pony 7. (a) Left tibial nerve: moderate lymphocytic and rare plasmacytic periarteriolar in®ltrate in the perineurium. H&E 175. (b) Left facial nerve: moderate perivascular lymphohistiocytic aggregate in the perineurium. H&E 350.
weeks after the ®rst vaccination. Bands became denser after the second vaccination. Bands also appeared in the 20 kDa regions. Additional bands were not seen after tick challenge. In contrast, no bands were seen in the non-vaccinated control ponies until 8±10 weeks after exposure to infected ticks when multiple bands appeared (Fig. 2). The B. burgdorferi growth inhibition assay with sera from the vaccinated ponies showed titers of 1:1280 at the time of challenge. Sera from the control ponies did not show growth inhibition before challenge (Fig. 3). 3.4. Histopathology
Fig. 5. Right metacarpophalangeal joint, pony 7. Severe periarteriolar lymphohistiocytic in®ltrate in ®brous layer of joint capsule. H&E 350.
Signi®cant histologic changes were con®ned to unvaccinated control ponies 1 and 7. Near the tick attachment site multiple nodular mononuclear cell aggregates were scattered about the deep dermis, within the cutaneous trunci muscle and the panniculus. These foci surrounded small arteries or nerves and consisted of lymphocytes mixed with variable numbers of histiocytes and plasma cells (Fig. 4). Signi®cant in¯ammatory changes in other tissues were found only in pony 7 (Fig. 5).
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in the thalamus (Fig. 7). Similarly, skeletal muscle from both front legs had multiple areas of moderate perivascular in¯ammation. Only mild in¯ammation was present in muscle from the left hind limb, while no in¯ammation was found in the right hind limb muscle. 4. Discussion
Fig. 7. Perivascular cung, pony 7. (a) Fifth lumbar dorsal spinal nerve root: mild lymphocytic perivascular aggregate. (b) Thalamus: mild lymphocytic perivascular cu. H&E 350.
A variety of structures were aected but changes were more numerous and of relatively greater severity in the cranial half of the body compared to the caudal half. Likewise, lesions were more frequent and severe on the left side compared to the right (i.e. closer to site of initial exposure). Nonsuppurative synovitis was present in the left elbow, left metacarpophalangeal joint, sti¯e and tarsus. Synovitis on the right side was limited to mild in¯ammation of the tarsus. Most aected joints had mild to moderate subsynovial perivascular lymphocytic aggregates (Fig. 6). Occasionally, the interstitium of the subsynovial tissue was in®ltrated by a small number of plasma cells. Perivascular aggregates were found infrequently in the ®brous layer of the joint capsule or in the periarticular adipose tissue. Perivascular mononuclear cell aggreagates also formed around small arteries adjacent to the perineureum of peripheral nerves. Aected nerves were primarily on the left side and included the ulnar, facial (Fig. 6), sciatic, tibial and ®bular nerves. The only aected nerves on the right side were the ulnar and tibial nerves. Very mild nonsuppurative perineuritis was also present in thoracic and lumbar dorsal spinal nerve roots and several light perivascular cus were present
Our criteria for rOspA vaccination eciency were a failure to isolate live B. burgdorferi either from monthly skin biopsies after tick challenge or from a variety of tissues taken at necropsy 3.5 months after exposure. The polymerase chain reaction (PCR) to detect B. burgdorferi DNA was not used because positive results can not dierentiate between viable and nonviable organisms. B. burgdorferi was isolated from skin biopsies from three of the control ponies and from other tissues from all control ponies challenged by tick exposure. Although we isolated B. burgdorferi from a lymph node from pony 6, this pony did not seroconvert. In contrast, isolation attempts were negative in all vaccinated ponies (Table 1). Histopathologic lesions were only found in ponies 1 and 7 in the skin where the ticks attached (Fig. 4). The observed changes are similar to lesions found in dogs infected with B. burgdorferi in our previous study [9]. Pony 7 also had nonsuppurative synovitis in various joints, perineuritis and neuritis (Fig. 6). The involvement of the facial nerve is noteworthy since facial paralysis has been reported in humans with Lyme disease [23]. However, we did not see any clinical signs in this pony. It is possible that the pony may have developed signi®cant clinical signs if it had been observed for a longer period of time. High KELA titers in vaccinated ponies prior to challenge apparently correlated with protection from infection (Fig. 1). However, KELA titers in vaccinated ponies, con®rmed by western blot, re¯ected antibody speci®c to the 32 kDa OspA which is not expressed by B. burgdorferi in mammalian hosts after tick exposure. In all vaccinated ponies, Western blots revealed a wide band at about 32 kDa OspA region. Additionally, we saw weaker bands in the 20 kDa region that likely were breakdown products of OspA [9]. The western blot pattern in vaccinated ponies did not change after tick exposure suggesting that infections did not become established in these vaccinated ponies. Strikingly, multiple bands consistent with infection by the etiologic agent of Lyme disease appeared in the non-vaccinated control ponies 8±10 weeks after challenge. Thus, western blotting is a reliable method to distinguish vaccinated from infected ponies [21]. The 32 kDa bands waned with time (Fig. 2, lanes 11±14), suggesting a drop in vaccinal antibody levels as a function of time.
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A correlation between serum growth inhibition of B. burgdorferi and protection from infection was found. All vaccinated ponies had high growth inhibition antibody titers after vaccination (Fig. 3) that gradually declined after tick challenge. Growth inhibition antibody in horses is similar to that of dogs and monkeys which were complement dependent [9,24]. When guinea pig complement was not added to the test, almost no growth inhibition titers occurred (data not shown). It is possible that the growth inhibition antibodies neutralized B. burgdorferi in the tick gut and prevented migration to the salivary gland and into the host [25]. However, we isolated B. burgdorferi from ticks after engorgement on vaccinated ponies, which indicated that the OspA antibodies did not kill all B. burgdorferi organisms in the tick gut. Following challenge, the nonvaccinated ponies lacked growth inhibition antibodies to B. burgdorferi even though they showed intermediate KELA antibody titers (Fig. 1). Western blot analysis also showed an antibody response to the B. burgdorferi antigens in unvaccinated control ponies (Fig. 2). However, no signi®cant anti-OspA antibodies were detected from these sera (data not shown). Thus, anti-OspA antibodies appear to be a critical factor in the inhibition of B. burgdorferi growth in vitro. Heterogeneity of OspA proteins in dierent Borrelia species has been reported mainly in Europe and Asia [26±29]. An OspA subunit vaccine protected animals only against homologous B. burgdorferi strains in Europe [14,30]. Therefore, a polyvalent OspA vaccine is necessary for protection against B. burgdorferi sensu stricto, B. garinii and B. afzelii infection [31]. The heterogenicity of OspA proteins must be considered in immunization strategies against Lyme disease. However, with few exceptions, only one serotype of B. burgdorferi prevails in the United States [26±29]. We, therefore, have not addressed the question of OspA heterogeneity in our vaccine trial. A recombinant OspA vaccine is a good candidate vaccine for ponies and one is currently being used in humans [10,11,32]. The recombinant OspA vaccine also protects dogs against infection [9]. Active and passive protection of mice by OspA against infection with B. burgdorferi when challenged by needle inoculation or tick exposure has been reported [15,25,33±35]. Our data show that ponies can also be protected by rOspA vaccination. Although the vaccinated ponies had a high titer of anti-OspA antibodies, one pony (pony 4) was infected by the Human Granulocytic Ehrlichiosis agent (HGE). Two weeks after tick challenge this pony developed a high fever (1058F) and ehrlichial inclusion bodies (morulae) were seen in approximately 20% of peripheral blood neutrophils during the febrile period. Thus, rOspA vaccination provides no cross protection against HGE. The presence of the HGE agent, B. burgdorferi and Babesia microti in the same ticks, I.
scapularis and I. paci®cus, further complicates the development of a vaccine against tick-borne diseases in both humans and animals [36±41]. This indicates that even if humans or animals have been vaccinated with Lyme vaccine, caution must still be taken to avoid tick bites in the ®eld they can be infected by agents other than Lyme disease. In summary, a rOspA vaccine protected ponies against B. burgdorferi infection. Further studies are needed to determine the duration of protection after vaccination, safety and cross protection against the possible heterogeneous OspA structures that may be found among new B. burgdorferi strains isolated in the United States [42]. Reportedly, vaccination with recombinant OspA can protect animals against infection, but can not eliminate the organisms if the animals were infected before vaccination [43]. A therapeutic Lyme vaccine is needed for this purpose. Attempts to develop a second generation Lyme vaccine using OspC [44±46], or decorin binding protein A (DbpA) [47±49] have been reported. It has been reported that patients may develop autoimmunity due to molecular mimicry between the dominant T-cell epitope of OspA and human-leukocyte-function-associated antigen I (hLFA-I) [50]. However, no clinical consequence using OspA as a subunit vaccine or bacterin have been reported in dogs since these vaccines became commercially available several years ago. Therefore, clinical signi®cance of this interesting in vitro phenomenon awaits further study. Acknowledgements We are grateful to Helen Bell for administrative assistance and to Patti Easton for technical assistance. We are grateful to Allyn Vondercheck, Dale Strickland, David Dietterich and John Daley for animal care. This work was supported by grants from the Zweig fund from Cornell University and the Cornell Biotechnology Program (CAT). References [1] Browning A, Carter SD, Barnes A, May C, Bennett D. Lameness associated with Borrelia burgdorferi infection in the horse. Vet Rec 1993;132:610±1. [2] Burgess EC, Mattison M. Encephalitis associated with Borrelia burgdorferi infection in a horse. J Am Vet Med Assoc 1987;191:1457±8. [3] Cohen D, Bosler EM, Bernard W, Meirs Dd, Eisner R, Schulze TL. Epidemiologic studies of Lyme disease in horses and their public health signi®cance. Ann NY Acad Sci 1988;539:244±57. [4] Kornblatt AN, Urband PH, Steere AC. Arthritis caused by Borrelia burgdorferi in dogs. J Am Vet Med Assoc 1985;186:960±4. [5] Lissman BA, Bosler EM, Camay H, Ormiston BG, Benach JL.
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diagnosis of infected and vaccinated symptomatic dogs. Semin Vet Med Surg 1996;11:172±82. Straubinger RK, Chang YF, Jacobson RH, Appel MJ. Sera from OspA-vaccinated dogs, but not those from tick-infected dogs, inhibit in vitro growth of Borrelia burgdorferi. J Clin Microbiol 1995;33:2745±51. Clark JR, Carlson RD, Sasaki CT, Pachner AR, Steere AC. Facial paralysis in Lyme disease. Laryngoscope 1985;95:1341±5. Nowling JM, Phillipp MT. Killing of Borrelia burgdorferi by antibody elicited by OspA vaccine is inecient in the absence of complement. Infect Immun 1999;67:443±5. Fikrig E, Telford SRd, Barthold SW, Kantor FS, Spielman A, Flavell RA. Elimination of Borrelia burgdorferi from vector ticks feeding on OspA-immunized mice. Proc Natl Acad Sci USA 1992;89:5418±21. Baranton G, Postic D, Saint Girons I, Boerlin P, Piaretti JC, Assous M, Grimont PA. Delineation of Borrelia burgdorferi sensu stricto, Borrelia garinii sp. nov., and group VS461 associated with Lyme borreliosis. Int J Syst Bacteriol 1992;42:378±83. Dykhuizen DE, Polin DS, Dunn JJ, Wilske B, Preac-Mursic V, Dattwyler RJ, Luft BJ. Borrelia burgdorferi is clonal: implications for taxonomy and vaccine development. Proc Natl Acad Sci USA 1993;90:10163±7. Peter O, Bretz AG, Bee D. Occurrence of dierent genospecies of Borrelia burgdorferi sensu lato in ixodid ticks of Valais, Switzerland. Eur J Epidemiol 1995;11:463±7. Wilske B, Preac-Mursic V, Gobel UB, Graf B, Jauris S, Soutschek E, Schwab E, Zumstein G. An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis. J Clin Microbiol 1993;31:340±50. Golde WT, Burkot TR, Piesman J, Dolan MC, Capiau C, Hauser P, Dequesne G, Lobet Y. The Lyme disease vaccine candidate outer surface protein A (OspA) in a formulation compatible with human use protects mice against natural tick transmission of B. burgdorferi. Vaccine 1995;13:435±41. Gern L, Rais O, Capiau C, Hauser P, Lobet Y, Simoen E, Voet P, Petre J. Immunization of mice by recombinant OspA preparations and protection against Borrelia burgdorferi infection induced by Ixodes ricinus tick bites. Immunol Lett 1994;39:249± 58. Keller D, Koster FT, Marks DH, Hosbach P, Erdile LF, Mays JP. Safety and immunogenicity of a recombinant outer surface protein A Lyme vaccine. JAMA 1994;271:1764±8. Fikrig E, Barthold SW, Kantor FS, Flavell RA. Protection of mice from Lyme borreliosis by oral vaccination with Escherichia coli expressing OspA. J Infect Dis 1991;164:1224±7. Fikrig E, Barthold SW, Flavell RA. OspA vaccination of mice with established Borrelia burgdorferi infection alters disease but not infection. Infect Immun 1993;61:2553±7. Schaible UE, Wallich R, Kramer MD, Gern L, Anderson JF, Museteanu C, Simon MM. Immune sera to individual Borrelia burgdorferi isolates or recombinant OspA thereof protect SCID mice against infection with homologous strains but only partially or not at all against those of dierent OspA/OspB genotype. Vaccine 1993;11:1049±54. Walker DH, Barbour AG, Oliver JH, Lane RS, Dumler JS, Dennis DT, Persing DH, Azad AF, McSweegan E. Emerging bacterial zoonotic and vector-borne diseases. Ecological and epidemiological factors. JAMA 1996;275:463±9. Dumler JS, Bakken JS. Ehrlichial diseases of humans: emerging tick-borne infections. Clin Infect Dis 1995;20:1102±10. Magnarelli LA, Dumler JS, Anderson JF, Johnson RC, Fikrig E. Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera. J Clin Microbiol 1995;33:3054±7. Magnarelli LA, Anderson JF, Staord 3rd KC, Dumler JS.
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Y. Chang et al. / Vaccine 18 (2000) 540±548 Antibodies to multiple tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in white-footed mice. J Wildl Dis 1997;33:466±73. Mitchell PD, Reed KD, Hofkes JM. Immunoserologic evidence of coinfection with Borrelia burgdorferi, Babesia microti, and human granulocytic Ehrlichia species in residents of Wisconsin and Minnesota. J Clin Microbiol 1996;34:724±7. Telford 3rd SR, Dawson JE, Katavolos P, Warner CK, Kolbert CP, Persing DH. Perpetuation of the agent of human granulocytic ehrlichiosis in a deer tick-rodent cycle. Proc Natl Acad Sci USA 1996;93:6209±14. Lovrich SD, Callister SM, Lim LC, DuChateau BK, Schell RF. Seroprotective groups of Lyme borreliosis spirochetes from North America and Europe. J Infect Dis 1994;170:115±21. Zhong W, Stehle T, Museteanu C, Siebers A, Gern L, Kramer M, Wallich R, Simon MM. Therapeutic passive vaccination against chronic Lyme disease in mice. Proc Natl Acad Sci USA 1997;94:12533±8. Gilmore Jr RD, Kappel KJ, Dolan MC, Burkot TR, Johnson BJ. Outer surface protein C (OspC), but not P39, is a protective immunogen against a tick-transmitted Borrelia burgdorferi challenge: evidence for a conformational protective epitope in OspC. Infect Immun 1996;64:2234±9. Preac-Mursic V, Wilske B, Patsouris E, Jauris S, Will G, Soutschek E, Rainhardt S, Lehnert G, Klockmann U, Mehraein
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P. Active immunization with pC protein of Borrelia burgdorferi protects gerbils against B. burgdorferi infection. Infection 1992;20:342±9. Probert WS, Crawford M, Cadiz RB, LeFebvre RB. Immunization with outer surface protein (Osp) A, but not OspC, provides cross-protection of mice challenged with North American isolates of Borrelia burgdorferi. J Infect Dis 1997;175:400±5. Hanson MS, Cassatt DR, Guo BP, Patel NK, McCarthy MP, Dorward DW, Hook M. Active and passive immunity against Borrelia burgdorferi decorin binding protein A (DbpA) protects against infection. Infect Immun 1998;66:2143±53. Cassatt DR, Patel NK, Ulbrandt ND, Hanson MS. DbpA, but not OspA, is expressed by Borrelia burgdorferi during spirochetemia and is a target for protective antibodies. Infect Immun 1998;66:5379±87. Hagman KE, Lahdenne P, Popova TG, Porcella SF, Akins DR, Radolf JD, Norgard MV. Decorin-binding protein of Borrelia burgdorferi is encoded within a two-gene operon and is protective in the murine model of Lyme borreliosis. Infect Immun 1998;66:2674±83. Gross DM, Forsthuber T, Tary-Lehmann M, Etling C, Ito K, Nagy ZA, Field JA, Steere AC, Hubert BT. Identi®cation of LFA-1 as a candidate autoantigen in treatment-resistant Lyme arthritis. Science 1998;281:703.
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Vaccination against Lyme Disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses Yung-Fu Chang a,*, Vesna Novosol a, Sean P. McDonough b, Chao-Fu Chang a, Richard H. Jacobson a, Thomas Divers c, Fred W. Quimby b, Sang Shin a, Donald H. Lein a a
Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA b Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA c Department of Clinical sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received 11 March 1999; received in revised form 21 April 1999; accepted 22 April 1999
Abstract Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the ®rst vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis ) collected from Westchester County, New York (tick infection rate r60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kineticELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA ®rst appeared in the sera of vaccinated ponies three weeks after the ®rst vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Immunogenicity; OspA; Aluminium hydroxide; Borrelia burgdorferi; Lyme borreliosis; Equine
1. Introduction Lyme Disease (LD) is the most important arthropod-borne bacterial infection in the United States. Aecting people, dogs, horses, cattle and cats, LD is caused by the spirochete Borrelia burgdorferi transmitted primarily by Ixodes ticks [1±7]. The incidence of equine Borrelia infection appears to be increasing in the northeastern United States, the Midwest, Texas and California [7]. The clinical features of Lyme dis* Corresponding author. Tel.: +1-607-253-3675; fax: +1-607-2532943. E-mail address: [email protected] (Y.F. Chang)
ease in horses including sporadic lameness, swollen joints, facial paralysis and encephalitis [7]. Results from a previous study in our laboratory indicated that ponies can be infected by exposure to B. burgdorferi-infected ticks [8]. In that study, 7 ponies were successfully infected after B. burgdorferi-tick exposure. Infection with B. burgdorferi was detected from skin biopsies and various tissues at post-mortem by culture and PCR. Also, these animals seroconverted. This indicated that we could use this equine Lyme model to evaluate the ecacy and safety of an equine Lyme vaccine. Because of the increasing risk of equine Lyme disease, the development of a safe and eective vaccine
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Table 1 B. burgdorferi isolation from skin biopsies, blood and tissues and histopathology from ponies immunized with 100 mg of rOspA plus adjuvanta Pony number
Vaccination
Histopathologyd
B. burgdorferi isolation skin biopsies months postchallenge
2 4 5 8 9 10 11 1 3 6 7
OspA+A OspA+A OspA+A OspA+A OspA+A OspA+A OspA+A A A A A
1
2
3
± ± ± ± ± ± ± + + ± ±
± ± ± ± ± ± ± ± + ± +
± ± ± ± ± ± ± + + ± +
bloodb
tissuesc
± ± ± ± ± ± ± ± ± ± ±
± ± ± ± ± ± ± L,M,T J,L, M L L, M
NL NL NL NL NL NL NL NS NL NL NS, NSPA, NSP, N
a
Vaccinated horses were inoculated with 100 mg of rOpsA in 1% aluminium hydroxide at day 0, 20 and 82 (A) and were challenged with B. burgdorferi-infected ticks at 30 days after the last vaccination (day 112 of the experiment). b Blood cultures were performed monthly and were all negative for B. burgdorferi. c Tissues positive for isolation of B. burgdorferi: J=joint capsules; L=lymph node; M=muscle; T = thyroid gland. d NL=no signi®cant lesion; NS=nonsuppurative deep dermatitis at tick attachment site; NSPA=nonsuppurative polyarthritis; NSP= nonsuppurative perineutritis; N=neuritis.
against this disease is urgently needed. In the past several years, it has been demonstrated that vaccination with outer surface protein A (OspA) from the organism could prevent B. burgdorferi infection in animal and human studies [9±17]. In this study, we demonstrate that a recombinant OspA vaccine can also protect ponies against infection.
2. Materials and methods 2.1. Animals Twelve SPF ponies, 1-year-old, (Table 1) from Cornell University, College of Veterinary Medicine were kept in P2 isolation units, fed a commercial ration and provided water ad libitum. The protocol of this study was approved by IACUC (Institutional Animal Care and Use Committee) at Cornell University to comply with Federal Law (PL99-198). All work was conducted in compliance with regulations, policies and principles of the Animal Welfare Act, the Public Health Service for Policy on Humane Care and Use of Laboratory Animals used in Testing, Research and Training, the NIH Guide for the Care and Use of Laboratory Animals and the New York State Department of Public Health regulations. All ponies were observed for clinical signs and their body temperatures were recorded daily. Body weights were measured weekly. One pony (12) was terminated
because of serious diarrhea at the beginning of this study. 2.2. Overexpression of recombinant OspA rOspA derived from B. burgdorferi strain B31 was overexpressed by a T7 promoter and puri®ed by immobilized metal ion anity chromatography as previously described [18]. Puri®ed rOspA (100 mg/ml) was kept at ÿ208C until used. 2.3. Vaccination of ponies Ponies were randomly allotted to either a vaccination group (eight ponies) or a non-vaccinated control group (four ponies). Each pony in the vaccinated group was injected intramuscularly three times at day 0, 20 and 82 with 100 mg of rOspA in adjuvant (1% v/ v aluminium hydroxide). The non-vaccinated control ponies were injected intramuscularly with adjuvant only. All ponies were challenged with B. burgdorferiinfected ticks 112 days after the ®rst vaccination (Table 1). 2.4. Ticks Adult ticks (Ixodes scapularis ) infected with B. burgdorferi were collected by ¯agging in a forested area of Westchester County, New York. Ticks were maintained at the Cornell Entomology Laboratory at 94% relative humidity and 108C for two months. To deter-
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mine the percentage of ticks infected with B. burgdorferi, 20 male or female ticks were ground and cultured individually in BSK-2 medium with 8 mg/ml kanamycin and 50 mg/ml rifampicin as previously described [9,19,20]. The cultures, examined weekly over a 6-week period for B. burgdorferi by dark®eld microscopy and IFA, showed a 55 to 60% infection rate. 2.5. Exposure of ponies to ticks One hundred and twelve days after the ®rst vaccination, ponies were exposed to 20 female and 10 male ®eld collected adult ticks (I. scapularis ) by placing the ticks onto the clipped side of each pony as previously reported [9]. Dexamethasone (0.2 mg/lb per day, Schering±Plough Animal Health, Kenilworth, NJ) was given intramuscularly for 5 consecutive days starting on the ®rst day of adult tick exposure. Ticks were allowed to feed and engorge for 7 days, when at least 50% of the female ticks were fully engorged; at this time all ticks were manually removed from the ponies. 2.6. Serum and tissue samples A serum sample was obtained from each pony before vaccination and then every two weeks for six months. Sera were tested by Western blotting, kineticELISA (KELA) and growth-inhibition test. After tick exposure (attachment), skin biopsies were taken at monthly intervals for isolation of spirochetes. Four months after challenge, all ponies were euthanized and tissues were harvested aseptically for culture of B. burgdorferi and for histopathology. 2.7. Isolation of B. burgdorferi To test for infection, attempts were made monthly to isolate B. burgdorferi from skin biopsies at the site of tick attachment and from various tissues at postmortem. Samples from skin punch biopsies (4 mm) and blood collected at monthly intervals after tick exposure and pieces of tissue (approx. 0.2±1 g) obtained aseptically at necropsy (Table 1) were homogenized in 5 ml BSKII medium in a tissue homogenizer (Stomacher: Tekmar, Cincinnati) and then transferred to 25 ml of prewarmed BSKII medium. For blood culture, 100 ml of blood was transferred to 6 ml of prewarmed BSKII medium. The cultures were checked weekly for up to six weeks for the presence of B. burgdorferi by dark ®eld examination and IFA. 2.8. Serology: KELA, immunoblots and growth inhibition tests KELA for measuring levels of serum antibody to B. burgdorferi was described previously [9,21]. Each unit
of slope was designated as a KELA unit. Correlation with western blotting analysis indicated that the cuto separating negative from positive sera was 100 KELA units [9,21]. Brie¯y, diluted serum was added to duplicate wells in microtiter plates containing antigens of French-pressed B. burgdorferi (B31) lysate. Bound antibody was detected with horseradish peroxidase conjugated goat anti-horse IgG (HRP; Cappel Research Products, Durham, NC). Color development using the chromogen tetramethylbenzidine with H2O2 as a substrate was measured kinetically and expressed as the slope of the reaction rate between enzyme and substrate solution. Western blot analysis was performed as previously described [9,21]. French-pressed B. burgdorferi lysate was used as an antigen and subjected to SDS-PAGE [9,21]. Western blot analysis was performed in a miniblotter [9,21]. Test sera from experimental animals were used as the primary antibody, followed by goat antihorse IgG conjugated to HRP as a second antibody. The growth inhibition assay was done as described elsewhere [9,22]. Brie¯y, serial dilutions of serum in microtitration plates were incubated with 106/ml of live B. burgdorferi in BSKII medium for 30 min and guinea pig complement was then added. Microtitration plates were sealed and incubated at 348C for four to six days. Bacterial growth was measured as a function of the pH indicated by a color change from red to yellow, which was determined in a microplate reader at 570/630 nm. 2.9. Gross pathology and histopathology All vaccinated and non-vaccinated ponies were euthanized approx. 4 months after tick exposure and examined for gross and histopathologic lesions. The following tissues were ®xed in 10% neutral buered formalin: joint capsules and synovial membranes (right and left elbow, shoulder, sti¯e, carpus, tarsus, fetlock), cerebellum, cerebrum, meninges, spinal cord, myocardium, urinary bladder, thyroid, liver, spleen, kidney, lung, stomach, intestine, skeletal muscles, aorta, eyes, nerves (left and right brachial plexus, trigeminal ganglion, cervical and thoracic nerve roots, median, ulnar, radial, sciatic, tibial, ®bular, facial) and lymph nodes (axillary, prescapular and popliteal). Tissues were embedded in paran wax, sectioned and stained with Hematoxylin & Eosin by conventional methods for histopathologic evaluation. 3. Results 3.1. Clinical signs No ponies showed any clinical signs (lameness, anor-
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Fig. 1. KELA antibody levels from vaccinated and unvaccinated ponies. The line at 100 KELA units represents the cuto between positive and negative sera. Solid symbols indicated that ponies were vaccinated. Open symbols indicated that ponies were nonvaccinated. Vaccinated ponies were inoculated three times with 100 mg rOspA plus 1% aluminium hydroxide intramuscularly at day 0, 20 and 82 and were challenged with B. burgdorferi-infected ticks on day 112 after the ®rst vaccination.
exia or depression) or elevated body temperature either following vaccination or tick challenge except for one of the vaccinates. Two weeks after tick exposure, pony 4 had a high fever (1058F) and was given Flunixin Meglumine intramuscularly (1 g daily) for two days. Examination of peripheral blood revealed that this
543
Fig. 3. Growth inhibition antibody titers from the vaccinated and unvaccinated ponies. Solid symbols indicated that ponies were vaccinated. Open symbols indicated that ponies were nonvaccinated. Sera were from the same group of ponies shown in Fig. 1. Data points oset to reveal superimposed lines.
pony was infected with Human Granulocytic Ehrlichiosis agent (HGE) with morulae clearly visible in neutrophils. 3.2. Isolation of B. burgdorferi B. burgdorferi was not isolated from any of the skin biopsies taken from the vaccinated ponies at monthly intervals after challenge or from any of the other tissues examined at necropsy (skeletal muscles, joint cap-
Fig. 2. Representative western blot analysis of antibody response in pony 2 vaccinated with 100 mg rOspA in adjuvant (A: lanes 1±14; Table 1) and in unvaccinated pony 1 (B: lanes 1±14; Table 1). Lane 1, preimmune serum; lane 2 to 14, two-week intervals after ®rst vaccination (A) or adjuvant control (B). Biotinylated SDS-PAGE standard-broad range molecular markers were used (Bio-Rad Laboratories, Richimond, CA). The numbers at the right indicate molecular weights.
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Y. Chang et al. / Vaccine 18 (2000) 540±548
Fig. 4. Skin, pony 7. Lymphocytic accumulation around a small muscular arteriole in the deep dermis.
sules, pericardium, peritoneum, lymph nodes and meninges). All 4 non-vaccinated, challenged control ponies were culture positive (Table 1). B. burgdorferi were isolated from lymph nodes (left prescapular and right popliteal), muscles (left and right triceps brachii, left and right vastus lateralis) and thyroid gland in pony 1; lymph nodes (right axillary and prescapular), muscle (right triceps brachii) and joint (right shoulder) in pony 3; left prescapular lymph node in pony 6 and left prescapular lymph node and muscles (left triceps brachii and left vastus lateralis) in pony 7. 3.3. Serology At the time of challenge, all vaccinated ponies had KELA titers to B. burgdorferi between 500 to 590 KELA units. These titers gradually declined after challenge (Fig. 1). KELA antibodies in the non-vaccinated, challenged control ponies were detectable by eight to ten weeks after challenge (Fig. 1). Western blot analysis showed OspA antibody at about 32 kDa three
Fig. 6. Nonsuppurative perineuritis, pony 7. (a) Left tibial nerve: moderate lymphocytic and rare plasmacytic periarteriolar in®ltrate in the perineurium. H&E 175. (b) Left facial nerve: moderate perivascular lymphohistiocytic aggregate in the perineurium. H&E 350.
weeks after the ®rst vaccination. Bands became denser after the second vaccination. Bands also appeared in the 20 kDa regions. Additional bands were not seen after tick challenge. In contrast, no bands were seen in the non-vaccinated control ponies until 8±10 weeks after exposure to infected ticks when multiple bands appeared (Fig. 2). The B. burgdorferi growth inhibition assay with sera from the vaccinated ponies showed titers of 1:1280 at the time of challenge. Sera from the control ponies did not show growth inhibition before challenge (Fig. 3). 3.4. Histopathology
Fig. 5. Right metacarpophalangeal joint, pony 7. Severe periarteriolar lymphohistiocytic in®ltrate in ®brous layer of joint capsule. H&E 350.
Signi®cant histologic changes were con®ned to unvaccinated control ponies 1 and 7. Near the tick attachment site multiple nodular mononuclear cell aggregates were scattered about the deep dermis, within the cutaneous trunci muscle and the panniculus. These foci surrounded small arteries or nerves and consisted of lymphocytes mixed with variable numbers of histiocytes and plasma cells (Fig. 4). Signi®cant in¯ammatory changes in other tissues were found only in pony 7 (Fig. 5).
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545
in the thalamus (Fig. 7). Similarly, skeletal muscle from both front legs had multiple areas of moderate perivascular in¯ammation. Only mild in¯ammation was present in muscle from the left hind limb, while no in¯ammation was found in the right hind limb muscle. 4. Discussion
Fig. 7. Perivascular cung, pony 7. (a) Fifth lumbar dorsal spinal nerve root: mild lymphocytic perivascular aggregate. (b) Thalamus: mild lymphocytic perivascular cu. H&E 350.
A variety of structures were aected but changes were more numerous and of relatively greater severity in the cranial half of the body compared to the caudal half. Likewise, lesions were more frequent and severe on the left side compared to the right (i.e. closer to site of initial exposure). Nonsuppurative synovitis was present in the left elbow, left metacarpophalangeal joint, sti¯e and tarsus. Synovitis on the right side was limited to mild in¯ammation of the tarsus. Most aected joints had mild to moderate subsynovial perivascular lymphocytic aggregates (Fig. 6). Occasionally, the interstitium of the subsynovial tissue was in®ltrated by a small number of plasma cells. Perivascular aggregates were found infrequently in the ®brous layer of the joint capsule or in the periarticular adipose tissue. Perivascular mononuclear cell aggreagates also formed around small arteries adjacent to the perineureum of peripheral nerves. Aected nerves were primarily on the left side and included the ulnar, facial (Fig. 6), sciatic, tibial and ®bular nerves. The only aected nerves on the right side were the ulnar and tibial nerves. Very mild nonsuppurative perineuritis was also present in thoracic and lumbar dorsal spinal nerve roots and several light perivascular cus were present
Our criteria for rOspA vaccination eciency were a failure to isolate live B. burgdorferi either from monthly skin biopsies after tick challenge or from a variety of tissues taken at necropsy 3.5 months after exposure. The polymerase chain reaction (PCR) to detect B. burgdorferi DNA was not used because positive results can not dierentiate between viable and nonviable organisms. B. burgdorferi was isolated from skin biopsies from three of the control ponies and from other tissues from all control ponies challenged by tick exposure. Although we isolated B. burgdorferi from a lymph node from pony 6, this pony did not seroconvert. In contrast, isolation attempts were negative in all vaccinated ponies (Table 1). Histopathologic lesions were only found in ponies 1 and 7 in the skin where the ticks attached (Fig. 4). The observed changes are similar to lesions found in dogs infected with B. burgdorferi in our previous study [9]. Pony 7 also had nonsuppurative synovitis in various joints, perineuritis and neuritis (Fig. 6). The involvement of the facial nerve is noteworthy since facial paralysis has been reported in humans with Lyme disease [23]. However, we did not see any clinical signs in this pony. It is possible that the pony may have developed signi®cant clinical signs if it had been observed for a longer period of time. High KELA titers in vaccinated ponies prior to challenge apparently correlated with protection from infection (Fig. 1). However, KELA titers in vaccinated ponies, con®rmed by western blot, re¯ected antibody speci®c to the 32 kDa OspA which is not expressed by B. burgdorferi in mammalian hosts after tick exposure. In all vaccinated ponies, Western blots revealed a wide band at about 32 kDa OspA region. Additionally, we saw weaker bands in the 20 kDa region that likely were breakdown products of OspA [9]. The western blot pattern in vaccinated ponies did not change after tick exposure suggesting that infections did not become established in these vaccinated ponies. Strikingly, multiple bands consistent with infection by the etiologic agent of Lyme disease appeared in the non-vaccinated control ponies 8±10 weeks after challenge. Thus, western blotting is a reliable method to distinguish vaccinated from infected ponies [21]. The 32 kDa bands waned with time (Fig. 2, lanes 11±14), suggesting a drop in vaccinal antibody levels as a function of time.
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A correlation between serum growth inhibition of B. burgdorferi and protection from infection was found. All vaccinated ponies had high growth inhibition antibody titers after vaccination (Fig. 3) that gradually declined after tick challenge. Growth inhibition antibody in horses is similar to that of dogs and monkeys which were complement dependent [9,24]. When guinea pig complement was not added to the test, almost no growth inhibition titers occurred (data not shown). It is possible that the growth inhibition antibodies neutralized B. burgdorferi in the tick gut and prevented migration to the salivary gland and into the host [25]. However, we isolated B. burgdorferi from ticks after engorgement on vaccinated ponies, which indicated that the OspA antibodies did not kill all B. burgdorferi organisms in the tick gut. Following challenge, the nonvaccinated ponies lacked growth inhibition antibodies to B. burgdorferi even though they showed intermediate KELA antibody titers (Fig. 1). Western blot analysis also showed an antibody response to the B. burgdorferi antigens in unvaccinated control ponies (Fig. 2). However, no signi®cant anti-OspA antibodies were detected from these sera (data not shown). Thus, anti-OspA antibodies appear to be a critical factor in the inhibition of B. burgdorferi growth in vitro. Heterogeneity of OspA proteins in dierent Borrelia species has been reported mainly in Europe and Asia [26±29]. An OspA subunit vaccine protected animals only against homologous B. burgdorferi strains in Europe [14,30]. Therefore, a polyvalent OspA vaccine is necessary for protection against B. burgdorferi sensu stricto, B. garinii and B. afzelii infection [31]. The heterogenicity of OspA proteins must be considered in immunization strategies against Lyme disease. However, with few exceptions, only one serotype of B. burgdorferi prevails in the United States [26±29]. We, therefore, have not addressed the question of OspA heterogeneity in our vaccine trial. A recombinant OspA vaccine is a good candidate vaccine for ponies and one is currently being used in humans [10,11,32]. The recombinant OspA vaccine also protects dogs against infection [9]. Active and passive protection of mice by OspA against infection with B. burgdorferi when challenged by needle inoculation or tick exposure has been reported [15,25,33±35]. Our data show that ponies can also be protected by rOspA vaccination. Although the vaccinated ponies had a high titer of anti-OspA antibodies, one pony (pony 4) was infected by the Human Granulocytic Ehrlichiosis agent (HGE). Two weeks after tick challenge this pony developed a high fever (1058F) and ehrlichial inclusion bodies (morulae) were seen in approximately 20% of peripheral blood neutrophils during the febrile period. Thus, rOspA vaccination provides no cross protection against HGE. The presence of the HGE agent, B. burgdorferi and Babesia microti in the same ticks, I.
scapularis and I. paci®cus, further complicates the development of a vaccine against tick-borne diseases in both humans and animals [36±41]. This indicates that even if humans or animals have been vaccinated with Lyme vaccine, caution must still be taken to avoid tick bites in the ®eld they can be infected by agents other than Lyme disease. In summary, a rOspA vaccine protected ponies against B. burgdorferi infection. Further studies are needed to determine the duration of protection after vaccination, safety and cross protection against the possible heterogeneous OspA structures that may be found among new B. burgdorferi strains isolated in the United States [42]. Reportedly, vaccination with recombinant OspA can protect animals against infection, but can not eliminate the organisms if the animals were infected before vaccination [43]. A therapeutic Lyme vaccine is needed for this purpose. Attempts to develop a second generation Lyme vaccine using OspC [44±46], or decorin binding protein A (DbpA) [47±49] have been reported. It has been reported that patients may develop autoimmunity due to molecular mimicry between the dominant T-cell epitope of OspA and human-leukocyte-function-associated antigen I (hLFA-I) [50]. However, no clinical consequence using OspA as a subunit vaccine or bacterin have been reported in dogs since these vaccines became commercially available several years ago. Therefore, clinical signi®cance of this interesting in vitro phenomenon awaits further study. Acknowledgements We are grateful to Helen Bell for administrative assistance and to Patti Easton for technical assistance. We are grateful to Allyn Vondercheck, Dale Strickland, David Dietterich and John Daley for animal care. This work was supported by grants from the Zweig fund from Cornell University and the Cornell Biotechnology Program (CAT). References [1] Browning A, Carter SD, Barnes A, May C, Bennett D. Lameness associated with Borrelia burgdorferi infection in the horse. Vet Rec 1993;132:610±1. [2] Burgess EC, Mattison M. Encephalitis associated with Borrelia burgdorferi infection in a horse. J Am Vet Med Assoc 1987;191:1457±8. [3] Cohen D, Bosler EM, Bernard W, Meirs Dd, Eisner R, Schulze TL. Epidemiologic studies of Lyme disease in horses and their public health signi®cance. Ann NY Acad Sci 1988;539:244±57. [4] Kornblatt AN, Urband PH, Steere AC. Arthritis caused by Borrelia burgdorferi in dogs. J Am Vet Med Assoc 1985;186:960±4. [5] Lissman BA, Bosler EM, Camay H, Ormiston BG, Benach JL.
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