Preparation and Purification of Recombinant Outer Surface Protein a (rospa) of Borrelia Burgdorferi Sensu Stricto and Borrelia Afzelii

The recombinant Outer surface protein A (rOspA) from Borrelia burgdorferi is a possible immunogen for protection of infected humans and animals against development of Lyme borreliosis (Lyme disease), a chronic tick-borne disease characterised by diverse dermatologic, neurologic, rheumatic, and cardiac manifestations. For several years, research and development have been directed towards a vaccine for the prevention of this debilitat-ing disease. Numerous animal studies demonstrate that pre-existing antibodies against the outer surface proteins of B. burgdorferi can prevent infection and disease caused by this organism. In this communication, using recom-binant DNA technology, genes from B. burgdorferi sensu stricto and B. afzelii were inserted into E. coli-expression vectors and the rOspA were produced. Our aim was to obtain rOspA protein in a purity and quantity desirable for immunization of experimental animals. rOspA is currently the most developed, molecularly-defined vaccine candidate for the prevention of Lyme borreliosis.


INTRODUCTION
Lyme borreliosis is a multisystem inflammatory disease caused by Borrelia burgdorferi a gram-negative spirochete 1 , transmitted by the bite of infected ixodes ticks.B. burgdorferi sensu lato has been divided in to 11 genospecies.Of these, three species are recognized as pathogenic for humans: all of which are present in Europe: B. burgdorferi sensu stricto, the only species causing Lyme borreliosis in the United States; B. afzelii; and B. garinii.These spirochetes alternate in nature between warm-blooded hosts (mammals) and poikilothermic vectors.The tick Ixodes ricinus is the main vector of these pathogens 2 .In the vector, the spirochetes replicate during the feeding process 3 , migrate through the gut wall, and invade various tissues, including the salivary glands, from where they are transmitted to the host blood via saliva 4 .OspA is abundantly expressed in unfed ticks, probably mediating adherence to midgut cells 5 and thus enabling borreliae to survive in the vector for prolonged periods without tick feeding.B. burgdorferi expresses OspA but not OspC when residing in the midgut of unfed ticks.However, when the tick starts feeding on mammals, OspC synthesis is induced and OspA is repressed 6 .The switch is in part due to the change in temperature 7 .
Outer surface protein A (OspA) (molecular weight 31 kDa), a species-specific surface lipoprotein that presents together with the flagellin approximately one third of the total spirochete protein.The crystal structure of OspA consists of a single-layer beta-sheet connecting N-terminal and C-terminal globular domains.The central beta-sheet consists largely of polar amino acids that are solvent-exposed on both faces; this seems to be unique among protein structures 8 .More than 100 other proteins have also been identified in the spirochete's genome, including OspB, OspC, OspD, OspE, and OspF(ref. 9).
The OspA protein has been used as most promising candidate for vaccine against infection with B. burgdorferi.The ospA gene of B. burgdorferi encodes an outer membrane protein which is a major antigen of the Lyme disease agent.OspA elicits immunity against B. burgdorferi infection and is being used as a human vaccine 10 .OspA antibody blocks spirochete transmission to the vertebrate host by binding to B. burgdorferi within the gut of engorging ixodes ticks 11 .
Active immunization studies with OspA immunogens shown high-level of protection in mice.Humoral immunity is sufficient for protection.Although binding to an antigenic determinant (conformational epitope) located within the carboxyl end of OspA appears to be important for protection, the mechanism of protection conferred by antibodies has not been established.A principal drawback of the vaccine is the heterogeneity of OspA protein among particular B. burgdorferi isolates 12 .
At present, two human Lyme borreliosis vaccines use recombinant OspA (rOspA) from B. burgdorferi sensu stricto.LYMErix, produced by SmithKline Beecham, and licensed for use in Canada, contains 30 µg of purified lipidated-rOspA protein combined with 0.5 mg of aluminium adjuvant 13 .The vaccine stimulates host immune system to produce OspA -specific antibodies.Studies suggest that when a tick bites a vaccinee, the antibodies enter and contribute to killing the bacteria in the tick.A second vaccine, ImuLyme, produced by Pasteur Merieux Connaught, contains 30 µg of purified lipidated-rOspA protein without adjuvant.The ImuLyme vaccine induces killing of the spirochetes within the tick before it can enter the human bloodstream 14 .

MATERIALS AND METHODS
Plasmid DNA was isolated from B. burgdorferi sensu stricto and B. afzelii.The DNA was used as a template for the PCR amplification of the OspA gene which was after purification (QIAEX II Gel Extraction kit, Qiagen) cloned into expression vector pDEST TM 17 (Invitrogen) using Gateway TM Technology (GT).His-tag-fused OspA protein was expressed in expression competent E. coli BL21 Star (DE3) (Invitrogen).BL21 Star (DE3) were grown for 12 h at 37°C in 2 liters of the LB medium un-til they reached mid-log (OD 600 = 0.6).IPTG (SERVA) was added at a final concentration of 0.1 mM, and the culture was incubated for another 4 h.Cells were collected by centrifugation (10.000RPMI, 10 min., 4 o C), resuspended in lysis buffer (lysis buffer: 50 mM NaH 2 PO 4 .H 2 O, 300 mM NaCl, 10 mM imidazol, pH = 8) containing lysozyme (0.5 mg/ml) and incubated for 1 h at 4 °C.After 10×10 s ultrasonic pulsions homogenisation lysate was centrifuged (10.000RPMI, 10 min., 4 o C) and 4 ml of the cleared supernatant was mixed with 1 ml of the 50 % Ni-NTA slurry (Qiagen) for 1 h at 4 o C. The supernatant -Ni-NTA mixture was loaded into a gravity column (Sigma) and washed twice with 4 ml washing buffer (washing buffer: 50 mM NaH 2 PO 4 .H 2 O, 300 mM NaCl, 30 mM imidazol, pH = 8).Protein was eluted with 4× 0.5 ml of elution buffer (elution buffer: 50 mM NaH 2 PO 4 .H 2 O, 300 mM NaCl, 250 mM imidazol, pH = 8).All buffers contained 5 mM β -mercaptoethanol, 0.5 mM PMSF, 2 µg/ml Leupeptin and 0.1 µg/ml Aprotinin.The fractions were analyzed using 12 % SDS-PAGE and Western immunoblot.Total protein was stained on gel with Coomassie brilliant blue.Protein blotted on PVDF membranes were incubated with Penta-His HRP antibodies (Qiagen) and detected by a color reaction using TMB Membrane Substrate (Amresco).Fractions with maximum concentration of rOspA were dialyzed against 200 mM NaCl at 4 °C overnight.

RESULTS
The rOspA proteins from B. burgdorferi sensu stricto and B. afzelii present 80 % of total protein in elution fractions and they were specifically recognized with Penta-His antibodies (Fig. 1, Fig. 2).The concentration of rOspA from B. burgdorferi sensu stricto (0.4 mg/ml) and B. afzelii (0.3 mg/ml) were measured using a densitometer.
Overdialyzed fractions with maximum concentration of rOspA were tested using polyclonal anti-OspA-IgG antibodies (by Bioveta, a.s.).The rOspA proteins reacted with this antibodies (data is not shown).

CONCLUSION
Purification of recombinant OspA from B. burgdorferi sensu stricto and B. afzelii by nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography under native conditions is highly effective.Proof of the 'principle' that rOspA is a protective antigen (as a possible immunogen to protect against Lyme borreliosis) will be confirmed by active immunization experiments in mice.