PT100980B - RECOMBINANT PROTEINS FROM BORRELIA, ITS PRODUCTION PROCESS, VACCINE AGAINST BORRELIA INFECTION AND IMMUNODIAGNOSTIC AGENT FOR ITS DETECTION - Google Patents

Abstract

Highly-purified immunologically-effected recombinant protein encoded by a gene of a full-length wild-type Borrelia lipoprotein, particularly the OspA encoded by the OspA gene of B.burgdorferi, useful in vaccines and test kits for Lyme disease, is prepared by inducing protein production from a host organism transformed by a plasmid vector containing the cloned gene and subsequently recovering and purifying the protein. The grown organism is lysed and contacted with a surfactant, particularly Triton X-114, which selectively extracts the desired protein from the lysed cells. Upon heating the resulting mixture to mildly-elevated temperature, a detergent phase separates out and is recovered separate from the other phases. The detergent phase is further purified from residual protein by column chromatography.

Description

DESCRIPTIVE MEMORY

FIELD OF THE INVENTION the present invention relates to the preparation of Borrelia recombinant lipoproteins, particularly the outer surface protein A (OspA) of Borrelia burqdorferi, the spirochete responsible for Lyme disease.

REFERENCE TO RELATED REQUESTS

This application is a continuation - in - part of patent application US copending ^Nos Serial 888,765, filed on May 27, 1992, which is itself a continuation - in - part of patent application US copending n ² , series 779 048, filed on October 18, 1991.

BACKGROUND OF THE INVENTION

Lyme disease is a zoonosis caused by the spirochete carried by ticks, Borrelia burqdorferi. The spirochete can cause serious dermatological, arthritic, neurological and other pathological disorders in an infected host. Recently, Lyme disease has become a serious epidemiological concern, particularly in North America, but also elsewhere in the world.

Attempts have been made to develop a vaccine against the disease and immunodiagnostic reagents useful in detecting antibodies against the spirochete. Much attention was focused on the main protein on the outer surface OspA, and antibodies against it were found to provide protection against the challenge with the spirochete in mice (ref. 1, 2 - identification of literature references appears at the end of the description ).

Previous attempts have been made to isolate soluble, purified Borrelia lipoproteins by growing and subsequently purifying Borrelia cell cultures. However, growth and subsequent purification //

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of proteins from crude extracts of Borrhea cells is very time consuming and expensive.

Recombination techniques have been suggested for the production of OspA and its derivatives, with a view to the potential for the production of large amounts of pure protein material. These techniques should involve expression of Borrelia genes in a suitable host / expression vector system, such as E. coli.

International Published Patent Application (PCT) WO 90/04411 discloses n a DNA fragment encoding OspA protein of B. burgdorferi of the New York strain B31 (ATCC 35210) which contains the ospA gene. The nucleotide sequence of the ospA gene, which encodes the full-length wild-type OspA, is described in the published PCT patent application, together with the obtained amino acid sequence.

Dunn et al described the preparation of a modified form of the OspA protein that is soluble, while still retaining specific reactivity for antibodies against wild-type B. burqdorferi OspA (ref. 3). The article describes the preparation of two plasmids, pET9-OspA and pET9-preOspA, the first containing a DNA sequence amplified by polymerase chain reaction (PCR) encoding a recombinant truncated version of OspA, and the last containing a sequence of PCR amplified DNA encoding wild-type, full-length OspA. Both sequences are expressed from the ió 10 promoter of bacteriophage T7.

The primary translation product of the full-length gene contains a hydrophobic N-terminal leader sequence, which is a substrate for the attachment of the lipid portion to the sulfhydryl side chain of the adjacent cysteine residue. Following this binding, cleavage by signal peptidase II occurs and the binding of the lipid moieties to the new N-terminus. On the other hand, the protein translated from the truncated gene is not lipidized, and is soluble in aqueous solution. It is said that the expression of the truncated, soluble OspA derivative overcomes certain problems

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-4 associated with the expression of recombinant versions of Borrelia burqdorferi full-length wild-type lipoproteins using E. coli, namely that the protein has poor solubility properties, due to the association of the protein with the host's outer cell membrane during expression, requiring the use of detergents to carry out the protein separation and making purification procedures difficult.

Plasmids were transferred to an E. coli expression strain, namely BL21 (DE3) (pLysS) (a host strain containing a chromosomal copy of the gene for T7 RNA polymerase under the control of the inducible lacUV5 promoter and a plasmid based on pACYC184, pLysS, which produces low levels of T7 lysozyme). With induction, plasmid pET9-preOspA was found to produce relatively smaller amounts of inducible protein than plasmid pET9-OspA. It was found that the latter product is soluble in the absence of detergent while the former requires treatment with Triton X-100 detergent to solubilize. Dunn et al's article contains no description of any subsequent isolation and purification of the detergent-soluble OspA protein.

Although the production of full-length recombinant OspA can be achieved in lower quantities than the truncated soluble variation of OspA and the use of detergents is necessary for the recovery of full-length protein (treatment of lipoproteins with detergents often impairs reactivity ), although the production of recombinant wild-type protein is considered desirable, since the lipidized wild-type protein is thought to produce an immune response superior to that of the truncated protein, due to the stimulation of B lymphocytes (ref. 4, 5) and cytotoxic T lymphocytes (ref. 6) by lipids covalently linked to the N-terminus of a peptide in a manner identical to that in which the lipid moiety binds to the B. burqdorferi OspA (ref. 7).

Long-lasting protective immunity was

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5 - challenge with spirochetes in mice by vaccination with OspA (ref. 1, 8). The antigen used in these studies was a fusion protein containing a large domain of glutathione-S-transferase at the amino end of OspA. This fusion protein is not likely to be a substrate for post-translational lipid bonding. The ability of this antigen to induce a protective response in these studies may be due to the fact that the antigen was delivered in complete Freund's adjuvant, with multiple secondary doses in incomplete Freund's adjuvant (ref. 1, 8), or in the form of E complete coli, living or dead, which expresses the fusion protein (ref. 9). In connection with this earlier activity, reference is also made to the published international patent application WO 92/00055.

As will be seen here, it has now been shown that the recombinant protein encoded by the full-length ospA gene, in the form absorbed by alum or without adjuvant, exhibits an immunogenicity that is not shown by the corresponding recombinant OspA protein without the bound lipid. It was shown that this difference in immunogenicity is not due to any difference between the antigenicity of the two proteins. The present invention represents the first description in which the inventors are aware of the increased immunogenicity of a large protein antigen by the expression of a bacterial lipoprotein.

SUMMARY OF THE INVENTION the present invention provides, in one aspect, a new, simple and effective way to produce a highly purified and immunologically effective recombinant protein encoded by the wild-type ospA full-length gene of B. burqdorferi and other Borrelia full-length lipoproteins encoded by the respective genes. The recombinant protein exhibits an immunogenicity that is the same as that of the native protein.

The present invention employs selective separation of the recombinant lipoprotein from other cellular constituents using particular detergents. The present procedure

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The invention allows the problems observed in the prior art associated with the production of the full-length OspA protein to be overcome, while providing a product with improved immunogenicity.

For the first time, a highly purified, immunologically effective recombinant protein is provided by the present invention, which is encoded by a wild-type Borrelia full-length lipoprotein gene, particularly the B ospA gene, burqdorferi, and which is formed by recombination from a host organism transformed by a plasmid containing the gene. These new proteins form an additional aspect of the present invention. Also provided here are certain new plasmids, useful in this transformation.

The new protein product provided herein is useful in vaccines against infection by Borrelia organisms, particularly those that cause Lyme disease, and these vaccines, comprising an immunologically effective amount of the protein, particularly that encoded by the full-length ospA gene of B. burqdorferi wild type, constitute an additional aspect of the invention.

In yet a further aspect of the invention, a method of immunizing a mammal against infection by Borrelia organisms, particularly those causing Lyme disease, is provided by administering an immunologically effective amount of a protein, particularly that encoded by the ospA gene of total length of B. burqdorferi wild type, in the absence of adjuvant.

The new proteins can also be used as immunodiagnostic agents for the detection of antibodies against a host's infection by a Borrelia organism, particularly one that causes Lyme disease, and therefore the presence of that infection.

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Here, as described below, immunologically effective purified recombinant protein encoded by the full length ospA gene was produced from several specific strains other than B. burqdorferi, specifically strains B31, ACA1 and Ip90. There are also full-length ospA genes that differ in the nucleotide sequence from the sequences of these specific genes, at most, in some nucleic acids, resulting in gene products that differ at most in some amino acids from those produced by the true ospA genes of strains B31, ACA1 and Ip90. These genes and the gene products are combined here within the B31, ACA1 and Ip90 strain families.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the PCR oligonucleotides used in cloning the B-31, ACA1 and Ip90 full length ospA gene of B. burgdorferi into the expression vector pET9a;

Figure 2 illustrates the cloning strategy for inserting the full-length ospA gene into the pET9a expression vector in order to place the ospA gene under the control of the T7 Φ 10 promoter, to form pOAl from the B31, pOA9 gene a from the ACA1 gene and pOAlO from the Ip90 gene;

Figure 3 is a predicted restriction map of the plasmid pOAl;

Figure 4 shows the results of several restriction digests of the pOAl plasmid, demonstrating that all predicted sites are present (M = markers (DNA ΦΧ 174 digested with Hae III); B = Bam Hl; E = Eco RI; H = Hind III; N = Nde I);

Figure 5 shows the PCR nucleotides used in cloning the full-length ospA gene of B-31, ACA1 and Ip90 of B. burqdorferi into the pCMBl expression vector;

Figure 6 illustrates the cloning strategy for inserting the full-length ospA gene into the pCMBl expression vector.

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in order to place the ospA gene under the control of the Trc promoter, to form pOA5 from the B31 gene, pOA7 from the ACA1 gene and pOA8 from the Ip90 gene;

Figures 7A, 7B and 7C show an elapsed time of induction with ITPG of OspA in two host strains containing pOAl (figure 7A), and a host strain containing pOA5 (figure 7B) and pOA6 (figure 7C);

Figures 8A, 8B and 8C are flow diagrams showing, respectively, the steps of cell growth and lysis, extraction with detergent, and purification involved in the production and purification of full-length recombinant OspA from E. coli according to an embodiment of the invention;

Figure 9 shows the solubilization of proteins in cell lysate complete with various detergents (Deo = sodium deoxycholate; Emp = Empyrene; Sarc = sodium lauryl sarcosinate; SDS = sodium dodecyl sulfate; TX-114 = Triton X-114), bands (M = markers (Bio-Rad Low Molecular Weight Standards); W = complete lysate; S = soluble fraction; P = pellet; A = aqueous phase; D = detergent);

Figure 10 is an immunoblot with the anti-OspA monoclonal antibody H5332, showing that the 31 kilodalton species in the detergent phase are OspA, Wh = complete cell lysate, Aq = aqueous phase after extraction with Triton X-114, Dt = phase detergent, Bx = aqueous phase back-extracted with Triton X-114, Pl = insoluble pellet;

Figure 11 illustrates the Triton X-114 phase partition of lipoprotein and non-lipid protein for pOAl and pOA2, in which bacteria containing pOAl that express OspA lipoprotein or that contain pOA2 that express non-lipidized OspA protein grew to the semi-logarithmic phase. and were induced with ITPG for 2 hours for pOAl and 4 hours for pOA2. After centrifugation, the cells were resuspended and lysis was performed by freezing and thawing. Triton X-114 was added and the

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phase partition by heating to 37 ° C.

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electrophoresis of an equal aliquot of each fraction in gel of 4 to 20% SDS-PAGE, and the gel was stained with Brilliant Blue of

Coomassie. The fractions are molecular weight markers (Μ), complete lysate (W), aqueous phase (A), detergent phase (D), and insoluble pellet (P), for pOAl and pOA2 as indicated;

Figures 12A, 12B and 12C illustrate the purification of OspA lipoprotein produced from pOAl (figure 12A), from pOA9 and pOAlO (figure 12B) and from pOA5, pOA7 and pOA8 (figure 12C), where an aliquot each fraction was electrophoresed on a 4 to 20% SDS-PAGE gel and the gel was tinted with Coomassie Brilliant Blue. Figure 12A: lane 1, low molecular weight markers; lane 2, complete lysate; lane 3, detergent phase; lane 4, DEAE-Sepharose; and lane 5, S-Sepharose elution; Figure 12B: P = pre-stained low molecular weight markers (106 kDa, 80 kDa, 49.5 kDa,

32.5 kDa, 27.5 kDa, 18.5 kDa), CL = complete cell lysate, D = Triton X-114 detergent phase, De = DEAE Sepharose pass-through fraction. S = Sepharose elution, pH 5.7 (purified OspA-2); Figure 12C: P = pre-stained low molecular weight markers (106 kDa, 80 kDa, 49.5 kDa, 32.5 kDa, 27.5 kDa, 18.5 kDa), 0 = OspA-L B31 purified by S -Sepharose, derived from pOAl (figure 12A), CL = complete cell lysate, D = Triton x-114 detergent phase, De = DEAE Sepharose passage fluid fraction.

Figure 13 is a silver-stained gel from three test vaccines used in an immunogenicity comparison study of purified full-length OspA with that of Borrelia burgdorferi proteins (M = markers; OA = purified recombinant OspA; E = fraction E of B. burgdorferi; SA = bovine serum albumin);

Figure 14 shows the IgG titers in the serum of mice boosted four weeks after the primary injection with 10 μφ of the purified full-length OspA and fraction B. burgdorferi of figure 13, with an ELISA assay with 100 ng of purified OspA in the solid phase, mouse serum as

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first goat anti-mouse IgG and alkaline phosphatase conjugated IgG as second antibody;

Figures 15A to 15E contain a graphical representation of the dose response of mice to lipoprotein. Figure 15A shows the dose response of C3H / He mice to unabsorbed lipoprotein; Figure 15B shows the dose response of BALB / c mice to non-adsorbed lipoprotein; Figure 15C shows the dose response of C3H / He mice to alum adsorbed lipoprotein; Figure 15D shows the dose response of CH3 / He mice to unabsorbed lipoprotein; Figure 15E shows the dose response of CH3 / He mice to non-lipid protein. Mice were vaccinated at week 0 with the indicated amount of protein, in PBS except where indicated, and boosted with the same vaccine at week 3. Serum IgG titer was determined at week 4 by ELISA using lipoprotein OspA as antigen;

Figure 16 contains a graphic representation of the antigenicity of lipoprotein and non-lipid protein; and

Figure 17 illustrates the production of plasmids pCMBl and pCMB2.

IDENTIFICATION OF PLASMIDS CONTAINING THE OSPA GENE

Vector Strain Plasmid

I.D. B. burqdorferi Expression

pOAL B31 pET9a pOA2 * B31 pET9a pOA5 B31 pCMBl pOA6 B31 pCMB2 pOA7 ACA1 pCMBl pOA8 Ip90 pCMBl pOA9 ACA1 pET9a porO Ip90 pET9a

* This plasmid contains truncated ospA from strain B31. All other plasmids contain full-length ospA from several strains.

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GENERAL DESCRIPTION OF THE INVENTION

The cloned ospA gene of B. burqdorferi from strain B31 (as described in WO 90/04411, mentioned above) (N-terminal region: SEQ ID NO: 1, c-terminal region: SEQ ID NO: 2. The rest of the sequence shown in WO 90/04411) was used as a template (pTRH44) and specifically designed oligonucleotides (PET-IN [CO1] (SEQ ID NO: 3) and PET-273 C [C03] (SEQ ID NO: 4)) in a polymerase chain reaction (PCR) to amplify the entire wild-type ospA gene, as shown in figure 1.

Similarly, the cloned ospA gene of B. burqdorferi from strains ACA1 and Ip90 (as described in reference 10 - the N-terminal region of ACA1 and Ip90 is: SEQ ID NO: 1; C-terminal region of ACA1: SEQ ID NO: 5; Ip90 C-terminal region: SEQ ID NO: 6) was used in a PCR reaction with pairs of primers (a) OspN2 (SEQ ID NO: 7) and BZ1 (SEQ ID NO: 8) and (b) OspN2 (SEQ ID NO: 7) and pK4 (SEQ ID NO: 9), at the N-terminal and C-terminal ends, respectively, to form the appropriate amplified fragments, as shown in figure 1.

The basic methods for amplifying a target nucleic acid sequence desired using oligonucleotide primers are generally known in the art and are described in US patent ^N2 4683202 , and 4800 159. One can refer to these patents for description of the techniques to employ.

The resulting fragments at the Ndel and Bam Hl sites of the plasmid vector pET9 were cloned to place the ospA gene under the control of a T7 promoter and effective translation initiation signals from the T7 bacteriophage, as shown in figure 2. The pET9 plasmids and pLysS, the bacterial hosts for cloning, the growth media and the processes used to direct the expression of the genes cloned by the T7 RNA polymerase were previously described in U.S. Patent No. ² 952 496 and can be referred to here. description. Although a T7 promoter system is a preferred expression system in the present invention, expression of the full-length ospA gene can be

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achieved using other expression systems compatible with the host organism, as described below.

The expression vector pET9 was used since it has a kan gene as its selective marker instead of the bla gene. Consequently, ampicillin is not used during cell growth and therefore there is no possibility of an immunogenic OspA / ampicillyl target protein conjugate. These conjugates are believed to be major antigenic determinants in penicillin allergy and may complicate immunological studies.

The resulting plasmids were called pOAl, pOA9 and pOAlO, containing the B. burqdorferi strains B31, ACA1 and Ip90 genes, respectively. The pOAl plasmid is virtually identical to the pET9-preOspA plasmid described by Dunn et al. (supra), except that the oligonucleotides used for the PCR reaction were different in the two cases. A predicted restriction map for the pOAl plasmid is shown in figure 3, while figure 4 contains the results of several restriction digestions of the pOAl plasmid, demonstrating that all predicted sites are present.

For protein production, plasmids pOAl, pOA9 and pOAlO were transformed into the E. coli expression strain or into another suitable host organism. Preferably, the E. coli strain is the E. coli T7 expression strain, as described in U.S. Patent No. ^B 4 952 496 mentioned above. Specifically, the strain can be the E. coli expression strain BL21 (DE3) (pLysS), as described above, or the E. coli strain HMS174 (DE3) (pLysS). The transformed host grew and the protein was induced with isopropyl-0-D-thiogalactoside (IPTG). Figure 7A shows an elapsed time of OspA induction from plasmid pOAl, after addition of IPTG. Results similar to those of pOAl were obtained using pOA9 and pOAlO. The synthesis of OspA protein from plasmid pOAl ended approximately one hour after induction, implying some toxicity of the protein to E. coli. although

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from this, protein production was up to an acceptable level of approximately 10 mg / 1 of cell culture.

In addition to the provision of plasmids pOAl, pOA9 and pOAlO and expression of lipoprotein OspA in E. coli using the T7 promoter, additional plasmids were constructed containing the full length ospA gene of B31, ACA1 and Ip90 under a different promoter and achieved expression of lipoprotein. To this end, plasmids pOA5 and pOA6 were prepared by cloning an ospA amplified PCR fragment of strain B31 at the Ncol and Bam Hl sites of plasmid expression vectors pCMBl and pCMB2 while plasmids pOA7 and pOA8 were prepared by cloning of an ospA PCR amplified fragment from the ACA1 strain (pOA7) and the Ip90 strain (pOA8) at the Ncol and Bam H1 sites of the pCMBl expression vector (see figure 6 for pOA5, pOA7 and pOA8).

As shown in Figure 5, the ospA genes cloned from the B. burgdorferi, B31, ACA1 and Ip90 strains were amplified by PCR reaction using pairs of primers (a) PK3 (SEQ ID NO: 10) and CO3 (SEQ ID NO : 3), (b) PK3 and (SEQ ID NO: 10) and BZ1 (SEQ ID NO: 8), and (c) PK3 (SEQ ID NO: 10) and PK4 (SEQ ID NO: 9), respectively in N- and C-terminal ends of the respective genes to form the appropriate amplified fragments.

Plasmids pCMBl and pCMB2 were constructed by digestion of plasmid pTrc99a (Pharmacia N ^a of Catalog 27-5007-01) and a kanamycin resistance gene (Pharmacia N ^a of Catalog 27-4897-01), isolation of the resulting fragments by Geneclean procedure, and by connecting the fragments to each other (figure 17). Plasmid pTrc99a, 4197 bp, contains a strong promoter adjacent to a multiple cloning site, followed by a strong transcription termination signal (rrnB). The expression of the target gene uses RNA polymerase from the host cell, allowing its use in a wide variety of E. coli strains. Expression is tightly controlled by the lactose suppressor gene (laclq) included in the vector. The lactose repressor protein prevents the transcription of the target gene in

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absence of the IPTG inductor. The kanamycin resistance gene is a 1282 bp double-stranded linear DNA fragment containing the Tn903 transposon gene flanked by enzyme restriction sites and encodes the aminoglycoside-3'-phosphotransferase enzyme, which confers resistance to kanamycin and to neomycin.

pCMBl, 5.5 kb, contains the kanamycin resistance gene oriented in such a way that its transcription is done in the same direction as the orientation from the Trc promoter, while pCMB2 contains the kanamycin resistance gene oriented in the direction opposite, so that transcription of the resistance gene and the gene of interest under the control of the Trc promoter results in the convergence of transcripts. Digestion with restriction enzymes of pCMBl and pCMB2 using Smal, HindIII and BamHI + Ncol showed the fragments to the exact predicted size.

Plasmids pOA5 and pOA6 were transformed into the E. coli expression strain or another suitable organism, preferably the competent DH5a cells. Transformed hosts were developed and the protein was induced with IPTG. The course of the induction time with pOA5 (figure 7B) was similar to that obtained with pOAl (figure 7A) while the levels of OspA produced by pOA6 (figure 7C) were several times lower. Similar results were obtained using pOA7 and pOA8.

The steps involved in the production and purification of the full-length recombinant OspA are shown schematically in Figures 8A to 8C. The specific conditions of the process are listed there as described in the following examples.

After the cell growth and protein induction step (figure 8A), the cells are subjected to freeze-thaw lysis. Treat the lysate with a detergent that is selective for the solubilization of the OspA protein, preferably in relation to other bacterial proteins

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present in the lysate. A series of experiments were conducted using different detergents to determine which detergent was selective for the OspA protein. Of those tested it was found that Triton X-114 selectively solubilizes a 31 kilodalton protein (figure 9), which was shown to be OspA by immunoblotting (figure 10).

The invention is not limited to the use of Triton X-114, but it also clearly includes other materials that exhibit a similar selective solubility for OspA as well as the phase separation property under moderate conditions as noted below.

After adding the selective detergent, the mixture is heated to a moderate temperature rise of about 35 ° to 40 ° C at which point the solution becomes cloudy as phase separation occurs. It is essential for the purification procedure of the present invention that this phase separation takes place under moderate conditions to avoid any denaturation or other damage to the immunological properties of the protein. (For example, if Triton X-100, mentioned by Dunn et al. (Ref. 3), is used as a detergent, while the selective separation of the OspA protein is carried out, much higher temperatures are required, around 60 to 65 ° C, to carry out phase separation, which is highly disadvantageous in relation to the usefulness of the product).

The centrifugation of the cloudy mixture results in the separation of the mixture into three phases, namely a detergent phase containing 50% or more of the OspA protein and a small amount (approximately 5% by weight) of the bacterial proteins, an aqueous phase containing the rest of the proteins bacteria and a solid pellet of cellular waste. The detergent phase is separated from the aqueous phase and the solid pellet.

By the steps of treatment with a detergent selective for OspA and the subsequent phase separation of the detergent phase, the substantially complete separation of OspA from the

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bacterial proteins (figure 12). There remains the final purification of OspA from the residual bacterial protein present in the detergent phase.

The final purification of the protein is carried out on a selective chromatographic column for the binding of bacterial proteins but not OspA, specifically DEAE-Sephacel, DEAE-Sepharose, or other equivalent chromatographic material. The detergent phase is loaded onto the column and the pass-through fluid, which contains all the purified OspA protein, is collected. The bound fraction contains all bacterial proteins in the detergent phase. After further purification using S-Sepharose or an equivalent chromatographic column (figure 12), in addition to being free of contaminating proteins, the passing fluid fraction is substantially free of liposaccharide (LPS), as indicated by the lack of pyrogenicity, as determined by lumen amebocyte lysate (LAL). The highly purified OspA solution can be freeze-dried or processed in another way.

Although the procedure described above was specifically directed towards the production of highly purified recombinant OspA protein, the described procedures and techniques are readily adaptable to the production of other highly purified recombinant Borrelia lipoproteins, by proper cloning of the appropriate gene, construction of a plasmid vector suitable containing the gene, transformation of a suitable host by the plasmid vector, and production and purification of lipoprotein, by appropriate choice of the selective surfactant.

EXAMPLES

Example 1

Plasmid pOAl was prepared as described above and used to transform E. coli BL21 (DE3) (pLysS) (pOAl) and HMS174 (DE3) (pLysS) (pOAl) strains. E. coli inoculated into LB media was inoculated with 25 µg / ml sulfate

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-17 kanamycin and 25 gg / ml of chloramphenicol at a rate of 12 ml of culture for each liter prepared. The culture was allowed to grow overnight in a mixing flask at about 37 ° C.

The following morning, 10 ml of the culture medium from the previous night was transferred to 11 ml of LB medium containing 25 µg / ml kanamycin sulfate and the culture was allowed to grow in a mixing flask at about 37 ° C to a level of D0 = 0.6 (although growth can be made to D0 = 1.5) in approximately 3 hours.

Isopropylthiogalactoside (IPTG) was added to the culture medium to a final concentration of 0.5 mM and the culture medium was allowed to grow for an additional two hours at about 37 ° C. At the end of this period, the culture medium was cooled to about 4 ° C and centrifuged at 10,000 x g for 10 minutes. The supernatant was discarded while the cell pellet was resuspended in 1/10 of the volume of PBS. The cell suspension was frozen in liquid nitrogen and can be stored indefinitely at -70 ° C, if desired.

After freezing the cell suspension, the cells were thawed to room temperature (about 20 ° to 25 ° C) which causes cell lysis. DNase I was added to the thawed material to a concentration of 1 Mg / ml and the mixture was incubated for 30 minutes at room temperature, which resulted in a decrease in the viscosity of the material.

The material incubated on ice was cooled to a temperature below 10 ° C and Triton X-114 was added in the form of a 10% by weight warehouse solution, to a final concentration of 0.3 to 1% by weight . The mixture was kept on ice for 20 minutes. The cooled mixture was then heated to about 37 ° C and left at that temperature for 10 minutes.

The solution became very hazy as phase separation occurred. The nebulous mixture was then centrifuged at about 20 ° C for 10 minutes at 12,000 x g, which caused

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separating the mixture into a lower detergent phase, an upper aqueous phase and a solid pellet. The detergent phase was separated from the other two phases and cooled to 4 ° C, without disturbing the pellet. Buffer A, namely 50 mM Tris, pH 7.5, 2 mM EDTA and 10 mM NaCl, was added to the cooled detergent phase to reconstitute 1/3 of the original volume. The resulting solution can be frozen and stored for further processing as described below or can be immediately subjected to such processing.

A DEAE-Sepharose CL-6B column was prepared in a volume of 1 ml / 10 ml of detergent phase and washed with 2 volumes of buffer C, namely 50 mM Tris pH 7.5, 2 mM EDTA, 1M NaCl , 0.3% by weight of Triton X-100, and then with 4 volumes of buffer B, namely 50 mM Tris pH 7.5, 2 mM EDTA, 0.3% by weight of Triton X-100.

The detergent phase was then loaded onto the column and the passage fluid containing OspA was collected. The column was washed with 1 volume of buffer B and the passage fluid was collected again. The combined pass-through fluid was a purified aqueous solution of OspA, which can be frozen for storage.

The column can be cleaned of bacterial proteins for reuse by eluting with 2 volumes of buffer C.

The additional and final purification of the DEAE-Sepharose column passage fluid can be performed by S-Sepharose Fast Flow chromatography. The DEAE-Sepharose column passage fluid was first acidified to pH 4.2 by the addition of 0.1 M citric acid. The S-Sepharose Fast Flow column was washed extensively with buffer C, and adjusted to pH 4.2 with citric acid.

The highly purified OspA was eluted from the column using buffer C, and adjusted to pH 5.7 with citric acid. The eluate was readjusted immediately to pH 7.5 by the addition of 2M Tris base.

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The highly purified aqueous OspA solution obtained by both chromatographic procedures was analyzed using Coomassie stained gels (figure 12A) and confirmed to contain OspA in highly purified form. The purity of the product produced by the last chromatographic procedure was superior to that formed by the first chromatographic procedure, exhibiting very low levels of endotoxin.

Example 2

The procedure of example 1 was repeated, with the exception that other surfactants, as well as Triton X-114, were used to form the detergent phase. The results obtained are shown in figure 9. As can be seen in figure 9, of the detergents tested, only Triton X-114 was able to provide a sufficiently selective solubilization of OspA to allow rapid purification by chromatographic column.

Example 3

Plasmids pOA5 and pOA6 were prepared as described above and used to transform E. coli strain DH5a. The procedure for the expression of the protein used in example 1 was repeated and the time elapsed in the expression of the lipoprotein OspA by pOA5 was identical to that observed for pOAl while the expression from pOA6 was found to be several times lower than that of pOA6. of pOA5 (figures 7B and 7C). Purification of the OspA protein was only continued in cells expressing pOA5.

The B31 OspA lipoprotein produced by the Trc expression system in this way was purified following a procedure similar to that described in example 1, with the exception that 0.1 mg / ml lysozyme was added to the cell pellet after collection and the cells were suspended for 30 minutes at room temperature before freezing.

fluid passing through the DEAE-Sepharose column contained

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OspA lipoprotein in a highly purified form (figure 9C).

Example 4

The procedures for the production of the plasmid pOAl (promoter pET) and pOA5 (promoter TRC) were repeated, as previously described, with the cloned ospA gene of the Asian strain (Ip90) of B. burqdorferi (ATCC ________), to form the plasmids pOA8 (Trc promoter) and pOAlo (pET promoter) containing the gene. Restriction digestion was performed with these plasmids, which showed that all predicted sites were present.

Procedures were also repeated with the ospA gene cloned from the European strain (ACA1) of B. burqdorferi (ATCC _____), to form the plasmids pOA7 (Trc promoter) and pOA9 (pET promoter) containing the gene. Restriction digestion was performed which showed that all predicted sites were present.

The growth and induction of the expression strains pOA7 and pOA8 proceeded identically to the strain of pOA5 (example 3) while the growth and induction of the expression strains pOA9 and pOAlO was carried out identically to that of the pOAl strain (example 1).

The ACA1 and lp90 OspA lipoproteins obtained by these operations were purified in an identical manner to the B31 OspA lipoprotein (pOAl) as described in example 1. The DEAE-Sepharose column passage fluid contained OspA lipoprotein in a highly purified form (figures 12B, 12C).

Example 5

The purified recombinant full-length OspA of E. coli, according to the procedure described in example 1, was compared with fraction E of B. burqdorferi, which is enriched in OspA (prepared as described in PCT N ² WO

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90/04411 mentioned above), and with bovine serum albumin (BSA) for immunogenicity in mice. C3H / He mice were injected with 10 pg of each vaccine (figure 13) in a form without adjuvant. Four weeks after the first injection, mice were boosted with the same antigen as used for the first injection. Serum titers were tested by an ELISA against purified, recombinant OspA lipoprotein (figure 14).

As can be seen, both immunogens elicited a very similar response, that is, a weak but detectable IgG response in the primary serum, with a 100-fold increase in serum titers after boost. For both immunogens, serum IgG titers were stable for at least 6 weeks. In no case was there any significant IgG response in the serum.

These experiments indicate that the recombinant OspA is also immunogenic to the purified protein of B. burqdorferi.

The immunological integrity of the recombinant OspA lipoprotein was further demonstrated by the fact that the sera from mice immunized with the recombinant lipoprotein were completely capable of inhibiting the growth of the B31 B. burqdorferi spirochete strain in vitro, as shown in Table 1 below. :

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TABLE 1. Titles of B. burgdorferi growth inhibiting activities in sera from mice vaccinated with lipoprotein and non-lipidized OspA protein

Strain | mouse 1 Antigen | 1 I 1 C3H / He | 1 I 1 OspA lipoprotein | 1 1 1 1 1 1 1 non-lipi- protein | 1 I given OspA | I 1 BALB / C | 1 I 1 OspA lipoprotein | 1 I 1 1 1 1 1 non-lipi- protein | 1 given OspA 1

Dose I Growth inhibition titre (Mg) | B.burgdorferi cement

---------------------------1 0 1 <8 0.1 I 32 0.5 | 256 2.5 | 1024 0.1 | <8 0.5 | <8 2.5 | <8 θ 1 <8 0.1 [ 4 0.5 | 16 2.5 | 512 0.1 | <8 0.5 | <8 2.5 I <8

As can be seen from this table, it was found that the serum inhibitory activity was proportional to the dose of lipoprotein administered to the mice and that it correlated very well with the results obtained by the ELISA assay for anti-OspA IgG. The serum from mice vaccinated with non-lipidized OspA protein, which did not contain detectable anti-OspA antibodies, had no effect on spirochete growth.

The growth inhibitory titers achieved in mice immunized with 2.5 µg of OspA lipoprotein in PBS were equal to or greater than those obtained in mice vaccinated with 20 µg of total B. burgdorferi protein in complete Freund's adjuvant and with a booster of total protein in PBS. The ability of the recombinant OspA lipoprotein to elicit a response in the

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serum capable of inhibiting the growth of spirochetes demonstrated that the critical protective epitopes in the protein are conserved during expression in E. coli and in subsequent purification.

Example 6

Plasmid pOA2 was prepared in a manner similar to that described above for plasmid pOAl, except that the primer PET-18N was used, having the sequence (SEQ ID NO: 11):

5 'CAG CAT ATG GCT AAG CAA AAT GTT AGC 3' together with the C-terminal primer PET-273C, in the PCR reaction to amplify the truncated form of the ospA gene devoid of the lipoprotein signal peptide. The region underlined in PET-18N is identical to nucleotides 51 to 67 of the OspA gene coding strand.

Plasmid pOA2 was used to transform E. coli strains BL21 (DE3) (pLysS) (pOA2) and HMS174 (DE3) (pLysS) (pOA2) and truncated OspA protein from transformed E. coli was expressed , as described in example 1. With the cell lysis and detergent extraction steps of example 1, the non-lipid protein was found to be present in the aqueous phase, as expected.

Example 7

The effect of lipid binding to OspA protein on immunogenicity was investigated.

C3H / He and BALB / C mice were vaccinated with both forms of OspA lipoprotein formed and isolated as described in example 1 or with the OspA non-lipid protein form prepared by Dunn et al. (ref 3) and reinforcement was given three weeks later. Sera were collected 1 to 2 weeks after boost, and tested by ELISA using purified OspA lipoprotein as antigen.

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Figures 15A and 15B show that the OspA lipoprotein in PBS induced a strong dose-dependent secondary IgG response in both C3H / He (figure 15A) and BALB / C mice (figure 15B). A small, if any, increase in lipoprotein immunogenicity was observed when the protein was absorbed in aluminum hydroxide in a protein: aluminum ratio of 1: 5 by weight prior to injection, as shown in Figure 15C.

In contrast to the response observed for OspA lipoprotein, the non-lipid protein was unable to induce any detectable anti-OspA antibody, even at the highest dose of 25 Mg / mouse, as seen in Figure 15E. Even the non-lipid protein absorbed in alum seems unable to produce an immune response.

It should be noted that the lipoprotein and non-lipid protein versions of OspA are identical in the deduced amino acid sequence except for the amino terminal amino acid residue which is cysteine modified for lipoprotein and methionine-alanine for the non-lipid protein, which suggests strongly that the difference observed in immunogenicity is due to the presence of the lipid portion.

Example 8

The inherent antigenicity of lipid and non-lipid OspA was also investigated to determine whether the difference in immunogenicity seen in example 7 was due to differences in its inherent antigenicity or differences in how the immune system recognizes and responds to the two proteins.

Sera from mice immunized with lipoprotein gave a positive signal in an ELISA using both versions of OspA as the test antigen, as shown in Figure 16. Similarly, sera from mice immunized with non-lipid protein failed to react with both antigens. Consequently, while both versions of OspA are equally antigenic, lipoprotein is well recognized by the immune system as

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-25 that non-lipid protein is not.

In addition, these results strongly suggest that, although the lipid group is essential for immunogenicity, the antibodies formed against the lipoprotein are directed mainly against the peptide portion of the molecule, and not against the lipid portion.

Example 9

The effect of the strain origin for the OspA protein on immunogenicity was observed.

Mice were vaccinated with purified OspA lipoproteins from B31, ACA1 and Ip90 derived from pOAl, pOA9 and pOAlO, respectively. The mice were vaccinated with 2.5 µg of protein at week 0, boosted with the same dose at week 3, and bled at week 4. Serum IgG titers were determined at week 4 by an ELISA assay using B31, ACA1 or Ip90 purified OspA lipoprotein as antigen. The titer was determined using the secondary goat anti-mouse IgG antibody.

The results obtained in these experiments are shown in figure 15D. As can be seen from these data, the recombinant OspA for all strains tested is immunogenic in mice and the polyclonal sera show partial cross-reactivity.

DESCRIPTION SUMMARY

As a summary of this description, the present invention provides a new and simple procedure for producing a highly purified and immunologically effective recombinant protein encoded by a wild-type Borrelia full-length lipoprotein gene, particularly the B. burgdorferi ospA gene, using a detergent to selectively extract the protein from the host strain and subsequently purifying the detergent solution by

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-26 Modifications are possible within column chromatography, within the scope of this invention.

LITERATURE REFERENCES

1. Fikrig, E., S.W. Barthold, F.S.Kantor and R.A. Flavell (1990). Protection of Mice Against the Lyme Disease Agent by Immunizing with Recombinant OspA. Science, 250: 553-556.

2. Simon, M.M., U.E. Schaible, M.D. Kramer, C. Eckerskorn, C. Museteanu, H.K. Muller-Hermelink, and R. Wallich (1991). Recombinant Outer Surface Protein A from Borreiia burqdorferi Induces Antibodies Protective Against Spirochetal Infection in Mice. J. Infect. Pis., 164: 123-132.

3. Dunn, J.J., B.N. Lade, and A.G. Barbour (1990). Outer Surface Protein A (OspA) from the Lyme Disease Spirochete, Borrelia burqdorferi: High Levei Expression and Purification of Soluble Recombinant Form of OspA. Protein Expression and Purification 1: 156-168.

4. Bessler, W.G., B. Suhr, H.-J. Buhring, C.P. Muller, K.-H. Wiesmuller, G. Becker, and C. Jung (1985). Specific Antibodies Elicited by Antigen Covalently Linked to a Synthetic Adjuvant. Immunobioloqy 170: 239-244.

5. Biesert, L., W. Scheuer, and Bessler, W.G. (1987). Interaction of Mitogenic Bacterial Lipoprotein and a Synthetic Analogue with Mouse Lymphocytes. Eur. J. Biochem .. 162: 651-657.

6. Deres, K., H. Schild, K.H. Wiesmuller, G. Jung, and H.G. Rammensee (1989). In vivo Priming of Virus-Specific Cytotoxic T Lymphocytes with Synthetic Lipopeptide Vaccine. Nature 342: 561-564.

7. Brandt, M.E., B.S. Riley, J.D. Radolf, and M.V. Norgard (1990). Immunogenic Integral Membrane Proteins of Borrelia burqdorferi Are Lipoproteins. Infection and Immunitv 58:

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983-991.

8. Fikrig, E., S.W. Barthold, F.S.Kantor, and R.A. Flavell (1992). Long-term protection of mice from Lyme disease by vaccination with OspA ”. Infect. and Immun. 60: 773-777.

9. Fikrig, E., S.W. Barthold, F.S.Kantor, and R.A. Flavell (1991). Protection of mice from Lyme borreliosis by oral vaccination with Escherichia coli expressing OspA. J. Infect. Pis. 164: 1224-1227.

10. Johnsson, Μ., L. Noppa, A.G. Barbour, and S. Bergstram (1992). Heteroantigenicity of Outer Membrane Proteins in Borrelia burgdorferi: Comparison of osp operons of three isolates of different geographic origins. Infect. Immun. 60: 1845-1853.

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28IDENTIFICATION OF SEQUENCES

1 1 1 | SEQ ID NO. | DESCRIPTION OF THE SEQUENCE | 1 1 L I FIGURE | 1 1 1 1 1 1 1 Γ | Coding string for the N- region | osdA terminal of strain B31, ACA1 and | | Ip90 | 1 1 1/ 5 | 1 1 1 1 2 1 I 1 1 [Coding string for region C- | [stromae B31 osdA terminal | I 1 1, 1 5 1 1 I 1 1 3 1 1 | PET-IN [CO1] primer oligonucleotide | 1 1 1 1 1 | 1 1 ⁴ 1 1 1 1 PET-273C primer oligonucleotide | | [C03], | I I 1/ 1 5 1 1 I 1 1 ⁵ 1 I 1 1 [Coding string for region C- | [osdA terminal of the stretch ACA1 | 1 1 1, 1 ⁵ 1 1 I 1 1 θ 1 I 1 1 [Coding string for region C- | osdA terminal of estiroe Io90 | I 1 1/ 1 5 | 1 I 1 1 7 | 1 1 [OspN2 primer oligonucleotide | 1 I 1 1 1 I 1 1 θ | 1 1 [BZ1 primer oligonucleotide | I I I, 1 5 1 1 1 9 1 1 | PK4 primer oligonucleotide | I I 1/ 1 5 1 1 | ίο I 1 1 [PK3 primer oligonucleotide | I I 5 1 1 I 1 1 11 1 1 [PET-18N primer oligonucleotide | —— 1 1

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SEQUENCE LISTING (1) INFORMATION FOR SEQ. ID NO. 1:

(i) SEQUENCE FEATURES:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 1:

ATATATTATG AAAAAATATT TATTGGG (2) INFORMATION FOR SEQ. ID NO. 2:

(i) SEQUENCE FEATURES:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 2:

GAGGAATAAA ATTTCGCAAA AATTAAA (3) INFORMATION FOR SEQ. ID NO. 3:

(i) SEQUENCE FEATURES:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

ΊΑ 487

MIS: jc 6102-98 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 3:

-30GCACATATGA AAAAATATTT ATTGGG (4) INFORMATION FOR SEQ. ID NO. 4:

(i) SEQUENCE FEATURES:

(A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 4:

CGGGGATCCC TCCTTATTTT AAAGCG (5) INFORMATION FOR SEQ. ID NO. 5:

(i) SEQUENCE FEATURES:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 5:

GAGGAATAAA TAAAGTTTCG ÇAAAAATTCA A (6) INFORMATION FOR SEQ. ID NO. 6:

(i) SEQUENCE FEATURES:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double

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MIS: jc 6102-98 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO. 6:

GAGGGATAAA ATTTCGTAGA AATTCAA (7) INFORMATION FOR SEQ. ID NO. 7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 7:

GGCCGCACAT ATGAAAAAAT ATTTATTGGG (8) INFORMATION FOR SEQ. ID NO. 8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 8 :.

CGGGGATCCC CTTATTTATT TCAAAGCG (9) INFORMATION FOR SEQ. ID NO. 9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 base pairs

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(B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID NO. 9:

CGGGGATCCC TATTTTAAAG CATC (10) INFORMATION FOR SEQ. ID NO. 10:

(i) SEQUENCE FEATURES:

(A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (Xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 10:

CGGCCATGGA AAAATATTTA TTGGG (11) INFORMATION FOR SEQ. ID NO. 11:

(i) SEQUENCE FEATURES:

(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID AT THE. 11:

CAGCATATGG CTAAGÇAAAA TGTTAGC

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Claims (21)

1 - Immunologically effective pure protein, characterized by being encoded by a full-length, wild-type Borreia lipoprotein gene and by being formed recombinantly from a host organism transformed by a plasmid containing the gene. 2 - Protein according to claim 1, characterized by being encoded by the wild-type, full-length, B. burqdorferi ospA gene. Protein according to claim 2, characterized in that said plasmid is plasmid pOAl, pOA5, pOA6, pOA7, pOA8, pOA9 or pOAlO. Protein according to claim 2 or 3, characterized in that said ospA gene is the isolate of the B31 strain family, Ip90 strain family or B. burqdorferi ACA1 strain family. Protein according to any one of claims 1 to 4, characterized in that said host organism is E. coli. Protein according to any one of claims 1 to 5, characterized in that said plasmid comprises the plasmid vector pET9 in which the gene is placed under the control of a T7 promoter and effective translation initiation signals from the T7 bacteriophage. Protein according to claim 6, characterized in that said host organism is an E. coli T7 expression strain. Protein according to claim 7, characterized in that said host organism is E. coli strain BL21 (DE3) (pLysS) or HMS174 (DE3) (pLysS). 74 487 MIS: jc 6102-98 Protein according to any one of claims 1 to 6, characterized in that said plasmid comprises the plasmid vector pCMBl or pCMB2 in which the gene is placed under the control of a TRC promoter. Protein according to any one of claims 1 to 9, characterized in that it is substantially free of bacterial protein and lipopolysaccharide. 11 - Process of producing a protein encoded by a wild-type Borrelia lipoprotein gene, of full length, characterized by comprising the steps of: (a) inducing Borrelia's lipoprotein from a host organism transformed by a plasmid containing said gene, (b) lysing the cells of said host organism, (c) treating the lysed cells with a surfactant that selectively solubilizes lipoprotein of Borrelia. preferably to bacterial and other types of proteins and which is capable of effecting the phase separation of a detergent phase under mild conditions, (d) effecting the phase separation in a detergent phase containing solubilized Borrelia lipoprotein, in an aqueous phase containing bacterial proteins and other types and in a solid phase containing cellular residues, (e) recovering said detergent phase from said solid phase and said aqueous phase, and (f) purifying said detergent phase so that it is free of proteins other than Borrelia's lipoprotein. Process according to claim 11, characterized in that the protein is an OspA protein encoded by the B. burqdorferi ospA gene. Process according to claim 12, characterized in that the plasmid is plasmid pOAl, pOA5, pOA6, pOA7, pOA8, pOA9 OR pOAlO. 74 487 MIS: jc 6102-98 Process according to any one of claims 11 to 13, characterized in that said surfactant is Triton X-114. Process according to any one of claims 11 to 14, characterized in that said treatment of said lysed cells is carried out at a temperature of about 0 ° to about 10 ° C, the resulting mixture is heated to a moderately high temperature from about 35 ° to about 40 ° C to effect the separation of said detergent phase, and said detergent phase is separated from said aqueous phase and said solid phase, by centrifugation. Process according to any one of claims 11 to 15, characterized in that said purification of said detergent phase is carried out by contacting said detergent phase with a chromatographic column, under conditions that result in the binding of proteins other than Borrelia's lipoprotein to the said column and recovering the passage fluid from said column containing Borrelia's lipoprotein. Process according to claim 16, characterized in that said column is further contacted with a buffer medium that displaces liquid containing Borrelia lipoprotein from said column, while the other proteins are retained by said column, and the fluid passing through said additional contact. Process according to any one of claims 11 to 17, characterized in that said lysis of the host organism is carried out by freezing and thawing the host organism. Process according to any one of claims 11 to 18, characterized in that said host organism is an E. coli strain that produces bacteriophage T7 RNA capable of directing high-level transcription from a T7 promoter and the said plasmid vector contains a Kan gene, such as 74 487 MIS: jc 6102-98 is its selective marker, and a T7 promoter. Borrelia infection vaccine, characterized in that it comprises an immunologically effective amount of a protein according to any one of claims 1 to 10. 21 - Immunodiagnostic agent for the detection of antibodies against infection of a host by a Borrelia organism, characterized in that it comprises a protein according to any one of claims 1 to 10.