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WO1994019697A1 - Procedes induisant une immunite a l'arthrite de lyme et titrage colorimetrique concernant l'activite borreliacide d'antiserums - Google Patents

Procedes induisant une immunite a l'arthrite de lyme et titrage colorimetrique concernant l'activite borreliacide d'antiserums Download PDF

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Publication number
WO1994019697A1
WO1994019697A1 PCT/US1994/002095 US9402095W WO9419697A1 WO 1994019697 A1 WO1994019697 A1 WO 1994019697A1 US 9402095 W US9402095 W US 9402095W WO 9419697 A1 WO9419697 A1 WO 9419697A1
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Prior art keywords
ospa
ospb
vaccine
burgdorferi
antisera
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PCT/US1994/002095
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English (en)
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Richard T. Coughlin
Jianneng Ma
Dante J. Marciani
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Cambridge Biotech Corporation
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Priority to EP94910185A priority Critical patent/EP0686265A4/fr
Priority to AU62728/94A priority patent/AU689466B2/en
Publication of WO1994019697A1 publication Critical patent/WO1994019697A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0225—Spirochetes, e.g. Treponema, Leptospira, Borrelia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/20—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1207—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention is in the field of medicinal chemistry.
  • the invention is related to vaccines for Borrelia burgdorferi comprising a saponin, and the use thereof to immunize animals.
  • Lyme disease is a tick-borne multisystemic disorder characterized by early erythema chronicum migrans, late arthritis, and cardiac and neurologic manifestations in humans and animals (Steere, A.C., N. Engl. J. Med.
  • Lyme disease is the most common tick-borne zoonosis occurring in humans and dogs (Steere, A.C., N. Engl. J. Med. 527:586-96
  • Lyme disease spirochetes have been recognized based on genetic and molecular determinants (Baranton et al, Int. J. Sys. Bacteriol.
  • Humoral immunity is a major protective mechanism against this bacterial infection or disease (Burgdorfer, W., et al. , Science 267:1317-1319
  • Convalescent human sera are able to kill the spirochete in the presence of complement in vitro, possibly by altering the bacterial outer membrane to allow the formation of an effective membrane attack complex (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Kochi, S.K., et al. , J. Immunol. 146:3964-3910 (1991)).
  • Heat-inactivated rat antisera to B. burgdorferi or mouse monoclonal antibody to a surface epitope of the spirochete in the absence of complement are also capable of lysing this spirochete (Pavia, C.S., et al , J. Infect. Dis. 163:656-659 (1991); Coleman and Benach, "Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria, " J. Infect. Dis. 765:658-666 (1992)).
  • in vitro borreliacidal activity of hamster immune sera to B. burgdorferi with complement is reported to correlate with in vivo protection (Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991)).
  • OspA and OspB based vaccine would stimulate protective immune responses in Lyme disease hosts needed to be addressed.
  • Borreliacidal activity of immune sera is usually determined by an in vitro assay adapted from an antileptospiral assay (Kochi and Johnson, Infect. Immun. 56:314-321 (1988)). Because B. burgdorferi does not reliably form isolated colonies on currently available agar medium, spirochete killing by the bactericidal antibody is determined by dark-field microscopy on the basis of loss of motility , refractility , or extensive surface blebbing (Kochi and Johnson,
  • the present invention is directed to a new colorimetric borreliacidal assay (CBA) for determination of the bactericidal activity of antiserum to B. burgdorferi, which is simple, reliable, and exhibits an easily measurable end-point.
  • CBA colorimetric borreliacidal assay
  • the results of the CBA correlated well with those of both direct dark-field microscopy and [ 3 H]-thymidine incorporation assay (TIA).
  • TIA [ 3 H]-thymidine incorporation assay
  • the invention relates to a method for the determination of the bactericidal activity of an antiserum to B. burgdorferi, which comprises
  • Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438.
  • the invention also relates to a vaccine, comprising OspA, OspB or fragments thereof; and a saponin adjuvant.
  • the invention relates to a vaccine, comprising full- length lipoproteins OspA and/or OspB; and a saponin adjuvant such as QS-21.
  • the invention also relates to a method of inducing immunity to bacteria causing lyme disease, e.g., B. burgdorferi and B. garinii, in an animal, comprising administering to the animal a vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant. More specifically, the invention relates to a method of inducing immunity to B. burgdorferi and other species of the genus Borrelia in an animal, comprising administering to the animal a vaccine comprising full- length lipoproteins OspA and/or OspB; and a saponin adjuvant such as QS-21.
  • An advantage of the present invention is that the immunogenicity of OspA- and OspB-based subunit vaccine is greatly enhanced by using the full- length lipoproteins OspA and OspB.
  • a further advantage of the present invention is that the immunogenicity of Osp subunit vaccine is further enhanced by the adjuvant QS-21.
  • Another advantage of the present invention is that a preferred vaccine formulation comprising OspA, OspB, and QS-21 displays borrelicidal activity against not only the homologous and closely related strains, but also against the heterologous and different genospecies of lyme disease spirochetes as well as other species of the genus Borrelia.
  • the OspA- and OspB-based vaccine of the present invention elicits synergistically higher functional humoral immune response than a single protein-based vaccine.
  • Figure 1 depicts the colorimetric borreliacidal assay of mouse antisera to B. burgdorferi strain B31 against the homologous strain. Borrelial culture at logarithmic growth phase were centrifuged for 8 min. at 9000 x g at 15°C and resuspended in fresh mBSK. Ninety five ⁇ l of the borrial suspension
  • Figure 2 depicts a graph showing the correlation of the colorimetric assay with the pHlthymidine incorporation assay.
  • B. burgdorferi strain B31 containing about 8 x 10 6 spirochetes
  • mBSK-PR for 30 h at 32°C.
  • Twenty ⁇ l of pH]thymidine in mBSK (2 ⁇ Ci) were added to each well and the plates were incubated for another 18 h for pulse-labelling the live spirochete (Pavia, C.S. et al, J. Infect. Dis 163:656- 659 (1991)).
  • FIG. 3 depicts a graph showing the changes of absorbance at 562/630 nm of the colorimetric borreliacidal assay with mouse antisera to OspA-B31 against the homologous strain. The assay was done as described in the description of Figure 1. The plate was incubated at 32°C for 120 h, and the absorbance measured using a microtiter reader before and every 24 h after incubation. Each serum dilution was performed in triplicate. The error bars represent the standard deviation of 3 measurements.
  • Figure 4 depicts a graph showing the significant inhibition of the decrease in absorbance at 562/630 nm of the colorimetric borreliacidal assay by mouse antisera to B. burgdorferi stain B31.
  • the serially diluted, heat- inactivated antisera or normal sera were incubated with the strain B31 in the presence of complement as described above in the description of Figure 1.
  • Each serum dilution was performed in triplicate.
  • the absorbance was measured after 48 h incubation. High absorbance indicates borrelial death; the low absorbance represents borrelial survival and growth.
  • the error bars represent the standard deviation of 3 measurements.
  • Figure 5 depicts a graph showing the average borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain.
  • Figure 6 depicts a graph showing the average borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87.
  • Figure 7 depicts a graph showing the borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain.
  • Figure 8 depicts a graph showing the borreliacidal activity of canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87.
  • Figure 9 depicts a graph showing the borreliacidal activity of C3H/Hej female mice that had been immunized twice with either 25 ⁇ g of truncated OspA, 25 ⁇ g of truncated OspB, or 25 ⁇ g of truncated OspA and 25 ⁇ g of truncated OspB.
  • Figure 10 depicts a graph showing the borreliacidal activity of
  • Figure 11 depicts a fluorograph of nitrocellulose membrane showing radiolabeling of FLOspA and FLOspB by [9,10- 3 H]palmitic acid.
  • Escherichia coli strain MZ-1 harboring ospA or ospB gene was grown in LB broth to log phase at 32 °C.
  • [9, 10- 3 H]palmitic acid was added to the culture and incubated for 2 h at 42 °C.
  • Bacteria were lysed, and OspA and OspB precipitated using specific MAbs described in the specification, by modification of the procedure (Katona et al, Infect. Immun. 60:4995-5003 (1992)).
  • Figure 12 depicts antibody isotype titers of canine antisera to various experimental vaccines. Beagles at age of 12 and 16 weeks were immunized subcutaneously twice with various experimental vaccines. Immune sera were isolated two weeks after last immunization, and assayed by ELISA using plates coated with B. burgdorferi antigens (Cambridge Biotech Corporation, Worcester, MA; Lindenmayer et al, J. Clin. Micwbiol 28:92-6 (1990)). Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX).
  • FIG. 13 depicts representative patterns of immunoblot with naturally exposed dog sera and antisera to experimental vaccines. Naturally exposed dog sera were isolated in New York area. Antisera to experimental vaccine containing 25 ⁇ g each FLOspA and FLOspB and 50 ⁇ g QS-21 were prepared as described above with respect to Fig. 12. Immunoblot was performed with antigen strips according to the instruction of manufacturer (Cambridge Biotech
  • Antisera to experimental vaccine containing 25 ⁇ g each of FLOspA and FLOspB and 50 ⁇ g of QS-21 were described above with respect to Fig. 12.
  • ELISA was done using plates coated with 0.2 ⁇ g OspA and 0.2 ⁇ g OspB, respectively.
  • Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX). Titer was defined as described above with respect to Fig. 12.
  • Figure 15 depicts antiborrelial activity against B.
  • Figure 16 depicts antiborrelial activity against different geographic Lyme disease spirochetes of B. burgdorferi sensu stricto and B. garinii sp. nov. of canine antisera to experimental vaccine formulated with 25 ⁇ g each of FLOspA and/or FLOspB and with or without QS-21. Antisera preparations are described above with respect to Fig. 12. Antiborrelial activity of antisera was tested as described in Table 1. Error bars represent the standard deviation of measurements of 32, 10, 10, 10, and 3 serum samples, respectively (from top to bottom of legend). High absorbance indicates high antiborrelial activity.
  • F ⁇ gure 17 depicts correlation of antiborrelial activity of canine antisera with isotypes IgGl and IgG2 antibody titer.
  • Antisera to experimental vaccine containing 25 ⁇ g each of FLOspA and FLOspB and 50 ⁇ g QS-21 are described above with respect to Fig. 12.
  • Antiborrelial activity and IgG antibody titers were determined as described in Table 1 and Fig. 12, respectively.
  • Figure 18 depicts expression of recombinant OspA and OspB in E. coli strain MZ-1.
  • Bacteria containing recombinant plasmids encoding OspA or OspB were grown to logarithmic phase at 32 °C, and then incubated at 42 °C for 1 h for induction of recombinant protein synthesis.
  • Whole cell lysates of bacteria before and after induction were analyzed by SDS-PAGE, and visualized by Coomassie blue staining. 1 , before induction of MZ-1 harboring pLCBCl (Beltz, G. A. et al , U.S. Patent No.
  • Figures 19 A, 19B and 19C depict SDS-PAGE and immunoblotting of whole cell lysates of B. burgdorferi.
  • Four to 6 ⁇ g of lysates of the bacteria were analyzed for protein composition on the 11 % SDS-PAGE gel, and visualized by Coomassie blue staining (Fig. 19A).
  • the antigenic properties of OspA and OspB were tested by transferring the separated proteins to the membrane and probing with 1:20 dilution of mouse antisera to OspA (Fig. 19B) or OspB (Fig. 19C) of strain B31. 1, strain B31; 2, strain CA-2- 87; 3, strain Fr; 4, strain G25. ⁇ indicates an approximately 22 KDa protein band in Fig. 19C.
  • Molecular weight markers are in KDa.
  • Figure 20 depicts antibody isotype titers of mouse antisera to OspA and OspB formulated with or without either QS-21 or alum.
  • Serially diluted mouse antisera were assayed by ELISA as described (Lindenmayer J. et al. , J. Clin. Microbiol 5:92-96 (1990)).
  • the isotype-specific goat anti-mouse IgG conjugated to horseradish peroxidase was used to measure antibody isotype.
  • the bars represent group means.
  • Figure 21 depicts OspA and OspB protein coding regions within B. burgdorferi.
  • Figure 22 depicts the basic features of the expression vector used to express B. burgdorferi recombinant OspA and OspB antigens.
  • Figure 23 depicts the entire DNA sequence of pLCBC10spA8+6 and the origin of each base.
  • the sequence information was obtained from the following publicly available sources: 1. pBR322 from Genebank Accession # J01749
  • Figure 24 depicts the entire DNA sequence of pLCBC10spB8+4 and shows the origin of each base.
  • the sequence information was obtained from the following publicly available sources:
  • Figure 25 depicts a restriction fragment profile of the OspA clone pLCBC10spA8+6. From left to right, each lane contains either a control marker or pLCBC10spA8+6 incubated with the indicated restriction enzyme(s). Lane 1: markers ⁇ Hindlll + XHaelll; lane 2: uncut; lane 3: BamHI; lane 4: PvuII; lane 5: EcoRI; lane 6: Hindlll; lane 7: Ndel; lane 8:
  • Figure 26 depicts a restriction fragment profile of the OspB clone pLCBC10spB8+4. From left to right each lane contains either a control marker or pLCBC10spB8+4 incubated with the indicated restriction enzyme(s).
  • Lane 1 markers ⁇ Hindlll + ⁇ XHaelll; lane 2: uncut; lane 3: BamHI; lane 4: EcoRI; lane 5: Ndel; lane 6: Pstl; lane 7: PvuII; lane 8: BamHI + EcoRI; lane 9: EcoRI + Hindlll; lane 10: EcoRI + Pstl; lane 11: Hindlll + Sspl; lane 12: PvuII + Sspl; lane 13: markers ⁇ Hindlll + XHaelll.
  • the present invention relates to a method for the determination of the bactericidal activity of an antiserum to B. burgdorferi, which comprises
  • Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438.
  • Radioactive incorporation assays and spirochete culture are used to verify the results (Kochi and Johnson, Infect. Immun. 56:314-321 (1988); Pavia, C.S. , et al , J. Infect. Dis. 163:656-659 (1991); Lovrich, S.D., et al , Infect. Immun. 59:2522-2528 (1991); Callister, S.M. , et al. , J. Clin. Microbiol 29: 1773-1776 (1991)). It is apparent that the existing bactericidal assays lack simplicity, reliability, and cannot handle a large number of serum samples.
  • the present microtiter CBA uses phenol red as an indicator of the accumulation of nonvolatile acid generated by spirochete metabolism.
  • the CBA detects borreliacidal activity of immune sera by measuring the absorbance at 562/630 nm. Significant decrease in absorbance represents the bacterial survival and growth; a small decrease in absorbance indicates borrelial killing.
  • phenol red final concentration 60 ⁇ g/ml
  • the dual wavelengths of 562/630 nm most sensitively reflected the absorbance changes in the presence of acid.
  • both the TIA and colorimetric assay using serially diluted spirochetes or using a concentration of 4 x 10 6 spirochetes and serially diluted antisera to lysates of strain B31 were performed. Correlation coefficients of these two assays were 0.977 and 0.935, respectively, and were linearly related over the useful range of most microplate readers.
  • the mouse antisera to lysates of strain B31 had a high borreliacidal activity against the homologous strain B31 (Fig. 4), strains Cr (a Wisconsin tick isolate), and Fr (a German tick isolate), but had little borreliacidal activity against the Swedish strain G25, a different genomic species in terms of rRNA gene restriction patterns (Postic, D. et al, Res. Micwbiol 141:465-415 (1990)).
  • the antisera to strain G25 possessed a high borreliacidal activity against the homologous strain G25, but did not against other strains tested. Normal mouse sera, compared with mBSK control, were not borreliacidal (Fig. 4).
  • burgdorferi or monoclonal antibody to a surface epitope of the spirochete in the absence of complement source are also borreliacidal (Pavia, C.S., et al. , J. Infect. Dis. 163:656-659 (1991); Coleman and Benach, "Characterization of antigenic determinants of Borrelia burgdorferi shared by other bacteria," J. Infect. Dis. 765:658-666 (1992)).
  • complement components are required for the destruction of the Gram-negative bacteria, including B. burgdorferi, by bactericidal antibody.
  • the borreliacidal titer was defined as the highest dilution of sera which inhibits absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes in the CBA.
  • the microtiter CBA for determination of borreliacidal activity of immune serum has been developed with the advantages of simplicity, reliability, and safety. Furthermore, the CBA can handle a large number of serum samples and is valuable in Lyme vaccine development and as a diagnostic method for Lyme borreliosis.
  • the invention also relates to a method for the detection of Lyme borreliosis, comprising
  • Color pH indicators are known in the art and may be readily obtained from the various suppliers. For example, various color pH indicators are described in the 1993 catalogue of Sigma Chemical Co., St. Louis, Missouri, at page 1438.
  • the invention also relates to a vaccine, comprising OspA, OspB or fragments thereof; and a saponin adjuvant.
  • This adjuvant significantly enhances both humoral and cellular immune responses to a variety of antigens and has been used in recombinant subunit vaccines including the experimental HIV-1 gpl60 protein vaccine and commercially available feline leukemia virus vaccine (Marciani D. J. et al , Vaccine 9:89-96 (1991); Newman, M. J. et al , J. Immunol 148:2351-2362 (1992); Wu, J.-Y. et al , J. Immunol. 745:1519-1525 (1992)).
  • QS-21 and aluminum hydroxide (alum) on the functional antibody responses to the recombinant OspA and OspB derived from B.
  • burgdorferi strain B31 was determined in mice. Only QS-21 induced high titers of IgG2a and IgG2b antibodies, the complement fixing isotypes (Kochi, S. K. et al , J. Immunol 146:3964-3910 (1991); Schmitz, J. L. et al , Infect. Immun. 60:2677-2682 (1992); Spiegelberg, H. L., Adv. Immunol 79:259-294 (1974)). QS-21 was shown to be superior to alum in enhancing functional antibody response to OspA and OspB. The experimental vaccine containing OspA and OspB formulated with QS-21 conferred complete protection against infection with either the homologous or the heterologous strains of B. burgdorferi in mice.
  • a saponin adjuvant, QS-21 significantly enhances immunogenicity of OspA and OspB.
  • aluminum hydroxide a widely used adjuvant for human vaccines, does not significantly influence OspA or OspB based experimental Lyme vaccines.
  • the impact of the adjuvants QS-21 and aluminum hydroxide (alum) on the immunogenicity of recombinant outer surface protein A (OspA) and B (OspB) of Borrelia burgdorferi was investigated. Both nonacylated OspA and OspB derived from strain B31 were expressed in Escherichia coli and purified by reversible citraconylation and anion exchange chromatography .
  • Antisera to OspA or OspB were prepared in mice with antigens formulated with QS-21 or alum, and evaluated for specific immunoglobulin G isotypes, agglutination, and borreliacidal activity.
  • QS-21 significantly enhanced IgG2a and IgG2b antibody responses to OspA and OspB, and IgGl response to OspA when compared with the formulation containing antigen alone.
  • alum significantly inhibited the induction of IgG2a and IgG2b responses to OspA.
  • Alum had no significant effect on IgGl response to OspA, or IgG2a and IgG2b responses to OspB, but significantly enhanced IgGl antibody response to OspB.
  • Antisera to OspA or OspB formulated with QS-21 possessed higher titers of agglutinating antibody than antisera to OspA or
  • OspA was superior to OspB and QS-21 superior to alum at eliciting functional antibody responses.
  • the vaccine containing OspA and OspB formulated with QS-21 was protective in mice against infection with 10 s infectious spirochetes of strains B31 or CA-2-87.
  • mice immunized with Osp vaccine formulated without adjuvant raised significantly higher titers of antibody to OspA than to OspB, indicating OspA was more immunogenic than OspB.
  • mice immunized with OspA or OspB formulated with QS-21 raised significantly higher antibody responses than those immunized with the antigens alone or antigens formulated with alum.
  • These antisera also possessed higher agglutination antibody titers and borreliacidal activity. It appeared that the borreliacidal activity of the antisera was correlated with the agglutination antibody titer and ELISA antibody titer with the exception of the low agglutination titer with strain G25.
  • Antisera to OspA formulated with QS-21 had a higher titer of agglutinating antibody than antisera to OspB formulated with QS-21. Those antisera reacted with not only strain B31, but also the heterologous California isolate CA-2-87, German isolate Fr, and Swedish isolate G25. Similarly, antisera to OspA formulated with QS-21 had significantly higher borreliacidal activity against the 4 strains than antisera to OspB formulated with QS-21. These results showed that OspA induced higher functional antibody responses than OspB.
  • antisera to OspB formulated with QS-21 also recognized an approximately 22 KDa protein band of strains B31, CA-2-87, and Fr, but not strain G25.
  • This OspB related lower molecular mass protein may be similar to a 21 KDa protein that Bundoc and Barbour have previously observed (Bundoc & Barbour, Infect. Immun 57:2733-2741 (1989)).
  • a premature stop codon in the ospB gene that terminates OspB 58 amino acids short of the full length protein has been observed by Rosa et al. (Rosa, P. A. et al , Mol. Microbiol 3:131-3040 (1992)). They indicate that this lower molecular mass protein may be a smaller OspB fragment.
  • strains B31 and G25 were designated as OspA serotypes 1 and 6, respectively, based on an OspA serotyping system (Wilske, B. et al. , J. Clin. Microbiol 57:340-350 (1993)).
  • strains B31 and G25 are included in B. burgdorferi sensu stricto and B. garinii sp. nov. , respectively (Milch, L. et al , J. Infect. Dis. 760:351-353 (1989)).
  • QS-21 significantly increased the agglutination antibody and borreliacidal activity against borrelial isolates with different OspA serotypes or genospecies. This functional increase in antisera may have resulted from a broadening of antibody response to outer surface exposed epitopes on OspA and OspB.
  • the major antibody isotypes enhanced by QS-21 in mice were IgG2a and IgG2b.
  • Alum increased only the IgGl antibody response, consistent with a previous report (Byars, N. E. et al , Vaccine 9:309-318 (1991)).
  • This characteristic of QS-21 in enhancing IgG isotypes is important because the isotypes of IgG antibody differ in immunoprotective efficacy for many infectious diseases (Spiegelberg, H. L., Adv. Immunol. 79:259-294 (1974); Briles, D. E. et al , J. Mol. Cell. Immunol. 7:305-309 (1984); Coutelier, J. P. et al. , J.
  • QS-21 also significantly induced OspA and OspB specific antibody IgGl and IgG2 responses in dogs and the borreliacidal activity appeared to be associated with IgG2.
  • QS-21 serves as an important component in a vaccine against Lyme disease.
  • OspA or OspB formulated with Freund's adjuvant (Fikrig, E. et al. , Science 250:553-556 (1990); Fikrig, E. et al , Infect. Immun. 60:651-661 (1992); Fikrig, E. et al , Proc. Natl. Acad. Sci. USA 59:5418-5421 (1992)) or OspA formulated with alum (Erdile, L. F. et al , Infect. Immun. 67:81-90 (1993)) have been shown to confer protection against experimental challenge in mice. The present study indicated that QS-21 was more potent than alum in enhancing the immunogenicity of OspA and OspB.
  • Antibody to OspA formulated with QS-21 was borreliacidal to all the strains tested, and antibody to OspB formulated with QS-21 borreliacidal to strains B31 and Fr. Some borrelial isolates do not produce OspA or OspB. Thus, the vaccine containing both OspA and OspB may provide better and broader protection. Our in vivo study showed that the vaccine containing OspA, OspB, and QS-21 was capable of conferring protection against the infection of the high doses of die homologous and heterologous spirochetes.
  • QS-21 has been used in a commercially available feline leukemia virus vaccine (Marciani, D. J. et al , Vaccine 9:89-96 (1991)) and in an experimental HIV-1 vaccine in rhesus macaques (Livingston, P. O., Annals New York Acad. Sci. 690:204-213 (1993)).
  • This adjuvant has been shown to significantly enhance both antibody and cell-mediated immune responses and has little or no toxicity in laboratory animals (Kensil, C. R. et al , J. Immunol 146:431-431 (1990); Marciani D. J. et al. , Vaccine 9:89-96 (1991);
  • OspA and OspB of B. burgdorferi possessed borreliacidal epitopes, and the adjuvant QS-21 was more efficient than alum in inducing antibody responses to OspA and OspB.
  • QS-21 significantly enhanced IgG2a, IgG2b, and functional antibody responses, and as shown below, it is an important component of a Lyme disease vaccine.
  • the experimental vaccine containing OspA and OspB formulated with QS-21 was shown to be highly protective in mice and as shown below, was found to be highly effective in the prevention of Lyme disease in dogs. TABLE 1. Agglutination with divergent borrelial strains of mouse antisera to OspA and OspB formulated with or without QS-21
  • OspB 200 100 ⁇ 50 100
  • Antisera induced by either OspA or OspB based vaccine had high titers of antibody and antiborrelial activity against B. burgdorferi sensu stricto strains B31 and CA-2-87 as those elicited by OspA and OspB based vaccine. However, only the latter were also antiborrelial to heterologous borrelial strain 24008 Fr and B. garinii sp. nov. strain G25, both European isolates.
  • Antisera to vaccine formulated with lipoproteins OspA and OspB possessed significantly (p ⁇ 0.005) higher antiborrelial activity than those to vaccine formulated with nonlipidated OspA and OspB. Among 63 naturally exposed B.
  • immunogenicity of OspA and OspB based subunit vaccine can be greatly enhanced using lipoproteins OspA and OspB; (2) immunogenicity of Osp subunit vaccine can be further enhanced by the adjuvant QS-21; (3) OspA and OspB based vaccine was more potent than a single protein based vaccine in eliciting functional humoral immune response in dogs.
  • lipidated OspA and OspB were significantly (p ⁇ 0.005) more immunogenic than nonlipidated OspA and OspB, and induced significantly (p ⁇ 0.005) higher functional antibody response in dogs.
  • QS-21 is superior to aluminum hydroxide in enhancing humoral immune response to truncated nonlipidated OspA and OspB in mice.
  • Aluminum hydroxide is incapable of enhancing antibody response to FLOspA (Erdile et al, Infect. Immun. 67:81- 90 (1993)).
  • the present data clearly demonstrated that QS-21 was also capable of significantly enhancing humoral immune response to FLOspA and FLOspB in dogs.
  • FLOspA and FLOspB based vaccine formulated with QS-21 was much more potent than either FLOspA or FLOspB based vaccine in inducing functional immune response.
  • OspA or OspB based
  • QS-21 formulated vaccine was capable of inducing an antibody response which was antiborrelial against an homologous B. burgdorferi sensu stricto strain B31 and a heterologous strain CA-2-87 (a California isolate), but not against two European isolates strain 24008 Fr (a French isolate) and B. garinii sp. nov. strain G25 (a Swedish isolate).
  • OspA and OspB based, QS-21 formulated vaccine elicited antibodies which were also antiborrelial to these two European isolates.
  • Cross antiborrelial activity against different genospecies and OspA serogroups of spirochetes of the antisera elicited by the experimental vaccine showed promise for developing an effective vaccine against Lyme disease for different geographic regions.
  • the high functional activity of antisera elicited by QS-21 formulated vaccine may have resulted from enhanced antibody titer and broadening antibody responses to functional epitopes on OspA and OspB of the spirochete.
  • the serum samples of the present invention were isolated from Westchester and Long Island, New York, a highly endemic region for Lyme disease (Alpert et al, NY State J. Med. 92:5- 8 (1992)). Repeat infection of dogs by Lyme disease spirochetes in this area may explain the different observations. Although relatively large number of sera samples contained OspA and OspB specific antibodies, their titers were low. It has been suggested that the quality of immune response to OspA and OspB is dependent on the antigen load (Schaible et al, Immunol. Let. 36:219- 26 (1993)). This poor antibody response to natural borrelial infection may be related to the quantity of spirochetes delivered by tick bite. In addition, only
  • the titer of borreliacidal activity was defined as the highest dilution of the antisera which inhibits absorbance change caused by 50% (2 x 10 6 bacteria) of spirochetes used in the assay.
  • the proteins OspA and OspB and fragments thereof may be obtained as described in the present specification or according to any other methods known in the literature. See, for example, Erdile, L. F. et al. , Infect. Immun. 67:81-90 (1993); Fikrig, E. et al , Science 250:553-556 (1990); Bergstrom, S. et al, Mol Microbiol. 5:479-86 (1989); Howe, T.R. et al, Science
  • OspA or "OspB” as used herein include lipidated and non- lipidated as well as acylated and non-acylated forms of the outer surface proteins A and B, unless indicated otherwise.
  • sustained release protein as used herein includes glycosidic triterpenoid compounds which produce foam in aqueous solution, have hemolytic activity in most cases, and possess immune adjuvant activity.
  • the invention encompasses the saponin per se, as well as natural and pharmaceutically acceptable salts and pharmaceutically acceptable derivatives.
  • saponin also encompasses biologically active fragments thereof.
  • Adjuvant saponins have been identified and purified from an aqueous extract of the bark of the South American tree, Quillaja saponaria Molina. See, U.S. Patent No. 5,057,540, the contents of which are fully incorporated by reference herein. At least 22 peaks with saponin activity were separable.
  • the predominant purified Quillaja saponins have been identified as QS-7, QS-
  • QS-21 designates the mixture of components QS-21-V1 and QS-21- V2 which appear as a single peak on reverse phase HPLC on Vydac C4 (5 ⁇ m particle size, 330 A pore, 4.6 mm ID x 25 cml) in 40 mM acetic acid in methanol/ water (58/42, v/v).
  • the component fractions are referred to specifically as QS-21-V1 and QS-21 -V2 when describing experiments or results performed on the further purified components.
  • the saponins of the present invention are purified from Quillaja saponaria Molina bark.
  • Aqueous extracts of the Quillaja saponaria Molina bark were dialyzed against water.
  • the dialyzed extract was lyophilized to dryness, extracted with methanol and the methanol- soluble extract was further fractionated on silica gel chromatography and by reverse phase high pressure liquid chromatography (RP-HPLC).
  • the individual saponins were separated by reverse phase HPLC as described in Example 1.
  • At least 22 peaks (denominated QS-1 to QS-22) were separable. Each peak corresponded to a carbohydrate peak and exhibited only a single band on reverse phase thin layer chromatography.
  • the individual components were identified by retention time on a Vydac C 4 HPLC column as follows:
  • the purified saponins are characterized by carbohydrate content, reverse phase and normal phase TLC, UV, infra red, NMR spectra, and fast atom bombardment - mass spectroscopy.
  • Carbohydrate content was used to quantitate the saponins in some instances.
  • the carbohydrate assay was the anthrone method of Scott and Melvin (Anal. Chem. 25:1656 (1953)) using glucose as a standard. This assay was used to determine a ratio of extent of anthrone reaction (expressed in glucose equivalents) per mg of purified saponin (dry weight) so that dry weight of a particular preparation could be estimated by use of anthrone assay. It must be noted that differences in reactivity with anthrone for different saponins may be due to carbohydrate composition rather than quantity as different monosaccharides react variably in this assay.
  • the substantially pure QS-7 saponin is characterized as having immune adjuvant activity, containing about 35% carbohydrate (as assayed by anthrone) per dry weight, having a uv absorption maxima of 205-210 nm, a retention time of approximately 9 - 10 minutes on RP-HPLC on a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 52-53 % methanol from a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 10 mM ID X 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of approximately .06% in water and .07%
  • the substantially pure QS-17 saponin is characterized as having adjuvant activity, containing about 29% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 35 minutes on RP-HPLC on a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol-water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 63-64% methanol from a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 10 mm ID x 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of .06% (w/v) in water and .03% (w/v)
  • the substantially pure QS-18 saponin is characterized as having immune adjuvant activity, containing about 25-26% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 38 minutes on RP-HPLC on a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 64-65 % methanol from a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 10 mm ID x 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, having a critical micellar concentration of .04% (w/v) in water and .02% (
  • the substantially pure QS-21 saponin is characterized as having immune adjuvant activity, containing about 22% carbohydrate (as assayed by anthrone) per dry weight, having a UV absorption maxima of 205-210 nm, a retention time of approximately 51 minutes on RP-HPLC on a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 4.6 mm ID x 25 cm L in a solvent of 40 mM acetic acid in methanol/water (58/42; v/v) at a flow rate of 1 ml/min, eluting with 69 to 70% methanol from a Vydac C 4 column having 5 ⁇ m particle size, 330 A pore, 10 mm x ID 25 cm L in a solvent of 40 mM acetic acid with gradient elution from 50 to 80% methanol, with a critical micellar concentration of about .03% (w/v) in water and .02% (w
  • the component fractions substantially pure QS-21-V1 and QS-21-V2 saponins, have the same molecular weight and identical spectrums by FAB-MS. They differ only in that QS-21 - VI has a terminal appose which is xylose in QS-21 -V2 (which therefore has two terminal xyloses and no appose).
  • the two components additionally contain the monosaccharides terminal arabinose, terminal appose, terminal xylose, 4-rhamnose, terminal galactose, 2-fucose, 3-xylose, and 2,3-gluc ⁇ uronic acid.
  • QS-21 significantly enhances the immunogenicity of truncated OspA (TOspA) and OspB (TOspB) of B. burgdorferi sensu stricto strain B31 and broadens immunoglobulin (Ig) G antibody responses in mice. Further, the immunological properties of QS-21 formulated, OspA and/or OspB based experimental Lyme disease vaccines were characterized in dogs. The data showed that QS-21 significantly enhanced antibody response to the experimental vaccines. Vaccine elicited antisera had high titer of antiborrelial activity.
  • the vaccines of the invention are useful as vaccines which induce active immunity toward antigens in individuals.
  • such individuals are humans, however the invention is not intended to be so limiting. Any animal which may experience the beneficial effects of the vaccines of the invention are within the scope of animals which may be treated according to the claimed invention.
  • the vaccines of the present invention induce active immunity when administered over a wide range of dosages and a wide range of ratios to the antigen being administered.
  • the saponin is administered in a ratio of adjuvant to OspA/OspB (w/w) of 3.0 or less, preferably 1.0 or less.
  • the OspA, OspB and saponin may be administered either individually or admixed with other substantially pure adjuvants to achieve the enhancement of the immune response.
  • the vaccines of the present invention may comprise a single saponin or mixtures of saponins.
  • the mixtures of the saponins may be purified saponins or crude mixtures of saponins.
  • saponin mixtures effective in the present invention are fractions QS-7 and QS-17, QS-7 and QS-18, QS-17 and QS-18, or QS-7, QS- 17, and QS-18 administered together.
  • Purified saponins may also be administered together with non-saponin adjuvants.
  • non-saponin adjuvants useful with the present invention are oil adjuvants (for example, Freund's Complete and Incomplete), liposomes, mineral salts (for example, AlK(SO 4 ) 2 , AlNa(SO 4 ) 2 , AlNH 4 (SO 4 ), silica, alum, Al(OH) 3 , Ca 3 (PO 4 ) 2 , kaolin, and carbon), polynucleotides (for example, poly IC and poly AU acids), and certain natural substances (for example, wax D from Mycobac- terium tuberculosis, as well as substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella), conjugates to the carrier proteins, such as bovine serum albumin, diphtheria toxoid, tetanus toxoid, edestin, keyhole-limpet hemocyanin, Pseudomonal Toxin A, choler- agenoi
  • the non- saponin adjuvant may comprise a protein fragment comprising at least the immunogenic portion of the molecule.
  • Other known immunogenic macromolecules which may be used in the practice of the invention include, but are not limited to, polysaccharides, tRNA, nonmetabolizable synthetic polymers such as polyvinylamine, polymethacrylic acid polyvinylpyrrolidone, mixed polycondensates (with relatively high molecular weight) of 4'4'- diaminodiphenyl-methane-3,3'-dicarboxylic acid and 4-nitro-2-aminobenzoic acid (See Sela, M., Science 766:1365-1374 (1969)) or glycolipids, lipids or carbohydrates.
  • a preferable adjuvant is alum, which gives a 3-fold increase in IgGl response.
  • the saponins may also be directly linked to the antigen or may be linked via a linking group.
  • linker group is intended one or more bifunctional molecules which can be used to covalently couple the saponin or saponin mixture to the OspA and OspB proteins and which do not interfere with the production of antigen-specific antibodies in vivo.
  • the linker group may be attached to any part of the saponin so long as the point of attachment does not interfere with the production of antigen-specific antibodies in vivo and thus interfere with the induction of active immunity.
  • Examples of linker groups which can be used to link the saponin to the OspA and OspB proteins may comprise
  • the saponins are linked to the OspA and OspB proteins by the preparation of an active ester of glucuronic acid, a component of the saponins, followed by reaction of the active ester with a nucleophilic functional group on the protein.
  • active esters which may be used in the practice of the invention include me glucuronate of N- hydroxysuccinimide, sulfo-N-hydroxysuccinimide, hydroxybenzotriazole, and p-nitrophenol.
  • the active esters may be prepared by reaction of the carboxy group of the saponin with an alcohol in the presence of a dehydration agent such as dicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide (EDC) , and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI).
  • DCC dicyclohexylcarbodiimide
  • EDC l-(3-dimethylaminopropyl)-3- ethylcarbodiimide
  • EDCI l-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide
  • the protein is then mixed with the activated ester in aqueous solution to give the conjugate.
  • the active ester of the saponin glucuronate is prepared as described above and reacted with the linker group, e.g. 2-aminoethanol, an alkylene diamine, an amino acid such as glycine, or a carboxy-protected amino acid such as glycine rt-butyl ester.
  • the linker contains a protected carboxy group
  • the protecting group is removed and the active ester of the linker is prepared (as described above).
  • the active ester is then reacted with the antigen to give the conjugate.
  • the antigen may be derivatized with succinic anhydride to give an antigen-succinate conjugate which may be condensed in the presence of EDC or EDCI with a saponin-linker derivative having a free amino or hydroxyl group on the linker. See WO91/01750.
  • the saponins retain adjuvant activity.
  • Those saponin derivatives prepared by reductive alkylation at the triterpene aldehyde do not appear to retain adjuvant activity at doses less than 40 ⁇ g.
  • derivatives in which the saponin triterpene aldehyde was reduced to an alcohol by sodium borohydride reduction did retain some activity.
  • a saponin conjugate comprising a linker with a free amino group (derived from an alkylene diamine) and crosslink the free amino group with a heterobifunctional cross linker such as sulf osuccinimidyl 4-(N-maleimidocyclohexane)- 1 -carboxy late which will react with the free sulfhydryl groups of protein antigens.
  • a heterobifunctional cross linker such as sulf osuccinimidyl 4-(N-maleimidocyclohexane)- 1 -carboxy late which will react with the free sulfhydryl groups of protein antigens.
  • the saponin may also be coupled to a linker group by reaction of the aldehyde group of the quillaic acid residue with an amino linker to form an intermediate imine conjugate, followed by reduction with sodium borohydride or sodium cyanoborohydride.
  • linkers include amino alcohols such as 2-aminoethanol and diamino compounds such as ethylenediamine, 1 ,2-propylenediamine, 1 ,5-pentanediamine, 1 ,6- hexanediamine, and the like.
  • the antigen may then be coupled to the linker by first forming the succinated derivative with succinic anhydride followed by condensation with the saponin-linker conjugate with DCC, EDC or EDCI.
  • the saponin may be oxidized with periodate and the dialdehyde produced therefrom condensed with an amino alcohol or diamino compound listed above.
  • the free hydroxyl or amino group on the linker may then be condensed with the succinate derivative of the protein in the presence of DCC, EDC or EDCI.
  • the ratio of saponin molecules per protein molecule may vary considerably according to the molecular weight of the antigen, the number of binding sites on the protein capable of being coupled to the saponin, and the antigenic characteristics of the particular saponin.
  • the ratio of saponin molecules to protein molecules may be about 0.1:1 to about 10: 1.
  • the ratio may range from about 1:1 to about 3:1.
  • Administration of the vaccines of present invention may be by parenteral, intravenous, intramuscular, subcutaneous, intranasal, or any other suitable means.
  • the dosage administered may be dependent upon the age, weight, kind of concurrent treatment, if any, and nature of the antigen administered.
  • the vaccines may be administered at a dosage of about 0.01 to about 1.0 mg/kg of protein and saponin per weight of the individual.
  • the initial dose may be followed up with a booster dosage after a period of about four weeks to enhance the immunogenic response. Further booster dosages may also be administered.
  • the effective compound useful in the method of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions.
  • any inert carrier is preferably used, such as saline, or phosphate-buffered saline, or any such carrier in which the compounds used in the method of the present invention have suitable solubility properties for use in the methods of the present invention.
  • the vaccines of the present invention may also be encapsulated within liposomes according to U.S. Patent No. 4,235,877 to Fullerton.
  • the invention also provides for a kit for the immunization of an animal comprising a carrier compartmentalized to receive in close confinement therein one or more container means wherein a first container contains the OspA and OpsB proteins and, optionally, the saponin.
  • the kit may instead include at least one other container means which contain a saponin adjuvant or other adjuvant as described herein.
  • the invention also relates to a method of inducing immunity to B. burgdorferi in an animal, comprising administering to the animal a vaccine comprising OspA, OspB or fragments thereof; and a saponin adjuvant.
  • Example 1 A Colorimetric Microtiter Assay for Borreliacidal Activity of Antisera
  • Antiserum to lysates of B. burgdorferi was raised by subcutaneous immunization of C3H/HeJ mice at age of 6 to 7 weeks.
  • One hundred ⁇ l of the spirochete lysates containing 25 ⁇ g of protein in saline was mixed with an equal volume of Freund's Complete Adjuvant for the first immunization.
  • the same dose of the antigen was mixed with an equal volume of Freund's Incomplete Adjuvant, and given for the next two immunizations at two week intervals.
  • Antiserum to a recombinant outer surface protein A derived from strain B31 (OspA-B31) (Hung, C.-H, et al , "Expression, purification and characterization of outer surface protein A (OspA) and B (OspB) from the Lyme disease spirochete, Borrelia burgdorferi," V Int. Con Lyme Borreliosis, Arlington, VA, USA (1992)) was produced by subcutaneous immunization of the mice 3 times with 200 ⁇ l of the antigen containing 25 ⁇ g of the recombinant protein and 20 ⁇ g of saponin adjuvant (QS-21) (Kensil, C.R., et al , J.
  • QS-21 saponin adjuvant
  • This CBA was performed in 96-well plate (Costar, Cambridge, MA). The appropriate concentration for each reagent used in the CBA was first determined by checkerboard titration. Each serum sample was prepared by mixing an equal volume of sera from at least ten mice, inactivated by heating at 56 °C for 45 min and sterilized by filtration through a 0.45 ⁇ m centrifuge filter (Spin-X, Costar, Cambridge, MA). Each sample was tested in triplicate. The sera were serially diluted in 100 ⁇ l of mBSK containing 120 ⁇ g/ml phenol red (SIGMA, St. Louis, MO) (mBSK-PR). Five ⁇ l of guinea pig complement were added to each well, and mixed.
  • SIGMA St. Louis, MO
  • the spirochete culture in mBSK at the logarithmic growth phase was centrifuged for 8 min at 9000 x g and resuspended in fresh mBSK medium.
  • Ninety-five ⁇ l of spirochete suspension (containing approximately 4 x 10 6 spirochetes) were added to each well (final volume 200 ⁇ l).
  • the plates were incubated at 32 °C for 2 to 5 days.
  • the absorbance at 562/630 nm of each well was measured using a microplate reader (Bio-tek Instruments, Burlington, VT) before and every 24 h after incubation.
  • 5 ⁇ l of mBSK were applied instead of guinea pig complement.
  • Controls consisting of mBSK instead of serum with or without complement were also included in each assay.
  • the absorbance at 562/630 nm of the bacterial growth controls containing different concentration of spirochetes in mBSK was also determined by incubation of 100 ⁇ l of mBSK-PR with 100 ⁇ l of the serially diluted spirochetes in mBSK at 32 °C.
  • the present CBA is based upon color changes that occur resulting from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes after incubation of a certain period of time in the presence of phenol red.
  • the color changes due to the accumulation of the acid could be detected qualitatively by the naked eye or quantitatively by a microplate reader.
  • a number of factors, such as concentration of phenol red, wavelength used for measuring the absorption, spirochete concentration, and buffering capacity of the growth medium could affect the CBA.
  • the CBA of mouse antisera to OspA-B31 is shown in Fig. 3.
  • the absorbance readings increased from the 0 to 24 h time points.
  • the absorbance readings decreased, and continued to decrease in a regular fashion through 120 h at all serum concentrations tested.
  • the absorbance decreased approximately 0.1 optical density every 24 h. The absorbance decreased more dramatically as antisera were diluted further.
  • normal mouse sera did not affect the growth of spirochete (Fig. 4), and was similar to borrelial growth controls in mBSK.
  • the borreliacidal TIA and CBA using serially diluted antisera to B31 lysates were also done simultaneously. These two assays correlated well (R 0.935).
  • each recombinant protein contained 16 amino acids (MVRABKRBEALRIAGS) (SEQ ID NO: 1) at its N-terminus derived from a bacterial leader and nucleotide linker sequence.
  • MVRABKRBEALRIAGS 16 amino acids
  • Expression of OspA and OspB was confirmed with specific monoclonal antibodies to OspA and OspB of strain B31 (Cambridge Biotech, Corp., Worcester, Mass).
  • OspA and OspB were purified by reversible citraconylation and anion exchange chromatography as described previously (Marciani, D.J. et al, in R. Burgess (ed.), protein purification, Alan R. Liss, Inc., New York, pp.443-458. ; Marciani, D.J. et al, Vaccine 9:89-96 (1991)).
  • Antisera were prepared by subcutaneous immunization of 10 month old Beagles twice at two week interval with one ml of the Lyme vaccine.
  • the vaccine contains 100 ⁇ g of truncated OspA and/or 100 ⁇ g of truncated OspB formulated with 50 ⁇ g of adjuvant QS-21.
  • Antisera were isolated two weeks after second immunization. Borreliacidal activity of the sera was determined by CBA. Borrelial culture at logarithmic growth phase was centrifuged for 8 min at 9,000 xg at 15 °C and resuspended in fresh mBSK.
  • Figure 6 shows the borreliacidal activity of canine antisera to truncated OspA and/ or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87.
  • Sera preparation and borreliacidal assay were described as above except that the incubation time of borreliacidal assay was 96 h.
  • High absorbance indicated borrelial death and high borreliacidal activity of the antisera.
  • the low absorbance represented borrelial survival and growth.
  • the error bars represent the standard error of 16 measurements of 8 serum samples for antisera and 8 measurements of 4 preimmune sera.
  • Figure 7 shows the borreliacidal activity of individual canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the homologous strain. Sera preparation and borreliacidal assay were described as above. This figure showed borreliacidal activity of individual dog antiserum and preimmune serum at 1:20 dilution. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth.
  • Figure 8 shows the borreliacidal activity of individual canine antisera to truncated OspA and/or OspB derived from B. burgdorferi strain B31 against the heterologous California strain CA-2-87. Sera preparation and borreliacidal assay were described above. The figure shows borreliacidal activity of individual dog antiserum and preimmune serum at 1:80 dilution. High absorbance indicated borrelial death and high borreliacidal activity of the antisera. The low absorbance represented borrelial survival and growth.
  • Figure 9 shows the borreliacidal activity of C3H/HeJ female mice that had been immunized twice with either 25 ⁇ g of truncated OspA, 25 ⁇ g of truncated OspB, or 25 ⁇ g of truncated OspA and 25 ⁇ g of truncated OspB.
  • Sera from ten mice in each group were pooled and tested for borreliacidal activity against the homologous strain B31 or the highly divergent Swedish strain G25, as described above.
  • Figure 10 shows the borreliacidal activity of C3H/Hej female mice that had been immunized twice with either 25 ⁇ g of truncated OspA + 20 ⁇ g of
  • E. coli, strain MZ-1 was used as the host strain for expression of recombinant antigens.
  • This strain was a gift from Takis Papas (National Cancer Institute, December 1984) and has the following genotype: galK ⁇ ⁇ BamN 7 M 53 CI857 ⁇ HI, his " , ilv, bio " , N + . (Nagai & Thogersen, Nature 509:810 (1984)).
  • the expression vector was used to transfect MZ-1 strain cells with replication deficient bacteriophage lambda DNA sequences containing a temperature sensitive C7S57 transcription repressor gene.
  • this repressor protein is active, inhibiting the synthesis of recombinant proteins.
  • 42°C protein synthesis is induced.
  • Use of this temperature sensitive mutant allows dense cell cultures to be achieved prior to the synthesis of recombinant proteins which may be toxic to the E. coli.
  • the plasmids for expression of OspA and OspB were each transfected into host cell line MZ-1 which were then used for induction of recombinant antigens.
  • OspA and OspB were produced according to Example 4 of the present specification and as further described below.
  • the genes encoding OspA and OspB of B. burgdorferi sensu stricto strain B31 were cloned using published DNA sequence (Bergstrom, S. et al , Mol. Microbiol. 5:479-486 (1989)) and standard molecular biological techniques (Maniatis, T. et al, Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982)).
  • Recombinant OspA starting at amino acid 17 and OspB starting at amino acid 20 were expressed in E. coli strain MZ-1 using expression vector pLCBCl (Beltz, G.
  • OspA and OspB genes were confirmed by DNA sequencing (Bergstrom, S. et al. , Mol. Microbiol. 5:479- 486 (1989)). Expression of OspA and OspB was confirmed with specific monoclonal antibodies to OspA and OspB of strain B31 and polyclonal antiserum to the lysates of strain B31 (Ma, J. et al , Program and abstracts of V Int. Conf. Lyme Borreliosis, Arlington, Virginia (1992)). OspA and OspB were purified by reversible citraconylation and/or anion exchange hromatography as described previously (Marciani, D. J.
  • B. burgdorferi strain B31 (Burgdorfer et al , Science 267: 1317-1319 (1982)) was received from H.J. Wellenski, Stammum Der Justus-Liebig- Universitat Giessen, Giessen, Germany. Total DNA extracted from this strain was used to subclone the OspA and OspB protein encoding regions separately. Figure 21 details each protein encoding region as they occur in B. burgdorferi strain B31.
  • OspA and OspB antigens was constructed by Cambridge Biotech Corporation (CBC) scientists and is based on published work (Shimatake & Rosenberg, Nature 297:128 (1981); Lautenberger et al , Gene 23:15 (1983); Lautenberger et al , Gene Analysis Techniques 7:63 (1984)).
  • the vector uses the bacteriophage lambda pL promoter, and a fragment of the lambda ell gene which provides a ribosome binding site, ATG initiation codon, and 12 additional codons.
  • a BamHI site follows which is used for cloning DNA fragments to be expressed, which in turn is followed by a polytranslation terminator.
  • the basic features are presented in Figure 22. Commercially obtained lambda and pBR322 DNA were used as starting materials.
  • the OspA BamHI-digested fragment was subcloned into The Cambridge Biotech expression vector, pLCBCl, also restricted with BamHI. Plasmids from colonies that contained die 907 base pair insert were also digested with EcoRI (located distally to die 5' end) to determine me orientation of the fragment within pLCBCl .
  • the correctly oriented clone was named pLCBC10spA8+6.
  • Figure 23 details die entire sequence of pLCBC10spA8+6 and die origin of each base.
  • the OspB BamHI-restricted fragment was also subcloned (separately) into pLCBCl restricted widi BamHI. Plasmids from colonies that contained die 945 base pair fragment when digested with BamHI were also digested with Hindlll to determine the direction of the insertion. The clone wi i the correct orientation was named pLCBC10spB8+4. Figure 24 details the entire sequence of pLCBC10spB8+4 and shows die origin of each base. Table 4. Validation of the OspA Clone
  • Murine monoclonal antibody (MAb) to OspA and OspB of B. burgdorferi sensu stricto strain B31 were produced widi modification of previously described procedures (Lane, R.D., J. Immunol. Methods 57:223-8 (1985); Kennett, R.H., "Hybridomas: A New Dimension in Biological Analysis," in Monoclonal Antibodies, Kennett et al , Plenum Publ. Corp. , New York (1980), pp. 365-7). Briefly, Balb/c mice were immunized at least two times with recombinant OspA, OspB, or borrelial lysate.
  • Primed spleen cells were isolated and fused witii the myeloma cell line SP2/0 by mixing at a 4:1 ratio in the presence of 50% polyethyleneglycol 1500 (Boehringer Mannheim, Indianapolis, IN). Hybridoma cultures producing antibodies of interest were cloned at least twice by limiting dilution.
  • MAbs to OspA and OspB were identified by enzyme-linked immunosorbent assay (ELISA) and immunoblotting.
  • Antibody isotypes were determined by ELISA using isotype-specific goat anti-mouse IgG conjugated to horseradish peroxidase (Pel-Freez, Rogers, AZ). MAbs were purified from culture supernatant using protein- A affinity column, and protein concentration determined by BCA assay (Pierce, Rockford, IL).
  • the supernatant was incubated witii mixture of purified OspA specific MAbs H3G4, L3B5, T1F6, and T2H12, and with OspB specific MAb P4D1, respectively, for 2 h at 22 °C with gentle agitation, and ti en incubated widi protein G-Sepharose 4FF (Pharmacia, Piscataway, NJ) in 0.25 M Tris-HCl, pH 6.8, for another 1 h at 22°C.
  • the mixture was microcentrifuged and washed 3 times, and resuspended in distilled water.
  • the isolated recombinant proteins were mixed with sample buffer (Laemmli, U.K. , Nature (London) 227:680-5 (1970)), boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.
  • Vaccine formulation was prepared as described in Example 4, below; also see Kensil et al, J. Immunol 746:431-7 (1990).
  • Each dose (1 ml) of vaccine contained 100 or 25 ⁇ g each of FLOspA and/or FLOspB formulated with or without 50 ⁇ g of QS-21 (Kensil et al, J. Immunol. 146:431-1 (1990), in saline, pH 6.5.
  • a vaccine containing 100 ⁇ g each of TOspA, TOspB and 50 ⁇ g of QS-21 was also prepared for comparative study.
  • Vaccination and antiserum Beagle dogs (Harlan Sprague Dawley, Indianapolis, IN) at age of 12 weeks were vaccinated subcutaneously with 1 ml of vaccine. Immune response was boosted once 4 weeks later with the same dose of vaccine. Antiserum was isolated 2 weeks after the second vaccination and stored at -20°C.
  • ELISA was performed using plates coated widi B. burgdorferi antigens, 0.2 ⁇ g OspA per well , and 0.2 ⁇ g OspB per well, respectively, as described (Lindenmayer et al, J.Clin. Microbiol 28:92-6 (1990)).
  • Antibody isotypes were determined using isotype-specific goat anti-dog IgG conjugated to horseradish peroxidase (Bethyl Laboratories, Inc., Montgomery, TX).
  • a colorimetric microtiter assay for antiborrelial activity of antisera was performed as described above. Briefly, heat-inactivated, filter-sterilized sera were serially diluted in 100 ⁇ l of mBSK containing 120 ⁇ g of phenol red per ml in 96-well plates. Five ⁇ l of sterile guinea pig complement (Gibco BRL, Gaithersburg, MD) and 95 ⁇ l of spirochete (containing approximately 6 x 10 7 of spirochetes) in fresh mBSK were added to each well. After 48 to 72 h incubation at 32 °C, the absorbance at 562/630 nm was measured using a microplate reader.
  • Antiborrelial activity was expressed by either absorbance change compared with those of preimmune sera or by titer.
  • the titer of antiborrelial activity was defined as me highest dilution of the antisera which inhibited absorbance change caused by 50% of spirochetes used in this assay.
  • Antiborrelial activity reflects borrelial killing and borrelial growdi inhibition by specific antibodies.
  • MAbs reactive with OspA and OspB of B. burgdorferi sensu stricto strain B31 were developed.
  • MAbs H3G11, L3B5, T1F6, T2H12 (all IgGl) are specific for OspA, and
  • MAb P4D1 (IgGl) is specific for OspB, as tested by ELISA and immunoblotting.
  • MAb P4D1 specific for OspB precipitated OspB from lysate of E. coli labeled with [9,10- 3 H]palmitic acid A mixture of 4 OspA specific MAbs described above were used for precipitation of OspA. Lipidation of the precipitated OspA and OspB was analyzed by SDS-PAGE and fluorography. both TOspA and TOspB are nonlipidated proteins, and FLOspA and FLOspB are lipoproteins as shown by me radiolabeled bands (Fig. 11). FLOspB had another radiolabeled band with a molecular mass of approximately 22 KDa, perhaps a recombinant fragment from OspB (see Example 4, below).
  • FIG. 12 Various experimental vaccines were evaluated in dogs for eliciting IgG isotype antibody responses (Fig. 12).
  • a QS-21 formulated vaccine containing 100 ⁇ g each of FLOspA and FLOspB induced 30-fold higher (p ⁇ 0.005) IgGl and 4-fold higher (p ⁇ 0.0005) IgG2 antibody responses than die same formulation containing TOspA and TOspB.
  • a vaccine containing 25 ⁇ g each of FLOspA and FLOspB was similar to the vaccine containing 100 ⁇ g each of the lipoproteins in terms of eliciting antibody titer.
  • QS-21 enhanced at least 4-fold higher (p ⁇ 0.1) IgGl and 8-fold higher (p ⁇ 0.05) IgG2 antibody responses when comparing FLOspA and FLOspB based vaccines formulated witii and widiout QS-21.
  • Either FLOspA and FLOspB based vaccine formulated wid QS-21 elicited antibody responses similar to bom FLOspA and FLOspB based vaccine formulated with QS-21 (p ⁇ 0.2 and p ⁇ 0.1, respectively). All antisera to vaccines formulated with nonlipidated or lipoproteins
  • OspA, OspB, and QS-21 contained antibodies to bodi OspA and OspB.
  • the representative immunoblot showed mat all 11 antisera to vaccine containing 25 ⁇ g each of FLOspA and FLOspB and 50 ⁇ g of QS-21 reacted with both OspA and OspB protein bands (Fig. 13, lane 13 to 23).
  • the antiborrelial activity of the canine antisera to me experimental Lyme disease vaccines was tested against die homologous strain B31 and heterologous strain CA-2-87, as well as the other Borrelial species G25 and 24008 Fr.
  • Antisera to QS-21 formulated vaccine containing TOspA and TOspB were antiborrelial to the homologous strain B31 and heterologous strain CA-2-87.
  • Their antiborrelial titers were 8-fold (p ⁇ 0.005) and 3-fold (p ⁇ 0.001) lower against B31 and CA-2-87, respectively, than those of antisera to the same formulation containing FLOspA and FLOspB (Fig. 15).
  • strain B31 OspA and OspB were cloned using published DNA sequence (Bergstrom, S. et al , Mol Microbiol. 5:479-486 (1989)) and standard molecular biological techniques (Maniatis, T. et al , Molecular Cloning, Cold Spring Harbor Laboratory, New York (1982)).
  • Recombinant OspA starting at amino acid 17 and OspB starting at amino acid 20 were expressed in E. coli strain MZ-1 using expression vector pLCBCl (Beltz, G. A. et al. , U.S. Patent No. 4,753,873 (1988)), a vector very similar to that described by Lautenberger (Lautenberger, J. A.
  • the vector makes use of die heat inducible pL promoter; the Shine-Dalgarno sequence and first N- terminal 16 amino acid codons are derived from the Lambda ell gene. Cloning sites within three codons of mature OspA and OspB were chosen for ligation into tiie expression vector. The cloned ospA and ospB genes were confirmed by DNA sequencing (Bergstrom, S. et al. , Mol.
  • OspA and OspB were purified by reversible citraconylation and/or anion exchange chromatography as described previously (Marciani D. J. et al. , Vaccine 9:89- 96 (1991); Marciani, D. J.
  • QS-21 was purified from the cortex of the tree Quillaja saponaria monlina by ultrafiltration, adsorption chromatography, and reverse phase HPLC (Kensil, C. R. et al , J. Immunol. 746:431-437 (1990)).
  • each dose (0.2 ml) of the experimental vaccine contained 25 ⁇ g of recombinant protein. This antigen dose used was found to induce significantly higher titers of antibody than either 1 ⁇ g or 5 ⁇ g of antigen (data not shown).
  • Formulated vaccines contained eitiier 20 ⁇ g of QS- 21 or 200 ⁇ g of alum (Alhydrogel, Accurate Chemical and Scientific Corp., Westbury, NY) in PBS, pH 6.5.
  • the adjuvant doses of both QS-21 and alum were previously optimized for the water soluble protein antigen, ovalbumin in mice (Kensil, C. R. et al , "The use of StimulonTM to boost vacine response," Vaccine Res. 2:273-281 (1993).)
  • the vaccine contained 25 ⁇ g each of OspA and OspB formulated with 20 ⁇ g of QS-21.
  • Antisera to OspA and OspB were prepared by subcutaneous immunization of 6 to 7 week old female C3H/HeJ mice (The Jackson Laboratory, Bar Harbor, ME) 3 times at 2 week interval as described in Example 1 (also see Fikrig, E. et al , Science 250:553-556 (1990).
  • SDS-polyacrylamide Gel Electrophoresis and Immunoblotting SDS- polyacrylamide gel electrophoresis (SDS-PAGE) was performed using an 11 % separating gel (Laemmli, U. K., Nature (London) 227:680-685 (1970)). The gel was stained witii Coomassie Brilliant blue R-250. Immunoblotting was conducted by modification of the procedure of Towbin et al. (Towbin, H. et al , Proc. Natl. Acad. Sci. USA 76:4350-5354 (1979)).
  • Bacterial lysates (4 to 6 ⁇ g) were subjected to SDS-PAGE, transferred onto nitrocellulose membrane, and probed with both 1:20 and 1:200 dilutions of mouse antisera.
  • Goat anti-mouse IgG conjugated to horseradish peroxidase (Fisher Scientific, Pittsburgh, PA) and 4-chloro-l-naphthol substrate was used to detect antigen-antibody binding on immunoblots.
  • ELISA die enzyme- linked immunosorbant assay
  • Borrelia burgdorferi culture was adjusted to a concentration which contained approximately 60 organisms per microscopic field.
  • One hundred ⁇ l of B. burgdorferi was incubated in 96-well plates witii an equal volume of heat- inactivated, serially diluted antisera or normal serum in mBSK medium at 32 °C for 2 hours. Agglutination was determined by dark-field microscopy. For each assay, 3 samples (10 ⁇ l) were taken and 9 fields of each sample were examined for individual spirochetes. The number of individual spirochetes per microscopic field was averaged.
  • Agglutination titer was defined as the highest dilution of the antisera that produced less than 15 individual spirochetes per microscopic field (i.e. caused > 50% of the spirochetes to agglutinate).
  • Colorimetric borreliacidal Assay was performed in duplicate in 96- well plates (Costar, Cambridge, MA) by the method of the present invention and as described in Example 1 of die present specification. Briefly, heat- inactivated, filter-sterilized test sera were serially diluted in 100 ⁇ l of mBSK containing 120 ⁇ g/ml of phenol red (SIGMA, St. Louis, Missouri) (mBSK- PR).
  • mice were subcutaneously immunized two or three times with the experimental vaccine containing OspA and OspB formulated with QS-21. Control mice were injected with sterile 0.15 M saline. Two weeks after last immunization, mice were challenged intradermally with 10 3 , 10 4 , and 10 5 infectious spirochetes of strains B31 or CA-2-87, respectively. In a separate experiment, all non-vaccinated mice were infected with as few as 10 spirochetes of either strain. Mice were sacrificed two weeks following the challenge, and the bladder, heart and blood samples were cultured for spirochetes as described (Schwan, T. G.
  • OspA and OspB in E. coli were induced by a shift of incubation temperature from 32°C to 42°C (lane 2 and 4, respectively, Fig. 18).
  • Recombinant OspA and OspB were purified from E. coli (lane 3 and 5, respectively, Fig. 18), and confirmed by immunoblotting using monoclonal antibodies to OspA and OspB of strain B31.
  • a few faint bands with low molecular weights in lanes 3 and 5 also reacted with the monoclonal antibodies on immunoblotting (data not shown), and are therefore not E. coli contaminants.
  • Neither OspA and OspB were acylated (data not shown).
  • strains B31 and Fr produced an approximately 31 Kda OspA and 34 Kda OspB (lane 1 and 3, Fig. 19A), respectively.
  • strain CA-2- 87 produced an approximately 31 KDa OspA and 34.5 KDa OspB (lane 2, Fig. 19A);
  • strain G25 produced an approximately 32 KDa OspA and 33.5 KDa OspB (lane 4, Fig. 19A).
  • QS-21 significantly enhanced the antibody responses of IgG2a and IgG2b to OspA (p ⁇ ⁇ 0.0001 for both) and OspB (p ⁇ 0.002 and 0.02, respectively) (Fig. 20).
  • QS-21 also significantly augmented the IgGl antibody response to OspA (p ⁇ 0.01), but not to OspB.
  • Alum had no significant effect on IgG2a and IgG2b responses to OspB (p>0.2), but significantly inhibited IgG2a and IgG2b antibody responses to OspA (p ⁇ 0.05 and p ⁇ 0.002, respectively) (Fig. 20, part A and part C).
  • the alum formulated vaccines enhanced IgGl responses to OspB (p ⁇ 0.01) but not to OspA.
  • Vaccines formulated with both QS-21 and alum augmented the antibody responses over those induced by alum alone, but less tiian QS-21 alone.
  • IgM and IgG3 antibody responses to OspA or OspB formulated with either QS-21 or alum were minimal when compared with other IgG isotype antibody responses. As a result, it was not possible to detect an adjuvant effect on either IgM or IgG3 induction.
  • Antisera to OspA formulated with QS-21 agglutinated not only the homologous New York strain B31, but also the heterologous California isolate CA-2-87 and German isolate Fr with agglutination titers of 1600 to 6400 (Table 1). This antisera also agglutinated die Swedish isolate G25 with a titer of 400.
  • Antisera from mice vaccinated with OspA formulated with alum or without an adjuvant had agglutination titers of ⁇ 50 to 1600.
  • Antisera to OspB formulated with alum or QS-21 or without an adjuvant had much lower agglutination titers ( ⁇ 50 to 400) with the strains tested.
  • OspA and OspB are shown in Table 2.
  • Antisera to OspA formulated without an adjuvant had low borreliacidal activity against strains B31, Fr, and G25 (titers of 40 to 80), and had undetectable activity against strain CA-2-87 (titer of ⁇ 10).
  • QS-21 enhanced the borreliacidal anti-OspA response 8- to 64-fold as tested against these 4 strains (titers of 320 to 1280).
  • Alum increased this response by less than 2-fold against the strains tested.
  • OspB formulated witii either QS-21 or alum induced a 16-fold increase in borreliacidal activity against strain B31 when compared with OspB alone.
  • a 16 fold increase in borreliacidal activity against strain Fr was also seen with antisera to OspB when formulated with QS-21, but not when formulated with alum. Neither of the antisera had borreliacidal activity against strain CA-2-87 or G25.
  • Vaccinated mice were protected from infection following challenge with as many as 10 5 infectious spirochetes (at least 4 log greater than the infectious dose) of either the strains B31 or CA-2-87. All vaccinated mice were free of spirochetes in bladder, heart, and blood. In contrast, all control mice were infected after challenge as shown by culturing spirochete from bladder tissue samples. Some control mice also had culture positive blood and heart tissue.

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Abstract

Un titrage colorimétrique simple permet de déterminer l'activité borréliacide d'un sérum immun à Borrelia burgdorferi. On décrit aussi un vaccin contre Borrelia burgdorferi et des procédés induisant une immunité aux bactéries provoquant l'arthrite de Lyme, y compris B. burgdorferi et B. garinii, par administration de ce vaccin. On décrit aussi des trousses comprenant un ou plusieurs flacons qui contiennent les éléments du vaccin contre B. burgdorferi, de même que des formulations de vaccins contre l'arthrite de Lyme.
PCT/US1994/002095 1993-02-26 1994-02-28 Procedes induisant une immunite a l'arthrite de lyme et titrage colorimetrique concernant l'activite borreliacide d'antiserums WO1994019697A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7008625B2 (en) 1993-11-01 2006-03-07 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US7060281B1 (en) 1999-06-18 2006-06-13 Research Foundation Of The State University Of New York Groups of barrelia burgdorferi and borrelia afzelii that cause lyme disease in humans
US8137678B2 (en) 2006-11-03 2012-03-20 Intervet Inc. Canine lyme disease vaccine
WO2012039614A1 (fr) 2010-09-21 2012-03-29 Stichting Katholieke Universiteit Nouveau procédé pour diagnostiquer la maladie de lyme en utilisant un test immunologique cellulaire
US8680236B2 (en) 2000-08-18 2014-03-25 Brookhaven Sciences Associates, Llc Altered OspA of borrelia burgdorferi
RU2660572C1 (ru) * 2017-04-26 2018-07-06 Федеральное Государственное Бюджетное Образовательное Учреждение Высшего Образования "Красноярский Государственный Медицинский Университет Имени Профессора В.Ф. Войно-Ясенецкого" Министерства Здравоохранения Российской Федерации Способ оценки вероятности исходов эритемной формы иксодового клещевого боррелиоза

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4721617A (en) * 1986-08-14 1988-01-26 Regents Of The University Of Minnesota Vaccine against lyme disease
US4806350A (en) * 1986-04-18 1989-02-21 Norden Laboratories, Inc. Vaccine formulation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993008306A1 (fr) * 1991-10-22 1993-04-29 Symbicom Aktiebolag AMELIORATION DANS LE DIAGNOSTIC ET LA PROPHYLAXIE DE LA BORRELIOSE PROVOQUEE PAR $i(BORRELIA BURGDORFERI)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4806350A (en) * 1986-04-18 1989-02-21 Norden Laboratories, Inc. Vaccine formulation
US4721617A (en) * 1986-08-14 1988-01-26 Regents Of The University Of Minnesota Vaccine against lyme disease

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
"GIBCO BRL LIFE TECHNOLOGIES. 1993-1994 CATALOGUE AND REFERENCE GUIDE, PASSAGE.", LIFE TECHNOLOGIES CATALOGUE AND REFERENCE GUIDE, XX, XX, 1 January 1993 (1993-01-01), XX, pages A + 1.02 - 1.04 + 1.94/1.95., XP002916173 *
378 - 383: "DELINEATION OF BORRELIA BURGDORFERI SENSU STRICTO, BORRELIA GARINIISP. NOV., AND GROUP VS461 ASSOCIATED WITH LYME BORRELIOSIS", INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, SOCIETY FOR GENERAL MICROBIOLOGY, READING, GB, vol. 42, no. 03, 1 July 1992 (1992-07-01), GB, pages 378 - 383, XP002948027, ISSN: 0020-7713 *
BARBOUR A G: "ISOLATION AND CULTIVATION OF LYME DISEASE SPIROCHETES", YALE JOURNAL OF BIOLOGY AND MEDICINE, NEW HAVEN, CT, US, vol. 57, no. 04, 1 August 1984 (1984-08-01), US, pages 521 - 525, XP002918330 *
DEVER L L, JORGENSEN J H, BARBOUR A G: "IN VITRO ANTIMICROBIAL SUSCEPTIBILITY TESTING OF BORRELIA BURGDORFERI: A MICRODILUTION MIC METHOD AND TIME-KILL STUDIES", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 30, no. 10, 1 October 1992 (1992-10-01), US, pages 2692 - 2697, XP002948028, ISSN: 0095-1137 *
DIFCO LABORATORIES.: "DIFCO MANUAL. DEHYDRATED CULTURE MEDIA AND REAGENTS FOR MICROBIOLOGY.", 1 January 1984, DETROIT, DIFCO., US, article "PHENOL RED CARBOHYDRATE MEDIA BACTO PHENOL RED BROTH BASE BACTO PHENOL RED DEXTROSE BROTH BACTO PHENOL RED LACTOSE BROTH BACTO PHENOL RED MALTOSE BROTH ", pages: 660 - 662, XP002948038, 016117 *
FIKRIG E, ET AL.: "BORRELIA BURGDORFERI STRAIN 25015: CHARACTERIZATION OF OUTER SURFACE PROTEIN A AND VACCINATION AGAINST INFECTION", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 148, no. 07, 1 April 1992 (1992-04-01), US, pages 2256 - 2260, XP002948018, ISSN: 0022-1767 *
FIKRIG E., ET AL.: "ROLES OF OSPA, OSPB, AND FLAGELLIN IN PROTECTIVE IMMUNITY TO LYME BORRELIOSIS IN LABORATORY MICE.", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 60., no. 02., 1 February 1992 (1992-02-01), US, pages 657 - 661., XP002001270, ISSN: 0019-9567 *
KENSIL C.R., ET AL.: "SEPARATION AND CHARACTERIZATION OF SAPONINS WITH ADJUVANT ACTIVITY FROM QUILLAJA SAPONARIA MOLINA CORTEX.", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 146., no. 02., 15 January 1991 (1991-01-15), US, pages 431 - 437., XP000671494, ISSN: 0022-1767 *
LORNE F. ERDILE ET AL: "Role of attached lipid in immunogenicity of Bprrelia burgdorferi OspA", INFECTION AND IMMUNITY, AMERICAN SOCIETY FOR MICROBIOLOGY., US, vol. 61., no. 01., 1 January 1993 (1993-01-01), US, pages 81 - 90., XP002007535, ISSN: 0019-9567 *
MA J, COUGHLIN R T: "A SIMPLE, COLORIMETRIC MICROTITER ASSAY FOR BORRELIACIDAL ACTIVITY OF ANTISERA", JOURNAL OF MICROBIOLOGICAL METHODS, ELSEVIER, AMSTERDAM,, NL, vol. 17, 1 January 1993 (1993-01-01), NL, pages 145 - 153, XP002948026, ISSN: 0167-7012, DOI: 10.1016/0167-7012(93)90008-6 *
MARCIANI D J, ET AL.: "GENETICALLY-ENGINEERED SUBUNIT VACCINE AGAINST FELINE LEUKAEMIA VIRUS: PROTECTIVE IMMUNE RESPONSE IN CATS", VACCINE, ELSEVIER LTD, GB, vol. 09, 1 February 1991 (1991-02-01), GB, pages 89 - 96, XP002948019, ISSN: 0264-410X, DOI: 10.1016/0264-410X(91)90262-5 *
MURPHY J R, BACHA P, TENG M: "DETERMINATION OF CORYNEBACTERIUM DIPHTHERIAE TOXIGENICITY BY A COLORIMETRIC TISSUE CULTURE ASSAY", JOURNAL OF CLINICAL MICROBIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 07, no. 01, 1 January 1978 (1978-01-01), US, pages 91 - 96, XP002948025, ISSN: 0095-1137 *
PAVIA C S, ET AL.: "ANTIBORRELIAL ACTIVITY OF SERUM FROM RATS INJECTED WITH THE LYME DISEASE SPIROCHETE", JOURNAL OF INFECTIOUS DISEASES. JID, UNIVERSITY OF CHICAGO PRESS., CHICAGO, IL., vol. 163, 1 March 1991 (1991-03-01), CHICAGO, IL., pages 656 - 659, XP002948029, ISSN: 0022-1899 *
See also references of EP0686265A4 *

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US7008625B2 (en) 1993-11-01 2006-03-07 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US7179448B2 (en) 1993-11-01 2007-02-20 Research Foundation Of The State Of New York Recombinant constructs of Borrelia burgdorferi
US7605248B2 (en) 1993-11-01 2009-10-20 Research Foundation Of The State University Of New York Recombinant constructs of Borrelia burgdorferi
US7060281B1 (en) 1999-06-18 2006-06-13 Research Foundation Of The State University Of New York Groups of barrelia burgdorferi and borrelia afzelii that cause lyme disease in humans
US7582304B2 (en) 1999-06-18 2009-09-01 Research Foundation Of The State University Of New York Groups of Borrelia burgdorferi and Borrelia afzelii that cause Lyme Disease in humans
US8680236B2 (en) 2000-08-18 2014-03-25 Brookhaven Sciences Associates, Llc Altered OspA of borrelia burgdorferi
US8992936B2 (en) 2000-08-18 2015-03-31 Research Foundation Of The State University Of New York Altered OspA of Borrelia burgdorferi
US8137678B2 (en) 2006-11-03 2012-03-20 Intervet Inc. Canine lyme disease vaccine
US8414901B2 (en) 2006-11-03 2013-04-09 Intervet Inc. Canine Lyme disease vaccine
WO2012039614A1 (fr) 2010-09-21 2012-03-29 Stichting Katholieke Universiteit Nouveau procédé pour diagnostiquer la maladie de lyme en utilisant un test immunologique cellulaire
US9316652B2 (en) 2010-09-21 2016-04-19 Stichting Katholieke Universiteit Method for diagnosing Lyme disease using a cellular immunological test
RU2660572C1 (ru) * 2017-04-26 2018-07-06 Федеральное Государственное Бюджетное Образовательное Учреждение Высшего Образования "Красноярский Государственный Медицинский Университет Имени Профессора В.Ф. Войно-Ясенецкого" Министерства Здравоохранения Российской Федерации Способ оценки вероятности исходов эритемной формы иксодового клещевого боррелиоза

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EP0686265A1 (fr) 1995-12-13
NZ262821A (en) 1997-03-24
CA2157004A1 (fr) 1994-09-01
AU6272894A (en) 1994-09-14
EP0686265A4 (fr) 2001-08-22
AU689466B2 (en) 1998-04-02

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