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WO1990011776A1 - Proteines et genes clones nouveaux utiles pour le diagnostic et la prophylaxie de la babesiose - Google Patents

Proteines et genes clones nouveaux utiles pour le diagnostic et la prophylaxie de la babesiose Download PDF

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Publication number
WO1990011776A1
WO1990011776A1 PCT/US1990/001812 US9001812W WO9011776A1 WO 1990011776 A1 WO1990011776 A1 WO 1990011776A1 US 9001812 W US9001812 W US 9001812W WO 9011776 A1 WO9011776 A1 WO 9011776A1
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lambda
dna
babesia
protein
fragment
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PCT/US1990/001812
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English (en)
Inventor
Terry F. Mcelwain
Stephen A. HINES
Travis C. Mcguire
Guy H. PALMER
Douglas P. Jasmer
David W. REDUKER
Will L. GOFF
Lance E. Perryman
William C. Davis
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University Of Florida
Washington State University
United States Department Of Agriculture
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Publication of WO1990011776A1 publication Critical patent/WO1990011776A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • 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

Definitions

  • Bovine babesiosis is a tick-transmitted, hemoparasitic disease caused by intraerythrocytic protozoa belonging to the genus Babesia.
  • the disease caused by Babesia manifests itself clinically by fever and extensive hemolytic anemia that often leads to hypotensive shock, cerebral involvement, and death. More than a half billion cattle are estimated to be at risk of acquiring babesiosis. This disease represents a primary impediment to food and fiber production in much of the world.
  • babesiosis has been partially successful through vaccination with attenuated strains of Babesia spp. or with more virulent strains followed by chemotherapeutic control.
  • Protective immunity in babesiosis may be directed against one or more surface antigens associated with sporozoites, infected erythrocytes, and/or merozoites.
  • Merozoite surface antigens are important in the pathogenesis and immunology of babesiosis due to their role in the parasite's recognition of, attachment to, and penetration of host erythrocytes and their accessibility to the immune system.
  • Bovine babesiosis can be caused by either Babesia bigemina or Babesia bovis. These parasites have antigenic similarities and differences that may have important functional roles in the induction of protective immunity and antibody- based diagnosis.
  • B. bovis isolates including the current Australian vaccine strain, are now known to consist of subpopulations that vary antigenically, in virulence, and in abundance within an isolate (Cowman, A.F., P. Timms, and D.J. Kemp [1984] Mol. Biochem. Parasitol. 11:91-103; Gill, A.C., A.F. Cowman, N.P. Stewart, D.J. Kemp, and P. Timms [1987] Exp. Parasitol. 63:180-188).
  • the attenuated vaccine provides the best protection against challenge with both homologous and heterologous strains, although there are a number of serious disadvantages, including a short shelf-life, variation in virulence, contamination with host erythrocyte stroma, and perpetuation of the life cycle by creation of a carrier state.
  • Inactivated vaccines induce protection against challenge with homologous strains; however, only partial protection occurs against challenge with heterologous strains.
  • proteins are known to be expressed on the surface of the merozoite and may be used to raise neutralizing antibodies. Thus, they can be used in the formulation of subunit vaccines for the prophylaxis of bovine babesiosis.
  • proteins described here raise antibodies to both Babesia bovis and Babesia bigemina, while others are species, or even isolate, specific.
  • monoclonal antibodies to bovine babesiosis antigens are used to identify merozoite surface antigens and may be used in the treatment and/or diagnosis of bovine babesiosis.
  • a further element of the invention is the identification of genes which code for Babesia proteins. These genes can be used to make recombinant proteins which can be utilized for vaccines.
  • the invention also provides a means of detecting the presence of disease- causing Babesia organisms.
  • the detection method involves the use of DNA probes which selectively identify the presence of these organisms.
  • Figure 1 is the translated DNA and amino acid sequence of lambda-Bo44.
  • Figure 2 is the DNA and amino acid sequence for rBv42.
  • Figure 3 is the DNA sequence for rBv60.
  • Figure 4 is the amino acid sequence for rBv ⁇ O.
  • the subject invention pertains to the identification of surface-exposed proteins of B. bovis merozoites.
  • the proteins of the invention have sizes of 16, 25, 37, 42, 44, 55, 60, 85, 98, 125, 145, 225, and 250 kDa.
  • the evidence that the proteins are surface exposed includes: (i) monoclonal antibody binding of live merozoites, (ii) labeling by surface iodination, and (iii) sensitivity to mild trypsinization.
  • B. bovis merozoite proteins that, by virtue of their surface location and their reactivity with immune bovine sera, are candidates for subunit vaccines.
  • six proteins appeared to be relatively immunodominant.
  • the 145 kDa protein was of parasite origin, but its location on the membrane surface was not directly apparent. This protein may have a small portion exposed at the surface of the merozoite that is sensitive to mild trypsinization but the epitope recognized by the monoclonal antibody located internally.
  • amino acids may be placed in the following classes: basic, hydrophobic, acidic, polar, and amide. Substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the antigenic activity of the compound.
  • bovis isolate The ability of antibodies against heterologous geographic isolates to iramunoprecipitate proteins from the Mexico B. bovis isolate indicates the conservation of at least one and probably more epitopes between proteins from the heterologous isolates. The conservation of these epitopes is extensive, as many Mexico B. bovis isolate proteins were precipitated by antisera against a different geographic isolate (Honduras). The 42,000 molecular weight Mexico B. bovis protein was precipitated by all five of the undiluted and three of the diluted Honduras antisera.
  • B. bovis proteins only one (the 42,000 molecular weight protein) is both isolate common and species specific. This protein can be used as an antigen for species-specific, antibody-based diagnosis.
  • MoAbs Monoclonal antibodies
  • a genomic library constructed in the lambda- gtll expression vector was screened with MoAbs for identification of clones expressing recombinant surface proteins. Four recombinant clones were identified.
  • lambda-Bo ⁇ is a good candidate for detecting Babesia infections in cattle and ticks
  • lambda-Bo25 can be used to distinguish geographic isolates of B. bovis.
  • the cloned line was derived from the Mexico isolate by limiting dilution cloning as previously described by Rodriguez et al. (Rodriguez, S.D., G.M. Buening, T.J. Green, and CA. Carson [1986] Infect. Immun. 42:15-18).
  • the parasites have been maintained in our laboratory by either repeated passages in splenectomized Holstein-Freisian bull calves or in vitro cultivation.
  • the contents of flasks containing >15% parasitized erythrocytes were centrifuged at 400 x g for 10 min at 4°G
  • the supernatant was centrifuged at 3,000 x g for 15 min at 4°C to pellet the merozoites.
  • the merozoites were suspended in Puck saline-glucose (saline-G), and 2 ml was overlaid on 10 ml of a preformed continuous gradient of 65% Percoll-35% Puck saline-G.
  • the gradient was centrifuged in a swinging bucket rotor at 3,000 x g for 20 min at 4°C
  • the merozoites were isolated from a band with an approximate density of 1.069 g/ml between erythrocyte ghosts at the Percoll-Puck saline-G interface and the residual intact erythrocyte pellet.
  • the merozoites were washed once in 0.15 M NaCl containing 0.01 M sodium citrate (CS), suspended in CS, and stored on ice until used (within 2 to 4 hr). Purification, Quantitation and Viability Estimation of Merozoites.
  • R bigemina. and Anaplasma marginale. were inoculated intravenously with approximately 6 x 10 8 R bovis-infected erythrocytes from the same blood stabilate used to initiate in vitro cultures.
  • each steer developed detectable parasitemia and a febrile response which persisted through day 13 postinoculation.
  • Antibody specific for R bovis was detected with the indirect fluorescent-antibody test on day 10 postinoculation.
  • the steers were challenge inoculated as before on days 48 and 80 postinoculation, and although the animals did not develop a fever or parasitemia, the antibody titer increased after each challenge.
  • Sera were collected and stored at -70°C after the final challenge, when the indirect fluorescent-antibody test titer was 1:10,000.
  • the cloned line was passed through a splenectomized calf whose blood at peak parasitemia was used to infect five 4-5 month old Holstein steers (5 x 10 7 infected erythrocytes each) and to initiate in vitro cultures.
  • the cattle were reinfected at 23 days post infection (DPI) with 10 8 infected erythrocytes (iRBC) from another splenectomized calf and at 77 and 99 DPI with 10 8 iRBC from culture.
  • DPI days post infection
  • iRBC infected erythrocytes
  • the samples were then suspended in the appropriate rhodamine-conjugated second antibody (1/40 dilution in PBS) (Kirkegaard and Perry, Inc., Gaithersburg, MD) and incubated on ice for 1 hr. After being washed, the merozoites were suspended in 6-CFDA and incubated for 20 min at room temperature. The merozoites were then centrifuged at 3,000 x g for 10 min at 4°C, suspended in 50 ul of PBS, and examined in wet mounts with appropriate filters for rhodamine (antibody binding) and fluorescein (6-CFDA viability) (546 to 590 nm and 450 to 520 nm, respectively).
  • nRBC erythrocytes
  • Metabolic Radiolabeling of Merozoites Metabolically radiolabeled parasite proteins from calf-derived merozoites were prepared for use in immunoprecipitation experiments according to the methods of McElwain et al. (1987, supra) except that cultures containing 100 uCi of [ 35 S]-methionine ( 35 S-Met; New England Nuclear, Boston, MA) per 3 x 10 9 erythrocytes were incubated at 37°C for 8-9 hr in a Forma Scientific water jacketed incubator instead of a candle jar. Parasites cultivated in vitro were metabolically radiolabeled using normal growth medium (Goff and Yunker, 1986) or D,L-methionine-free medium, addition of 20-400 uCi/ml 35 S-Met,
  • Washed iRBC's from 35 S-methionine labeled cultures were lysed in 10 mM Tris, 154 M NaCl pH 7.4, 1% (v/v) TRITONTMX-114, 1 mM phenylmethylsulfonyl fluoride (PMSF) at 0-4°C and frozen at -20°C
  • PMSF phenylmethylsulfonyl fluoride
  • phase separation was repeated twice by adding 200 ul of 15% (v/v) TRITONTMX-100 in 10 mM Tris, 154 mM NaCl to the aqueous phase, dissolving the detergent on ice, and re-extracting at 37°C over the same sucrose cushion.
  • the three detergent phases resulting from centrifugation were mixed with 10 mM Tris, 154 mM NaCl at 0-4°C, combined, and TCA-precipitable radioactivity counted along with the aqueous phase.
  • Immunoprecipitation Immune sera were used either unadsorbed or adsorbed three times with an equal volume of packed intact nRBC's and three times with an equal volume of nRBC ghosts.
  • Radiolabeled R bovis or bacterial lysate was processed as described previously (Palmer and McGuire [1984], supra) and incubated overnight at 4°C ith 15 ul of bovine serum or 15 ul of serum diluted in Veronal buffered saline (VBS) pH 7.4, 1% (v/v) Nonidet P-40 (NP-40). 150 ul of 10% (v/v) formalinized Protein G-bearing Streptococcus (Omnisorb.
  • C-labeled protein standards used for molecular weight determination were myosin, 200 kDa; phosphorylase b, 92.5 kDa; bovine serum albumin, 69 kDa; ovalbumin, 46 kDa; carbonic anhydrase, 30 kDa; and lysozyme, 14.3 kDa (Amersham Corp., Arlington Heights, IL).
  • gels were fixed in 30% (v/v) methanol, 10% (v/v) acetic acid, vacuum dried, and exposed to Kodak XAR-2 X-ray film with an intensifying screen at -70°C.
  • Immunoblotting Immunoblotting of merozoite and recombinant lysogen proteins using monoclonal antibodies was performed as outlined (McElwain et al., 1987) using standard procedures (Towbin, H. and J. Gordon [1984] J. Immunol.
  • Parasite antigen for immunoblotting was prepared from MASP culture flasks with approximately 25% parasitemia. Briefly iRBC's and nRBC controls were collected, washed two times in cold Puck's saline-G and two times in cold PBS, resuspended in PBS, counted, and frozen at -20°G To remove hemoglobin from lysed cells, the samples were thawed and washed in cold PBS
  • Immunoblotting using bovine antisera was performed as follows: The nitrocellulose was washed three times quickly in VBS pH 7.4, 0.25% (v/v) TWEENTM20, 0.25% (w/v) gelatin (blocking buffer), incubated 4-6 hr in blocking buffer, cut into strips, and each strip reacted overnight at room temperature with immune serum diluted in blocking buffer. The nitrocellulose strips were then washed three times in blocking buffer and two times in VBS 0.1% (w/v) gelatin prior to incubation for 2 hours at room temperature with 125 I-Protein G (Amersham Corp.) in VBS pH 7.4, 0.1% (w/v) gelatin (Akerstrom, B., T. Brodin, K. Reis, and L. Bjorck [1985] J.
  • 6-CFDA-positive merozoites were obtained from frozen blood stabilates, lysed in buffer containing 50 mM Tris, 5 mM EDTA, 5 mM iodoaceta ide, 1 mM PMSF, 0.1 mM N-alpha-p-tosyl-L-lysyl chloromethyl ketone (TLCK) and 1% NP-40 (lysis buffer), and frozen at -70°C until use. Aliquots of 1 ul containing either 10 7 , 10 6 or 10 5 merozoites were spotted onto nitrocellulose filters and air dried. Corresponding numbers of similarly lysed noninfected bovine erythrocytes were spotted on for control. Nitrocellulose filters with spotted antigen were washed three times (10 min each) in buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 0.05%
  • TWEENTM20, and 0.1 mM PMSF then incubated in TNTP with 5% nonfat dry milk for 1 hr to block unbound sites.
  • Filters were washed three times in TNTP plus 5% milk, incubated in the same buffer containing 2 ug/ml of specific surface- binding MoAb (1/2 hr), washed three times, incubated for 1/2 hr in a 1:5000 dilution of rabbit anti-mouse immunoglobulin (prepared in our laboratory) in TNTP plus 5% milk.
  • the filters were incubated for 1/2 hr in TNTP plus 5% milk containing 5 x 10 6 CPM of 125 I-labeled Protein A, washed sequentially with TNTP, TNTP plus 0.1% TRITONTMX, and TNTP, then dried and examined by autoradiography.
  • Partially purified merozoites of B. bovis obtained from frozen blood stabilates were used to immunize BALB/c mice for hybridoma production. Each mouse received an initial subcutaneous immunization of 10 7 6-CFDA-positive organisms in Freund's complete adjuvant followed by 3-4 subcutaneous immunizations of 10 7
  • mice with high titers > 1:1000 received an intravenous booster immunization of 10 6 6-CFDA- positive organisms in sterile PBS.
  • Hybridoma supernates were screened first for convenience by IFA-fixed (Ross, J.P.J and K.F. Lohr [1968] Res. Vet. Sci. 9:557-562). Positive supernates were then screened by IFA-live using stabilate-derived merozoites in order to identify surface reactive MoAbs (McElwain et al. [1987]). The MoAbs were first screened on fixed infected erythrocyte preparations, and four MoAbs were selected for further evaluation because of their distinctive patterns of fluorescence.
  • BABB35A 4 precipitated a major protein of 42 kDa and a minor protein of 37 kDa.
  • BABB75 and BABB90C 4 precipitated single proteins of 60 and 85 kDa, respectively.
  • the 250 kDa protein does not enter the resolving gel in a standard 14 cm 7.5-17.5% polyacrylamide gel but is clearly resolved in a 25 cm gel.
  • An eighth protein of 25 kDa is immunoprecipitated by immune sera from two calves. Control immunoprecipitation of identically radioiodinated intact nRBC and nRBC ghosts revealed no specific bands on SDS-
  • Adsorbed immune sera was used to immunoprecipitate 35 S-methionine metabolically labeled parasite proteins which were run alongside immunoprecipitated surface-iodinated merozoite proteins in a polyacrylamide gel.
  • the immunoprecipitable 35 S antigen profile is identical in all five protected animals.
  • the 125, 98, 85, 55, 42, and 37 kDa antigens comigrate perfectly with metabolically labeled proteins.
  • the 25 kDa surface protein that is not identified by immunoprecipitation of methionine labeled antigen does comigrate with a glycoprotein that is metabolically labeled with 3 H-glucosamine.
  • An 35 S-methionine labeled 25 kDa protein can be precipitated from other 35 S-antigen preparations.
  • the immunodominant 42 kDa merozoite surface protein was further characterized as an integral membrane protein based on its hydrophobic nature in phase separated TRITONTMX-114 solution.
  • integral membrane proteins have a hydrophobic domain that allows interaction with the hydrophobic core of the lipid bilayer and with non-ionic detergents.
  • Parasite proteins were metabolically labeled in culture with 35 S-methionine and solubilized in 1% TRITONTMX-114 at 0-4°C
  • the antigen preparation was warmed above the detergent's cloud point (20°C) and separated into aqueous and detergent phases by centrifugation. Immunoprecipitation from each phase and the starting solution shows that the 42 kDa antigen partitions into the detergent phase.
  • Antiserum C151 which was used for immunoprecipitations, was collected from a spleen-intact cow 60 days after experimental infection with a cryopreserved Mexico isolate blood stabilate of R bovis.
  • R bovis proteins were metabolically labeled in microaerophilus stationary-phase culture by incubation in methionine- deficient medium for 18 to 24 hr with 10 uCi of [ 3S S]methionine per ml.
  • Antiserum C151 immunoprecipitated homologous Mexico isolate proteins biosynthetically labeled with [ 35 S]methionine with molecular weights ranging from 14,500 to greater than 200,000.
  • Proteins reactive with serum diluted 1:40 had relative molecular weights of 145,000, 120,000 (doublet), and 42,000, while the 42,000 molecular weight protein was still recognized by serum diluted 1:80.
  • Example 1 - B. bovis Proteins with Isolate-Common Epitopes Five different antisera obtained from cattle after recovery from acute infection with R bovis in Honduras were able to immunoprecipitate most of the Mexico isolate R bovis proteins precipitated by C151 antiserum. The 120,000 and 42,000 molecular weight proteins recognized by 1:40 dilutions of C151 antiserum were also recognized by 1:25 dilutions of the Honduran antisera.
  • Antiserum B85 was collected from a spleen-intact calf 25 days after experimental infection with a cryopreserved Mexico isolate of R bigemina. This antiserum, which had an indirect fluorescent-antibody titer of 1:1,600 against the Mexico R bigemina isolate, reacted with the Mexico R bovis isolate at a titer of
  • Antiserum C151 (anti-R bovis Mexico isolate) had indirect fluorescent- antibody titers of 1:5,120 and 1:640 against R bovis and R bigemina. respectively.
  • Four of the eight R bovis proteins immunoprecipitated by B85 antiserum (120,000, 59,000, 53,000, and 19,000 molecular weight) also had isolate-common epitopes.
  • the 120,000 molecular weight protein was one of the proteins recognized by C151 serum antibodies diluted 1:40.
  • Example 9 Identification of Monoclonal Antibodies Using similar techniques, additional MoAbs specific for surface-exposed epitopes on live merozoites were identified. All of the identified MoAbs are listed in Table 1. The MoAbs reacted with the outer surface of culture- or stabilate- derived merozoites in either a punctate (restricted to a discrete region on the merozoite surface) or a homogenous (over the entire surface of the merozoite) pattern when examined by IFA-live.
  • the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures.
  • the depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposit(s). All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
  • merozoites Partially purified and viable merozoites free from contaminating bovine leukocytes were obtained from frozen stabilates.
  • Merozoites (1.4 x 10 8 6-CFDA- positive) were lysed in 10 mM Tris, 1 mM EDTA (TE buffer, pH 7.4) containing 2% SDS, and the suspension was treated with DNAse-free RNAse A (100 ug/ml) followed by Proteinase K (100 ug ml).
  • Genomic DNA was isolated from the suspension by sequential phenol, phenol/chloroform, chloroform, and ether extractions followed by ethanol precipitation at 0- °C in the presence of 2 M ammonium acetate.
  • the DNA pellet was washed once with 70% ethanol, lyophilized, resuspended in TE buffer and stored at 4°C The concentration and purity of the DNA were assessed by spectrophotometry and agarose gel electrophoresis. The DNA was sheared into fragments of between 4-8 kb by repeated passages through a 25 gauge hypodermic needle (Young, R.A., B.R.
  • fragments were methylated with EcoRI methylase (Promega Biotec) and blunt-ended using the large fragment of E. coli DNA polymerase I (Klenow Fragment, Bethesda Research Laboratories, Gaithersburg, MD).
  • EcoRI linkers (BRL, Gaithersburg, MD), end-labeled with 32 P transferred from 5'-[gamma- 32 P]ATP in a kinase reaction (Huyhn et al., 1985), were ligated to fragment termini using T4 DNA ligase (Bethesda Research Laboratories). Free linkers were separated from fragments with EcoRI termini by size fractionation on a Sephacryl
  • Fragments were then ligated into the EcoRI site of the lambda-gtll expression vector which resulted in the insertion of parasite DNA into the ⁇ -galactosidase structural gene (lacZ) of the bacteriophage (Young, R.A. and R.W. Davis [1983] Proc. Natl. Acad. Sci. USA 80:1194-1198).
  • Ligated DNA was packaged into gamma phage heads (Gigapack Gold Packaging Extract, Stratagene Cloning Systems, San
  • the amplified library was stored in sterile SM buffer (0.1 M NaCl, 8 mM MgS0 4 -7H 2 0, 50 mM Tris [pH 7.5], 2% gelatin) at 4°G
  • Recombinant phage expressing proteins with surface-exposed epitopes were identified by immunoscreening plaques with MoAbs. Enough recombinant phage to give 10 5 plaque forming units (pfu)/150 mm diameter petri dish were used to infect E. coh host Y1090 by incubation at 37°C for 20 min in LB medium. Infected cells were added to LB top agar (55°C) containing 100 ug/ml ampicillin and 10 mM MgCl 2 and plated out on 150 mm diameter LB agar plates. Plates were incubated at 42°C for 4 hr to allow plaque formation without concomitant expression of fusion protein.
  • LacZ-directed gene expression was then switched on by overlaying each plate with a dry nitrocellulose filter saturated previously with 10 mM IPTG and incubating the plates at 37°C for 8-10 hr. After incubation, nitrocellulose filters with bound proteins were marked, removed from the plates, and processed as described previously for dot blot immunoassay. Single plaques expressing recombinant surface epitopes of interest were identified by autoradiography, picked from plates, and rescreened and picked three more times to insure purity of the recombinant phage, stability of the DNA insert, and reliability of recombinant protein expression.
  • Other hosts, such as Salmonella can be transformed by suitable procedures well known to those in the art.
  • E. coli host strain Y1089 was lysogenized with lambda-gtll (control) and each of the recombinant clones using standard procedures (Huyhn, T , R.A. Young, and R.W. Davis [1985] "Constructing and Screening cDNA Libraries in lambda-gtlO and lambda-gtll," In: DNA Cloning. Vol. 1 : A Practical Approach
  • Lysogenized bacteria were examined by dot blot immunoassay in order to determine the ability of clones to produce recombinant protein after induction with IPTG.
  • MoAb 23.70.174.83 was purified from ascitic fluid by ammonium sulfate precipitation and DEAE cellulose chromatography and then coupled to Sepharose 4B for immunoaffinity purification of rBo44-15. Solubilized and sonicated rBo44-15 lysogen preparations were applied to the affinity column, the column was washed repeatedly, then adherent recombinant protein was eluted with 0.1 M diethylamine (pH 11.5) containing 0.5% deoxycholate. Elutes were collected directly into 1 M Tris (pH 8.5) then dialyzed against PBS to remove detergents.
  • rBo44-15 from affinity column chromatography contained several high M,. proteins ranging from approximately 94 Kd to >165 Kd as well as several lower M,. proteins ranging from 26 Kd to 50 Kd.
  • Western blot analysis of column-purified protein preparations revealed two major bands of reactivity at M r 165 Kd and 150 Kd that correspond to two major bands present in silver-stained gels.
  • several bands of lower M r 26-31 Kd
  • Hyperimmune bovine serum (KLK C151) and preimmune sera were used as positive and negative controls, respectively.
  • Sera from calves immunized with either rBo44-15 or ovalbumin were used to immunoprecipitate metabolically radiolabeled merozoited proteins (McElwain et al., 1987) in order to verify the specificity of the antibody response.
  • Antibody titers in serum from calves immunized with partially purified rBo44- 15 varied from 10 "2 to 10 "5 as evidenced by IFA-live.
  • preimmune serum B452-pre
  • serum from ovalbumin-immunized calves showed reactivity with live merozoites at dilutions of 10 "1 and 10 "2 , respectively.
  • Example 15 Methods and Materials for Construction of DNA Probe
  • Parasites and DNA Isolation Strains of parasites used in this study include a Mexico (M) and Australia (S strain) isolate of R bovis and a Mexico isolate of R bigemina.
  • Babesia bovis (M) DNA for both the genomic library preparation and analysis of clones was derived from infected bovine erythrocyte cultures washed three times in phosphate buffered saline (PBS), pH 7.2, followed each time by centrifugation at 400xg.
  • Babesia bovis (S) and R bigemina DNA was similarly derived from infected calf blood depleted of buffy coats by three washes in PBS.
  • Infected erythrocytes for isolates were differentially lysed in nine volumes of 0.42% NaCl, infected ghosts were pelleted at 400xg, lysed in 5 volumes of 10 mM Tris-HCl (pH 7.5), 10 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, and 1% sodium dodecyl sulfate (SDS), incubated 16 hr with proteinase K (100 ug/ml), extracted with phenol:chloroform:isoamyl alcohol (24:24:1).
  • DNA in the aqueous phase was spooled after addition of 2 volumes of cold ethanol, spooled DNA was dried and resuspended in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA (TE), treated with RNases A and Tl (15 ug/ml, 15 units/ml, respectively). The solution was reextracted, spooled, dried, and resuspended in TE for use.
  • bovine leukocyte DNA cells in the buffy coat of uninfected blood were processed similar to infected erythrocytes.
  • DNA from phage plaques was adsorbed onto nitrocellulose and replicate filters were differentially hybridized to nick translated DNA (2X 10 6 cpm/ml) from either R bovis (M) or R bigemina in 6X SSPE (IX SSPE:150 mM NaCl, 10 mM NaH 2 P0 4 , 1 mM EDTA, pH 7.4), 100 ug/ml denatured salmon sperm DN and 1% SDS at 65°C for 16 hr. Filters were washed twice in 2X SSPE and 1% SDS at room temperature for 20 min, and twice in 0.1X SSPE and 0.1% SDS at 65°C for
  • restriction fragments were separated electrophoretically on 0.7% agarose gels and transferred to nylon filters. Filters were then hybridized, as described above, in the presence of 10% dextran sulfate, to lambda-gtll recombinant DNA or preparatively isolated insert DNA that was radioactively labeled. Final wash stringency was either 65°C or 50° as indicated.
  • DNA extracted as described above was spotted onto nylon membranes. Membranes were dried at room temperature, saturated with 0.5 M NaOH and 1.5 M NaCl, neutralized in 1 M ammonium acetate and 0.02 M
  • DNA-DNA hybridization assays are based on the fact that single-stranded DNA will reanneal only with a complementary strand of DNA whose sequence is homologous. DNA probes have been used as a means of detecting various infectious agents, and some are now used routinely in clinical microbiology laboratories. The identification of DNA sequences of Babesia spp. makes it possible to create DNA probes for the identification of these species. Therefore, one application of the identification and isolation of genomic sequences which encode babesial antigens is the use of the DNA fragments as DNA probes.
  • lambda-gtll genomic DNA library of Babesia bovis was screened to identify DNA probe candidates for direct detection of the parasite in blood or ticks infected with the parasite.
  • Two sequences (lambda-Bo6 and lambda-Bo25) demonstrated superior sensitivity and were analyzed in more detail.
  • the insert size of lambda-Bo6 is 2.75 kilobase pairs (kb).
  • Bo25 insert was not possible since one of the EcoRI insert sites was lost during cloning.
  • digestion of lambda-Bo25 with EcoRI and Kpnl produces a fragment of 2.2 kb which is specific to this clone compared to wild type lambda- gtll. Since the Kpnl site in lambda-gtll occurs approximately 1 kb from the EcoRI cloning site, a minimum estimated size of the insert is 1.2 kb.
  • Inserts from the lambda clones were excised with EcoRI (lambda-Bo6) or SstI and Kpnl (lambda-Bo25) and cloned into the plasmid BS + (Stratagene), producing the two clones pBo6 and pBo25. Insert fragments preparatively isolated from these plasmid clones were radioactively labeled and used in dot and Southern blot analyses.
  • pBo ⁇ detected 100 pg of both a Mexico and an Australia isolate of R bovis, but pBo ⁇ also detects 1.0 ng of B. bigemina DNA under identical conditions.
  • a unique characteristic of pBo ⁇ is that it hybridizes to a 7.4 kilobase band in uncut genomic DNA of both R bovis and R bigemina. Similarity of restriction enzyme patterns of the pBo6 sequence in genomic DNA from both geographic isolates suggests that this sequence is well conserved between geographic isolates of R bovis. Thus, this sequence is a candidate DNA probe for detecting R bovis infections in cattle and ticks.
  • pBo25 exhibited no detectable hybridization to bovine or R bigemina DNA.
  • This sequence detected 100 pg of homologous Mexico isolate DNA but under identical conditions the sensitivity was reduced to 1 ng for Australia isolate DNA. Restriction enzyme analysis of the pBo25 sequence showed major differences in the number, size, and intensity of bands between the two R bovis geographic isolates tested. Thus, this sequence can distinguish geographic isolates of R bovis.
  • DNA probes can be labeled in a variety of ways.
  • the DNA fragment preparation is adjusted to a concentration of 1 mg/ml (TE) and is mixed with photo-activatable biotin (PAB) at a ratio of 1:3 (DNAPAB) in a 1.5 ml Eppendorf tube.
  • the tube is placed in an ice bath 10 cm below a 275 W (GE RSM) sunlamp and the DNA + PAB is irradiated for 15 min.
  • the DNA solution is then mixed with an equal volume of 0.1 M Tris-Cl (pH 9.0) and the volume adjusted to 100 ul with H 2 0.
  • the unincorporated PAB is extracted from the DNA by the addition of an equal volume of 2-butanol, vortexing, centrifuging briefly, and withdrawing the lower aqueous phase with a Pipetman. The extraction can be repeated to remove any traces of unbound PAB.
  • 3 M NaOAc pH 5.6
  • the labeled DNA is precipitated by the addition of 3 volumes of ethanol. After the sample is cooled at -70°C for 15 min, the precipitated DNA is recovered by centrifugation for 10 min.
  • the DNA pellet is dissolved in 10 mM Tris (pH 7.9) and 0.1 mM EDT The labeled probe DNA remains stable for 1 year if stored at -20°C
  • a non-radioactive method of labeling the DNA probes may be desirable because: 1) the photoactivatable reactions are simple and rapid, 2) the sensitivity is as high as 32 P-labeled probes, 3) the PAB-labeled probes have a long storage life, 4) these probes are relatively inexpensive, and 5) detection of bound probes is by simple colorimetric methods.
  • the radioactive labeling of probes requires the use of 32P, which has a very short half-life (14 days) and is thus unstable and expensive. The use of radioactive probes would be limited because of cost, the dangers of radioactivity, strict requirements for disposal, and the need for licensing. However, if for some reason the biotin-HRP method of labeling is not acceptable, the DNA fragments can be labeled with [gamma-P] 32 deoxy CTP by standard nick translation methods.
  • the cloned insert DNA was excised from the lambda-gtll vector and recloned into the plasmid BS + (Stratagene), producing the clone pBo44-15.
  • DNA templates for sequencing the insert of pBo44-15 were obtained by creating deletion libraries of this clone using ex ⁇ nuclease III and mungbean nuclease. A different deletion library was obtained starting at each end of the clone, which allowed sequencing of both strands of the insert DNA. The DNA sequence was obtained using the Sanger dideoxy method.
  • the sequence of Bo44-15 insert DNA is shown in Figure 1.
  • the insert is
  • the amino acid sequence shown represents the one long open reading frame identified in the sequence.
  • the open reading frame begins at position 1 and encodes a stop codon (TAA) beginning at position 568.
  • TAA stop codon
  • This reading frame is in correct register for expression as a fusion protein of ⁇ -galactosidase in lambda-gtll, provided the clone is in the correct orientation.
  • a notable feature of this open reading frame is that it would encode a 24 amino acid sequence beginning at AA position 85 which is tandemly repeated beginning at AA position 109. Comparison of these two putative repeats shows only two positions that differ between the repeats as shown below: 85 PQRPAETQQTQDSAAPSTPAAPSP 108 109 PQRPAETQQTQDSTAPGTPAAPSP 132
  • Numbers represent the beginning and ending amino acid position in the open reading frame for each repeat. Letters are the single letter code for amino acids. Asterisks below the aligned repeats indicate amino acid differences between the two repeats.
  • repeat amino acid sequence is that such repeats are often immunodominant epitopes in surface proteins from a variety of other protozoan parasites, and they induce antibodies that protect against the diseases.
  • the DNA sequences coding for two other of the R bovis proteins have also been discovered.
  • the DNA sequence for the R bovis surface proteins of 42 kDA and 60 kDA are shown in Figures 2 and 3, respectively. These sequences, or portions of the sequences, can be used as DNA probes as described in Examples 16 and 17. Also, the proteins produced from cells transformed with these sequences, or portions of these sequences, can be used for vaccines or in the preparation of monoclonal antibodies as described in the examples which follow. The procedure for obtaining these sequences are described below:
  • B. bovis cDNA Expression Library Erythrocytes from asynchronous R boyis-infected blood cultures were washed three times in Puck's saline G and stored frozen in liquid nitrogen. Cells were thawed in lysis buffer containing 0.2 M NaCl, 0.2 M Tris-HCl pH 7.5, 1.5 mM MgCl 2 , 2% SDS (w/v), and 200 ⁇ g/ml Proteinase K and then incubated in lysis buffer at 46°C for two hours (Bradley, J.E., G.A.
  • Lambda rBv42 phagemid DNA was isolated from bacteria by anion exchange chromatography (Qiagen Inc., Studio City, CA) and restriction enzyme digested by standard methods (Maniatis, T, E.F. Fritsch, and J. Sambrook [1982] Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
  • Vaccines may be produced from the polypeptides expressed by the parasites themselves or by cells which have been transformed with DNA fragments from Babesia. By introducing these polypeptides, along with a pharmacologically suitable vehicle or adjuvant, into the animal host, that host can be induced to generate immunological protection against Babesia. The preparation of such a vaccine composition is within the skill of one trained in the medical and immunological sciences. Vaccines may utilize entire polypeptides or epitopes with immunological activity.
  • mice can be immunized with antigens of, or cells expressing antigens of, Babesia.
  • the antigens used for this immunization can be those which are identified and described in the previous examples.
  • the techniques employed to accomplish this immunization procedure are familiar to those skilled in this art.
  • the spleens can then be removed from the immunized mice and the cells therefrom fused to SP-2 myeloma cells using polyethylene glycol.
  • the desired hybrid cells can then be selected by adding hypozanthine-aminopterin-thymidine to the medium.
  • the surviving cells can then be tested for antibody production.
  • the testing for antibody production can be accomplished using IFA, ELISA, immunoblot, and/or immunoprecipitation procedures.
  • the monoclonal antibodies can be used to test for the presence of Babesia antigens in a sample of biological fluid.
  • Other monoclonal antibodies to Babesia antigens can also be used.
  • the testing procedure involves contacting the biological fluid with a composition containing one or more of the monoclonal antibodies. If Babesia antigens are present in the biological fluid, then a reaction will occur and this reaction can be detected and quantified by fluorescence or other means.
  • Anti-Babesia antibodies can be detected in a fluid sample from a bovine suspected of containing these antibodies by performing ELISA procedures on the clinical samples.
  • Generalized ELISA procedures are well known to those skilled in the art.
  • the ELISA procedures or other simple diagnostic procedures of the subject invention could utilize as antigens, for example, whole cell or cell lysate using recombinant microorganisms which express Babesia antigens.
  • the biological sample When the biological sample is contacted with the whole cell or cell lysate microorganisms, this contacting is done under conditions which will promote antigen/antibody immunocomplex formation between antigens expressed by the microorganism and antibodies present in the sample.
  • the resulting immunocomplex can be readily detected utilizing standard labeling procedures.
  • the deposit was accompanied by: a scientific description a proposed taxonomic description indicated above.
  • the strain will be made available if a patent office signatory to the Budapest Treaty certifies one's right to receive, or if a U.S. Patent is issued citing the strain.
  • the strain will be maintained for a period of at least 30 years after the date of deposit, and for a period of at least five years after the most recent request for a sample.
  • the United States and many other countries are signatory to the Budapest Treaty.
  • the deposit was accompanied by: a scientific description X a proposed taxono description indicated above.
  • the strain will be made available if a patent office signatory to the Budapest Treaty certifi one's right to receive, or if a U.S. Patent is issued citing the strain.
  • the culture should die or be destroyed during the effective term of the deposit, it shall be y responsibility to replace it with a living culture of the same.
  • the strain will be maintained for a period of at least 30 years after the date of deposit, and a period of at least five years after the most recent request for a sample.
  • the United States a many other countries are signatory to the Budapest Treaty.

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Abstract

Identification de nouvelles protéines de la surface des mérozoïtes de Babesia bovis, anticorps monoclonaux de ces protéines, gènes de codage de ces protéines, ainsi que l'utilisation de ces nouvelles protéines, des clones d'ADN recombinant et des anticorps monoclonaux pour détecter, traiter et prévenir la babésiose.
PCT/US1990/001812 1989-04-04 1990-04-04 Proteines et genes clones nouveaux utiles pour le diagnostic et la prophylaxie de la babesiose WO1990011776A1 (fr)

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EP0834567A3 (fr) * 1996-10-01 1999-03-31 Corixa Corporation Composés et méthodes pour le diagnostique et le traitement d'infections à Babesia microti
WO1999029869A1 (fr) * 1997-12-11 1999-06-17 Corixa Corporation COMPOSES ET PROCEDES DE DIAGNOSTIC ET DE TRAITEMENT DE L'INFECTION AU $i(B. MICROTI)
US6183976B1 (en) 1996-10-01 2001-02-06 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
US6451315B1 (en) 1996-10-01 2002-09-17 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
US6569433B1 (en) 1996-10-01 2003-05-27 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
WO2005026199A3 (fr) * 2003-09-14 2005-09-15 Univ Utrecht Holding Bv Vaccin a base de piroplasmide
CN101974621A (zh) * 2010-09-17 2011-02-16 浙江大学 一种牛巴贝斯虫lamp检测方法
WO2013059795A1 (fr) * 2011-10-20 2013-04-25 Immunetics, Inc. Dosage sensible et spécifique pour babesia spp.

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FR2655853B1 (fr) * 1989-12-20 1994-06-10 Centre Nat Rech Scient Procede de culture intensive, in vitro, de souches de babesia divergens, procede de preparation d'exoantigenes et vaccin contenant ces antigenes.
AU645641B2 (en) * 1990-12-21 1994-01-20 Commonwealth Scientific And Industrial Research Organisation Babesial protease antigen
ZA93236B (en) * 1992-01-15 1993-08-16 Commw Scient Ind Res Org Babesial rhoptry antigens.

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6569433B1 (en) 1996-10-01 2003-05-27 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
US6183976B1 (en) 1996-10-01 2001-02-06 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
US6214971B1 (en) 1996-10-01 2001-04-10 Corixa Corporation Compounds and methods for the diagnosis and treatment of Babesia microti infection
US6306396B1 (en) 1996-10-01 2001-10-23 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
US6451315B1 (en) 1996-10-01 2002-09-17 Corixa Corporation Compounds and methods for the diagnosis and treatment of B. microti infection
EP0834567A3 (fr) * 1996-10-01 1999-03-31 Corixa Corporation Composés et méthodes pour le diagnostique et le traitement d'infections à Babesia microti
WO1999029869A1 (fr) * 1997-12-11 1999-06-17 Corixa Corporation COMPOSES ET PROCEDES DE DIAGNOSTIC ET DE TRAITEMENT DE L'INFECTION AU $i(B. MICROTI)
US7799330B2 (en) 2003-09-14 2010-09-21 Universiteit Utrecht Holding B.V. Piroplasmid vaccine
US7465459B2 (en) 2003-09-14 2008-12-16 Universiteit Utrecht Holding B.V. Piroplasmid vaccine
JP2007527707A (ja) * 2003-09-14 2007-10-04 ユニバーシテイ・ユトレヒト・ホールデイング・ベー・ベー ピロプラスミドワクチン
WO2005026199A3 (fr) * 2003-09-14 2005-09-15 Univ Utrecht Holding Bv Vaccin a base de piroplasmide
CN101974621A (zh) * 2010-09-17 2011-02-16 浙江大学 一种牛巴贝斯虫lamp检测方法
WO2013059795A1 (fr) * 2011-10-20 2013-04-25 Immunetics, Inc. Dosage sensible et spécifique pour babesia spp.
US10254293B2 (en) 2011-10-20 2019-04-09 Immunetics, Inc. Sensitive and specific assay for Babesia spp

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