WO1988007374A1

WO1988007374A1 - Vaccine against brucella abortus

Info

Publication number: WO1988007374A1
Application number: PCT/US1988/001000
Authority: WO
Inventors: Leslie G. Adams; Richard P. Crawford; Donald S. Davis; Thomas A. Ficht; Roger Smith, Iii; Blair A. Sowa; Joe W. Templeton; John D. Williams; Albert W. Wu
Original assignee: The Texas A & M University System
Priority date: 1987-03-30
Filing date: 1988-03-29
Publication date: 1988-10-06
Also published as: GB2227936A; NZ224065A; CA1337269C; AU623854B2; JPH02503196A; MX166766B; AU1573288A; EP0357642A1; GB2227936B; GB8921650D0

Abstract

The present invention relates to an improved vaccine against $i(Brucella abortus). Specifically, the invention is a novel vaccine in which specific antigens of $i(Brucella abortus) are combined to induce an immunological response which provides protective immunity yet permits differentiation between field strain infected and vaccinated cattle.

Description

VACCINE AGAINST BRUCELLA ABORTUS

The present invention relates to an improved vaccine against Brucella abortus. Specifically, the invention is a novel vaccine in which specific antigens of Brucella abortus are combined to induce a-n immunological response which provides protective immunity yet permits differentiation between field strain infected and vaccinated cattle.

Brucella abortus is a bacterial organism which causes bovine brucellosis which is characteri2ed by spontaneous abortion and chronic infection within the lymph nodes and in the mammary glands of cattle. This disease causes extensive economic loss due to abortion, the birth of weak debilitated calves, decreased milk production and infertility.

To reduce the incidence and economic loss caused by bovine brucellosis, several different vaccines have been used in the past. The vaccines are generally prepared using either live or killed strains of Brucella. The use of these vaccines has several disadvantages. One disadvantage is that currently there is no consistently effective means for distinguishing strain 19 vaccinated cattle from those infected by pathogenic strains of Brucella abortus. Both the vaccine and the pathogenic strain cause the production of cross-reacting antibodies which serve as the basis for current serological tests for brucellosis.

Other disadvantages of some of the killed vaccines include: the need to inject the vaccine twice; the instability of several of the strains used to create the vaccines; the vaccines frequently cause large tissue reactions at the site of injection; the immunity induced is also short lived; and the protective immunity is suboptimal.

Crude protein derivatives have also been used experimentally to produce protective, immunity. These crude extracts have the same disadvantages as the killed and live vaccines. Additionally, the extracts are crude and contain cellular components not required to induce protective immunity. This can cause further confusion in differentiating between vaccinated and field strain infected cattle.

Finally, the currently approved live vaccine strain S19 is pathogenic to humans requiring that the vaccine be administered only by veterinary professionals.

The present invention is an improved vaccine against Brucella abortus which permits differentiation between vaccinated and field strain infected cattle. The vaccine consists of a combination of the major antigens from Brucella abortus as immunizing agents with at least one of the major antigens absent from the combination. The antigen combination can take several forms to include purified cellular proteins, either extracted from B. abortus cells or produced through recombinant techniques; synthetic peptides containing the appropriate antigenic epitopes; transposon mutants of IL_±. abortus and modified viral vectors.

There are several key antigens which are common to the pathogenic strains of Brucella abortus. The antigens are the outer membrane proteins (Omp) I, II, III, the 0 polysaccharide, and the 7kd and 8kd envelope proteins. Each of these antigens, either alone or in combination, induces the production of antibodies in vaccinated cattle. Additionally, cattle injected with vaccines having the major O polysaccharide antigen deleted from the immunizing agent do not produce antibodies which react with standard serological assays. Therefore, vaccinated cattle can be differentiated from field strain infected cattle.

The immunizing agent used in the vaccines can take several forms. The immunizing agent can consist of purified antigens isolated from: killed, attentuated, or pathogenic Brucella abortus strains; synthetic peptides constructed to present the optimal antigenic epitopes contained in Omps I, II and III and envelope proteins 7kd and 8kd; crude or purified antigens extracted from genetically modified E^. coli expressing these antigens; modified live B^. abortus strains having the DNA encoding one or more of the antigens deleted from its genome; or a live recombinant viral vector with the genetic material for one or more of the major Brucella antigens inserted into its genome. The important feature of each of these immunizing agents is that it can induce an immunogenic response that protects against B^ abortus infection and provide a means of distinguishing vaccinated cattle from field strain infected cattle.

The killed or synthetic vaccines have .the additional advantages of sterile immunity and nonpathogenicity to humans and animals. Therefore, individuals other than certified veterinarians can use the vaccines safely. Similarly, a live recombinant viral vector also presents a minimal biological hazard if an appropriate (nonpathogenic) vector is used.

The use of modified live Bj_ abortus has the advantage of providing longer-lived immunity than killed bacteria. Vaccines using modified live Brucella abortus may still exhibit some pathogenic activity in humans and should be handled by certified professionals.

As noted above, the immunizing agent of the present invention can be in several forms. The following is a discussion of the preferred methods which can be used to create different immunizing agents of this invention. It will be obvious to those skilled in the art that deviations from the procedures discussed below are possible without departing from the basic scope of the invention.

I. Purified Proteins From Brucella abortus

A vaccine against Brucella abortus was prepared from killed B__Ϊ_ abortus as follows.

Cultures of Brucella abortus strain S2308, obtained from Dr. Billy Deyoe, USDA/NADC, Ames, IA were fermenter grown in Trypticase soy broth to an OD_55Q of 106-133. The cells were harvested, rendered nonviable by irradiation with 1.38 Mrad of Co gamma radiation at 4 C, and frozen at -20°C. The cells were then thawed and subjected to osmotic and sonic shock to rupture the cells and purified cell envelopes were collected by the method described by J.F. Lutkenhaus, 131 J. Bacteriol. 631-637 (1977), with the following modifications: 10 milliliter aliquots of thawed suspended cells were centrifuged at 12,000 rpm for 5 minutes at 4°C to remove the cells from suspension. The resulting supernatant was then discarded. The pelleted cells were resuspended in 12 milliliters of Lutkenhaus buffer and subjected to 20,000 Hz sonic disruption for 6 consecutive periods of both 3 minutes at 300 watts and 2 minutes at 50 watts of power. During the sonication process, the suspension was held in an ice bath. The suspension was centrifuged at 12,000 rpm for 5 minutes to remove any unlysed cells and the. resulting pellet was discarded.

The supernatant from above was centrifuged at 30,000 rpm at 4°C for 45 minutes and the resulting supernatant was discarded. The pellets were resuspended in 1.5 milliliters of Lutkenhaus buffer by sonication at 20,000 Hz at 100 watts of power for 1-2 minutes. The process was repeated and the protein concentration of the resulting suspension was determined using the BCA protein assay kit available from The Pierce Chemical Company and comparing the readings to the mean of values obtained using controls of bovine serum albumin and ovalbumin standards. The process yielded 6 to 9 milligrams of protein per milliliter.

Envelope proteins 8kd and 7kd were isolated by excising bands of proteins from sodium dodecylsulfate polyacrylamide gel electrophoretograms (SDS-PAGE) of cell envelope preparations. The following procedure was used:

Cell envelope preparations prepared by the same method described above containing 12 milligrams of protein were added to 2 volumes of 2x concentration SDS-PAGE sample buffer containing: 12.6% v/v glycerol; 10.0% v/v β-mercaptoethanol; 0.004% w/v bromophenol blue; 5% w/V recrystalized SDS in 0.236 M Tris-HCL buffer pH 6.8. The resulting solutions were mixed and boiled for 5 minutes. The sample buffer was mixed with 5 milliliters of molten 2% agarose and divided into 10 equal parts that were loaded uniformly onto each of 10, 1.5 millimeters x 18 centimeters x 18 centimeters ISO-DALT electrophoretic gel plates. The plates were manufactured by Electro

Nucleonics of Oalc Ridge, Tennessee and contained an 11.5% to 18% exponential gradient of acrylamide and bisacryla ide which were prepared as described by Sowa et al., 153 J. Bacteriol. 962-968 (1983). The reagents for SDS-PAGE electrophoresis were prepared as described by

O'Farrell, 250 J. Biol. Chem. , 4007-4021 (1975), with the exceptions noted above.

After electrophoresis for 17 hours at 140 volts constant voltage, the acrylamide gels were removed from the plates and gently stained with Coomassie Brilliant Blue using the procedure outlined by Hunkapiller et al., 91 Methods in Enzymology 227-236 (1983). The gels were destained in 20% ethanol.

Stained bands of the appropriate molecular weight containing the desired proteins were carefully excised -from the gels with a sharp blade and quickly frozen at -20 C. The Omps were identified as the dominant bands on the gel and were further identified by their apparent molecular weights: Omp I = 88,000 daltons, Omps II = 36,000 daltons, and Omp III = 26,000 daltons. Proteins 8kd and 7kd were identified as intense bands at apparent molecular weights of 8,800 and 7,500 daltons, respectively. Proteins 8kd and 7kd had no corresponding bands in cell envelope preparations from S19 Brucella abortus cells. Proteins were electroeluted from the excised gel bands by the method of Hunkapiller, infra, and frozen at -4°C. Each batch of excised protein was quantified by laser densitometry. Using Coomassie Brilliant Blue stained SDS-PAGE gels containing aliquots of each protein isolate, the results were compared to protein standards using bovine serum albumin and soybean trypsin inhibitor. Standard curves were constructed using the values obtained for each standard protein and the amounts of isolated experimenta^'l protein were calculated by comparing optical density values for each stained band to the standard curve. Values obtained using two standard curves were averaged.

A vaccine containing 7kd and 8kd envelope proteins was prepared by pooling 1680 micrograms of each protein in electroelution buffer. The proteins were then frozen and evaporated to dryness in a vacuum centrifuge. The resultant mixture of lyophilized Coosmassie Brilliant Blue stained proteins and electroelution buffer salts was then incorporated with an adjuvant comprising 0.25 micrograms monophosphoryl lipid A, 0.25 micrograms cell wall skeleton, 0.25 micrograms of trehalose dimycolate, 0.02 milliliters squalane and 0.002 milliliters of Tween 80 obtained from RIBI Immuno Chem Research, Inc. of Hamilton, Montana. A second vaccine was also prepared using the above procedure to isolate and purify Omp I, Omp II and Omp III from the O antigen deficient B_^_ abortus strain described below.

Tests were conducted using both of the above vaccines. In each test, 27 nonpregnant heifers were vaccinated with 30 micrograms of vaccine containing purified Brucella antigens Omp I, Omp II and Omp III intramuscularly (IM) with a follow-up injection of 30 micrograms IM 60 days after the initial inoculation.

After 18 weeks following the first inoculation, production of antibodies to vaccine proteins was detected by the enzyme-linked immunosorbent assay (ELISA) in the inoculated cattle; however, only one heifer inoculated with the Omp I, Omp II and Omp III vaccine had antibodies detected by standard serological tests. None of the cattle inoculated with the 7kd and 8kd vaccine had antibodies detected by standard serological tests.

Nonpregnant heifers were vaccinated with a vaccine using cell envelopes of the O deficient strain in the same manner as the purified proteins described above. After 18 weeks, antibody production was detected by the ELISA procedures in two-thirds of the cattle vaccinated. None of the cattle had antibody activity for brucellosis using standard USDA serological assays.

In the same experiment, 27 nonpregnant heifers were inoculated with normal strain 19. Each heifer received g one subcutaneous injection of 5x10 CFU (colony forming units) as a control. After 18 weeks, only four cattle exhibited antibody production as detected by ELISA tests, and six of the cattle had antibody activity as detected by standard serological tests for Brucella abortus.

The above vaccines are merely illustrative of the types of vaccines that can be prepared using this process. Those skilled in the art will recognize that it may be possible to selectively combine various Brucella abortus antigens to produce effective vaccines that can be used to produce immunological responses in cattle that are vaccinated and still be differentiated from those of field strain infected cattle. For example, it may be desirable to produce a vaccine which contains the 0 antigen and induces the production of antibodies which react with standard serological tests. By deleting one of the other Brucella antigens, vaccinated cattle can still be distinguished from field strain infected cattle using tests to detect the absence of antibodies corresponding to the deleted antigen.

II. Fusion Proteins

An alternate method for producing Brucella immunizing agents has been developed using Escherichia coli to produce selected antigens. The genetic sequence encoding one or more of the Brucella antigens is inserted into the E. coli and production of antigens is induced. The E. coli cells are lysed and the vaccine is prepared using whole cell extracts. Alternately, the fusion proteins can be purified and used as an immunizing agent for the vaccine.

Modified E^ coli strain MC 4100 have been developed which can be used to produce the improved vaccine of the instant invention. These organisms have been found to express Brucella DNA encoding portions of antigens 7kd, Omp I, Omp II and Omp III. The organisms have been deposited with The American Type Culture Collection and have ATCC Deposit Numbers as follows:

STRAIN ATCC DEPOSIT NO.

E. coli MC 4100 Omp 1.11 67355

E. coli MC 4100 Omp 2.63 67356

E. coli MC 4100 Omp 3.10 67354 Ej. coli MC 4100 Omp 7.01 67357

The recombinant E. coli described above were produced as follows. Pathogenic S2308 and attenuated S19 strain Brucella abortus were grown as described by Alton et al., Laboratory Techniques in Brucellosis, World Health

Organization, Monograph Series, No. 55, 2nd Ed.., 11-63 (1975). The cells were then harvested and the DNA extracted and purified-by CsCl banding using the method described by Maniatis et al., Molecular Cloning: A Laboratory Manual, 1st ED., 269-294 (1980). Random-sized fragments of Brucella abortus DNA with an average length of 1 to 2kd were generated by partial digestion using pancreatic DNAse as described by Young et al., 80 Proc. Nat'l. Acad. Sci. USA, 1194-1198 (1983).-

EcoRI linkers were ligated to the ends of the DNA fragments and then treated with EcoRI restriction endonuclease. Then the fragments were electrophoresed on a 1% agarose gel and fragments between l.Okd and 2kd were purified by electroelution in the manner described by Smith, 65 Methods in Enzymology, 371-380 (1980).

The eluted fragments were then inserted into EcoRI and phosphatase treated Lambda gtll DNA with T4 DNA ligase in the method described by Young et al. The Lambda gtll DNA was obtained from Pro-Mega Biotec, Madison, Wisconsin. Lambda gtll is used because of its unique EcoRI site which interrupts the Lac Z. gene encoding β-galactosidase 53bp upstream from the termination codon. Phages containing antigenic inserts generate an inactive B-galactosidase fusion protein and can be distinguished from nonrecombinant phages by their inability to produce blue plaques on a Lac Z- host grown on X-Gal plates.

Phage stocks were then grown at 42°C on E___ coli Y1088 as described by Young et al., 222 Science 778-782 (1983). The ability of the Lambda gtll expression vector to form lysogens was exploited to maximize the yield of the fusion protein by using the host E^ coli MC 4100.

Specific antigen producing clones were screened by infecting Ej_ coli MC 4100 with the Lambda gtll recombinants. The lysogen colonies were screened as described by Young et al. , 80 Proc. Nat'l. Acad. Sci. USA, 1194-1198 (1983). Phage plaques were also screened on a lawn of E_j_. coli Y1090 using the method described by Young et al., 222 Science 778-782 (1983). These strains of E. coli are preferred because they are deficient in the Ion protease. Antigen degradation is thereby reduced. Lysogen colonies were grown at 32 C, followed by induction at 42 C. An increased antibody binding was generally observed, however, by screening phage plaques wherein the cceellllss weerre. incubated at 42 C for 3-4 hours following infection,

The following formula was used to calculate the number of phages needed to assure production of the desired sequence: N = In (l-P)/ln (1-f) where P is the probability of finding a particular sequence, f is the frequency of this sequence in the genome of B_^ abortus and assuming a genome size of 4x10 bp with an average gene size of 1000 bp, and n is the number of plaques required. A library of 120,000 recombinant phages is required to ensure complete recovery and expression of the Brucella genome; (assuming fragments can be inserted in only one out of three reading frames and one of two orientations to provide proper expression) . The entire library of recombinants can be easily screened on five to ten 15 centimeter plates.

A dry nitrocellulose filter which was previously saturated with IPTG (isopropyl thio-β-D-galactopyranoside) was placed over the plates and the plates were incubated at 38°C for 2 to 8 hours. The filter was previously saturated with IPTG which is an inducer of Lac Z. transcription and will also cause the transcription of the foreign DNA inserts of the recombinant phage. The position of the filter was marked and the filter removed and washed with TBST (50mM Tris HC1, pH8.0/150mM NaCl/0.05%(v/v)Tween 20). The filter was incubated with TBST containing 3% gelatin for 30 minutes at room temperature. Identification of the recombinant phage which express B^_ abortus surface antigens was performed by incubating the filter in TBST plus 1% gelatin containing antisera (1:500) raised against outer membrane proteins of B. abortus. The filters were washed three times with TBST and incubated with alkaline phosphatase conjugated second antibody (1:7500) in TBST plus 1% gelatin. Following this incubation, the filters were washed four times for 15 minutes each in TBST and transferred to color development solution as described by Schuurs et al., 81 Clin. Chim. Acta. , 1-40 (1977) . Lysates were prepared using the procedure outlined below from cells induced to produce recombinant antigen production and were examined by western blot analysis in order to confirm the identity of the expressed DNA sequences. Aliquots containing 10 to 50 micrograms of crude cell extract were subjected to SDS-PAGE electrophoresis and the proteins were then transferred to nitrocellulose, Towbin et al., 76 Proc. Nat'l. Acad. Sci. USA, 4350-4354 (1979). Nonspecific binding sites on the nitrocellulose were blocked by incubation in TBST + 3% gelatin. The nitrocellulose filters were rinsed with TBST, then incubated for at least four hours in TBST + 1% gelatin containing the appropriate antisera at a 1:250 dilution. E^_ coli lysates of various concentrations in TBST were preincubated for 30 minutes with the antibody prior to incubation with blots. The mixture was removed and the blots were washed twice with TBST, and alkaline phosphatase linked second antibody (1:7500 dilution) was added. Following a 30 minute incubation at room temperature (approximately 25 C), the blots were washed extensively as above and color developing reagents were added.

After a third incubation of the blots at room temperature for 30 minutes, the amount of bound second antibody was measured. Membrane preparations containing B. abortus outer membrane proteins were used as positive controls and nonrecombinant β-galactoside protein was used as a negative control. The recombinants containing the outer membrane protein epitopes were successful in binding with these antibodies. Recombinants expressing the majority of the protein epitopes will be used first in experiments to determine protective immunity. Recombinants expressing fewer epitopes can be utilized to determine which portions of the protein are dominant in protective immunity. This can be achieved by examining the immune response in cattle vaccinated with the larger fusion proteins. Cattle which are protected from subsequent challenge can be examined via Western blot analysis and blastogenic response to individual fusion products of reduced size or partially digested proteins purified from B_i_ abortus. In this fashion, specific epitopes responsible for inducing protective immunity can be identified and the mechanisms involved in immune protection elucidated.

To produce the vaccine and the lysates needed to determine which gene sequences were being expressed by the modified E__s_ coli. Lambda lysogens were grown in a liquid culture (Luria broth) at 32°C to a cell density of 2x10 per milliliter. The lysogens were then incubated at 42°C for 30 minutes and then at 38 C for 2 hours ^'with vigorous aeration. The cells were pelleted by centrifugation at 5000 x g for 15 minutes and resuspended in 1/20-1/50 original volume of TEP buffer [lOOmM Tris HCl, pH 7.4/10mM EDTA/lmM Phenyl methyl sulfonyl fluoride (PMSF) ] . The cell suspension was immediately frozen in dry ice-ethanol and can be stored at -70°C. Thawing of the cells resulted in lysis; however, to ensure lysis, the cells were also sonicated. The sonicated extract was centrifuged at 10,000 rpm for 10 minutes. The supernatant was then mixed with 3 volumes of saturated ammonium sulfate and chilled on ice for 60 minutes. The resulting slurry can be stored at 4°C indefinitely.

The lysates that are produced by this method can be directly inoculated into cattle with or without an adjuvant. Alternatively, the fusion proteins can be further purified by size fractionation, ammonium sulfate precipitation or gel filtration or by taking advantage of the charge properties of the β-galactosidase which binds tightly to DEAE cellulose in the method described by Craven, et al., 240 J. Biol. Chem. , 2468-2477 (1965). The fusion proteins can also be purified using an anti-β- galactosidase immuno-affinity column. Alternatively, because β-galactosidase is large, the fusion proteins can be purified by extraction from a preparative SDS-PAGE gel as described previously in a modification of the procedure described by Hunkapiller.

Cells isolated to date, produced using the above technique, exhibit the ability to produce the antigenic epitopes expressed in Omp I through III and the antigen 7kd. Through the use of different antisera, it is possible to identify and culture other lysogens coding for different combinations of antigens.^* Thes.e different lysogens can then be used to prepare alternate immunizing agents for use in preparing brucellosis vaccines.

III. Transposon Mutants of Brucella abortus

A third method for producing immunizing agents against Brucella abortus which will induce an immunological reaction against Brucella abortus and still permit differentiation between vaccinated and field strain infected cattle can be achieved through the development of transposon mutants. The mutants can be developed through PI and Mu phage infections of Brucella abortus.

PI and Mu phages are isolated from Ej_ coli SF800 Pl::Tn5 lacZ kan^R and str^S and E^ coli CT151 kan^R str^S.

The E_j_ coli CT151 contains the lysogenic bacteriophage Mu::Tn5(dl) kan R and strS. The strains are isolated by streaking them out on LB [lOg Bacto-tryptone, 5g yeast extract, 5g NaCl, pHed to 7.5] plates containing 40 micrograms/milliliters of kana ycin sulfate. The plates are then incubated at 30°C for 24 hours. A single colony is selected and used to inoculate a 50 milliliter culture of LB broth containing 40 micrograms/milliliters of kanamycin sulfate. The flasks are incubated overnight with a gentle agitation at 30°C. The overnight culture is used to inoculate 500 milliliters of LB broth containing 5 mM CaCl_. Non-pHed LB broth is used to avoid calcium precipitation. CaCl- is also required for the CT151 strain growth.

The cultures are grown to an OD_g00 of 0.4 and then warmed to 42°C by immersing the cultures in a 90 C water bath. The temperature is monitored using an ethanol rinsed thermometer. 'The culture flasks are placed in an air shaker and vigorously aerated at 42 C for 30 minutes and then cooled to a temperature of 38 C. The vigorous aeration is continued for approximately 90 minutes or until the cells are lysed. Temperatures should be maintained above 37 C during this process to ensure that sufficient lysis occurs. Following lysis, the cultures are adjusted to 2% in CHC1., and agitated an additional 10 minutes to ensure complete lysis.

The cell supernatant is then adjusted to 0.5 M in NaCl and chilled to 4°C. The supernatant is kept at that temperature for at least 60 minutes. Bacterial debris is removed by centrifugation at 8000 rpm for 10 minutes. To ensure increased stability of the Mu bacteriophage, the following salts are added: 1-3 mM MgSO. and 1-3 mM Pb(OAc)₂- Solid polyethylene glycol 8000 (PEG) is added to a final concentration of 10% (w/v) and the solution is incubated at 4°C for at least 60 minutes. The PEG precipitate is then pelleted by centrifugation at 8000 rpm for 20 minutes. The pellet containing bacteriophage is resuspended in an ice cold PI buffer [10 mM Tris-HCl, pH 7.6/10 mM CaCl₂] or Mu buffer [10 mM Tris-HCl, pH 7.6/1 mM MgSO./ ImM Pb(OAc)₂] at 1/50 the original volume and kept on ice. For each 3.5 milliliters of resuspended PEG pellet, 2.4 grams of solid CsCl is added and the resulting solution is centrifuged to equilibrium. The resuspended PEG pellet is centrifuged for 24 hours in an 80Ti rotor at 38,000 rpm at a temperature of 5 C. The bacteriophage bands are located by their opacity using a high intensity lamp and collected by side puncture of the tubes. The harvested bacteriophage are dialyzed against three changes of 500 milliliters of PI and Mu buffers and stored at 4°C over CHC1₃.

B. Infection of Brucella abortus With PI and Mu Phage

A confluent plate of Brucella abortus S-19 strain is incubated for 48 hours at 37 C on potato infusion agar (PIA) or trypticase soy agar (TSA) . The cells are then harvested from the plate into 5 milliliters of non-pHed tryptose broth adjusted to 10 mM CaCl-. The S-19 cell suspension is then diluted 100-fold prior to infection. To the tube containing the 1:100 dilution of the S-19 cells, 0.1 milliliters of the phage prepared as described above is added and incubated without agitation at 38°C for 30 minutes. The reaction is diluted two-fold with 1.0 milliliters of non-pHed tryptose broth (without CaCl_). The solution is transferred to a screw cap jar in a shaking water bath at a temperature of 38°C. The mixture is incubated for 2 hours with vigorous agitation. Following the shaking, 0.4 milliliter aliquotes are spread onto PIA or TSA plates containing 25-40 microgra s/milliliters of kanamycin. The plates are incubated at 38°C. Negative controls without bacteriophage were used in the experiment. The plates are checked beginning at day 3 and for as long as 14 days. Any colonies observed are picked and restreaked for isolation. Colonies so isolated are characterized via standard biotyping procedures as outlined by Alton et al., Laboratory Techniques in Brucellosis, World Health

Organization, Monograph series, No. 55, 2nd ED., 64-56 (1975). Stock suspensions are stored in 50% glycerol at -70°C.

B. Identification of Mutant Genotype

To identify the genetic lesion caused by transposon mutagenesis, hybridization analysis using Tn5 DNA as a hybridization probe is performed as described by Southern 98 J. Mol. Biol. 503-517 (1975). Once the size of the restriction fragment bearing the transposon insertion is known, this size fragment is isolated from a second digest following size separation on an agarose gel. The DNA is then cloned into a plasmid vector and recombinants carrying the transposon are selected again using Tn5 as a hybridization probe. Flanking Brucella DNA sequences are characterized by DNA sequence analysis and are used to identify the genetic lesion.

A mutant form of strain S-19 Brucella abortus has been created using the above technique. The organism identified as Brucella abortus S19 LPS::Tn5 has been deposited with the American Type Culture Collection and been assigned ATCC Deposit No. 53593. This organism has been found to be lacking the 0 antigen common to smooth strains of Brucella abortus. This organism can be used as an immunizing agent to create a Brucella abortus vaccine which provides protective immunity, yet still permits differentiation between field strain infected and vaccinated cattle.

The organism can be used as an immunizing agent in one of three forms. First, a stable deletion mutant recovered following deletion of the transposon can be used as a live immunizing agent in the same manner that «strain S-19 is currently used. Second, the organism can be lysed as described in Section I above and the cell envelopes can be used as an immunizing agent.

IV. Viral Vector Immunizing Agent

A fourth type of immunizing agent that can be used to induce an immunogenic response against brucellosis, yet still provide a means for differentiating between field strain infected and vaccinated cattle is the use of a modified viral .vector. In this case a recombinant pox or herpes virus is developed having the genetic material encoding one or more of the common antigens of Brucella abortus incorporated into the viral genome and expressed by the viral vector.

A. Identification of DNA

Fragments to be Expressed

DNA fragments expressing portions of the outer membrane proteins of Brucella abortus in Lambda gtll are used as hybridization probes to detect larger 20kb fragments which contain the entire gene for the outer membrane protein. Construction of a genomic library is performed as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, 1st ed., 269-294 (1980) which is hereby incorporated by reference. The process involves partial digestion of Brucella abortus DNA extracted from either strain 19 or S2308 which has been partially digested with Sau 3AI. Fragments of approximately 20kb in size are purified by gel electrophoresis and ligated into the vector Lambda 2001. Phage containing inserts of foreign DNA are grown on E. coli P2 392 where nonrecombinant phages are unable to grow. Selection of the recombinants containing the genes encoding the outer membrane proteins or other immunogenic proteins of B__s_ abortus is performed by hybridization using the Lambda gtll inserts as probes. This is accomplished by plating out a library of Lambda 2001 on E^. coli P2 392. After the plaques are developed, plaque lifts are performed onto nitrocellulose. The nitrocellulose filters are hyridized with various DNA probes coding portions of the outer membrane proteins or other immunogenic proteins of Bj_ abortus. Positive plaques are selected and the screening procedure is repeated three or four times until the purity of the plaques is ensured. Mapping of the recombinant phage is performed as described by Maniatis et al. Hyridization analysis using the original hybridization probe and an oligonucleotide probe derived from amino-terminal sequence of the proteins purified from B. abortus cell envelopes are used to verify the " restriction fragment carrying the gene desired. Once identified, this fragment can be purified by gel electrophoresis prior to cloning in the viral expression system. B. Cloning the Outer Membrane

Protein and Other Immunogenic Protein Genes of B. abortus

Restriction fragments identified as described above are cloned into viral vectors as described by Mackett et al., 49 J. Virol. 857-864 (1984). In the procedure, the restriction fragments are cloned into plasmid vectors directly downstream of and fused to viral promotors. The plasmids are used to transfect cells which are infected with the virus to be used. Following several rounds of plaque purification of the recombinant virus, restriction endonuclease analysis of the recombinant virus is performed and the structure of the recombinant virus is verified using the original Brucella DNA fragment as a hybridization probe. The recombinant virus is then present as a homogeneous stock which can be used to infect tissue cultures which can be subsequently used to check the levels of Brucella antigen production.

Using the procedures outlined above, it is possible to construct modified herpes virus or pox virus which can carry the genes for one or more of the common antigens of Brucella abortus. These viruses can then be used as an immunizing agent in a vaccine against brucellosis using the standard procedures for inoculating cattle with pox or herpes viruses. For example, the genetic sequences coding for outer membrane proteins I through III can be isolated and inserted into the genome of either a pox or herpes virus. When the pox or herpes virus is inoculated into cattle, the translated antigen will stimulate T lymphocytes and induce the production of antibodies which will provide protective immunity for the cattle. The antibodies produced will not react with standard serological assays for brucellosis, thereby providing a means for differentiating between vaccinated and field strain infected cattle.

V. Synthetic Peptide Immunizing Agent

A fifth type of immunizing agent having the ability to induce an immunogenic response against brucellosis while retaining a means for serologically differentiating between field strain infected and vaccinated cattle is the use of one or more synthetic peptides constructed to contain the dominant antigenic epitopes found in the Omps and 7kd and 8kd proteins. In this case, the synthetic peptides will be treated as are the purified antigens isolated from B_j_ abortus in Section I. These synthetic antigens may be administered either alone or in combination with suitable adjuvants.

The synthetic Peptide Immunizing Agents are created by first analyzing the amino.acid sequences of the major Brucella antigens to include Omp I through III and the 7kd and 8kd proteins and then determining the antigenic regions of the antigens using the algorithms described by Hopp, T.P. et al., 78 Proc. Nat'l. Acad. Sci. USA 3824- 28(1981) and Kyte, J. et al., 157 J. Mol. Biol. 105-132 (1982) and then determining the secondary structure using the algorithm described by Chou, P. et al., 47 Advances in Enzymology 45-148 (1978). Alternatively, the peptide sequence can be determined by using the method described in Section II above to identify dominant protective epitopes for fusion proteins. By determining the DNA sequences which code for the epitopes, it is possible to develop the a ino acid sequences for the antigenic proteins. Once the amino acid sequences are identified, the antigenic peptides can be synthesized'using standard techniques. One possible method involves the use of a Biosearch^® model 9500 peptide synthesizer using the standard solid phase t-Boc synthesis described in the instruction manual published by Biosearch, Inc. (A New Brunswick Scientific Co., 2980 Kerner Blvd., San Rafael, CA 94901). This method is a modification of that described by Merrifield, R.B., 85 J. Amer. Che . Soc. 2149 (1963) which is hereby incorporated by reference..

Once the peptides are synthesized, they can be used as an immunologic agent as described in Section I above.

VI. Identifying Sequences

For purposes of product identification and to aid in identifying vaccinated cattle, where possible all products will contain the antigenic peptide sequence TEAGEPST (for Texas Agricultural Experiment Station) as a result of insertion of the appropriate DNA sequence to be expressed in cloned products or by inclusion of at least one repeat unit of a synthetic peptide having this sequence in a vaccine composed of purified or synthetic products.

From the above, it will be clear to one skilled in the art that several different approaches can be used to develop or to reach the same end result, namely a vaccine which provides protective immunity against Brucella abortus, yet provides a means for differentiating between field strain infected and vaccinated cattle. By the deletion of one or more antigens from the vaccine, tests can be developed to differentiate between the two types of cattle. While the preferred embodiment calls for the deletion of the O antigen from the immunizing agent used, it is readily apparent that deletion of antigens other than the O antigen can be used to achieve the same results. The deletion of the 0 antigen is preferred because the differentiation between vaccinated and field strain infected cattle can be readily accomplished using standard serological assays for brucellosis.

Claims

CLAIMS :

1. A vaccine which provides protective immunity against Brucella abortus infection comprising an immunizing agent having one or more, but not all, of the following Brucella antigens: O, Omp I, Omp II, Omp III, 7-kd and 8kd.

2. The vaccine of claim 1 wherein said immunizing agent comprising a mixture of purified antigens isolated from membranes of Brucella abortus.

3. The vaccine of claim 3 wherein said purified antigens are the 7kd and 8kd antigens.

4. The vaccine of claim 4 wherein said 7kd and 8kd antigens are isolated from membranes of a strain of Brucella abortus strain 2308.

5. The vaccine of claim 2 wherein said Omp I, Omp II and Omp III antigens are isolated from membranes of a strain of Brucella abortus.

6. The vaccine of claim 5 wherein said Brucella abortus strain is strain 0 antigen minus mutant.

7. The vaccine of claim 1 wherein said immunizing agent comprises a mixture of Brucella antigens Omp I, Omp II and Omo III.

8. The vaccine of claim 7 wherein said Omp I, Omp II and Omp III antigens are isolated from E^. coli strains MC 4100 Omp 1.11, MC 4100 Omp 2.63 and MC 4100 Omp 3.10 having ATTC Deposit Nos. 67355, 67356 and 67354, respectively.

9. The vaccine of claim 1 wherein said immunizing agent comprises the antigens isolated from mutant E^_ coli strain MC 4100 Omp 7.01 having ATCC Deposit No. 67357.

10. The vaccine of claim 1 wherein said immunizing agent comprises Omp I.

11. The vaccine of claim 10 wherein said Omp I antigen is isolated from membranes of Brucella abortus.

12. The vaccine of claim 11 wherein said Brucella abortus is strain S19.

13. The vaccine of claim 10 wherein said Omp I is isolated from Ej_ coli strain S19 LPS::Tn5 having ATCC Deposit No. 53593.

14. The vaccine of claim 1 wherein said immunizing agent is a viral vector having the genetic material encoding for one or more of the Brucella antigens inserted into said viral vector's genome.

15. The vaccine in claim 14 wherein said viral vector is herpes virus.

16. The vaccine in claim 15 wherein the genetic code for Omp I is incorporated into the genome of said herpes virus.

17. The vaccine in claim 14 wherein said viral vector is pox virus..

18. The vaccine of claim 1 wherein the immunizing agent comprises intact cell envelopes isolated from O deletion

Brucella abortus strain S19 LPS::Tn5 having ATCC Deposit No. 53593.

19. The vaccine of claim 1 wherein said immunizing agent is a synthetic mixture of peptides resembling one or more of the Brucella antigens: Omp I, Omp II, Omp III, 0, 7kd and 8kd.

20. A vaccine which provides protective immunity to Brucella abortus comprising a strain of Brucella abortus as an immunizing agent having one or more of the genes for the following antigens deleted from its genome: Omp I, Omp II, Omp III, 7kd and 8kd.

21. The vaccine of claim 20 wherein said Brucella abortus is identified as strain S19 LPS::Tn5 having ATCC No. 53593.

22. The vaccine of claim 21 wherein said Brucella abortus ATCC No. 53593 is attenuated.