CA2125426A1 - Rhodococcus equi gene sequence - Google Patents

Abstract

This invention relates to Rhodococcus equi bacteria immunogenic proteins on the bacterial cell surfaces, genetic information as it relates to the proteins and diagnostics and vaccine components based on the use of such proteins. The DNA sequence encoding the virulent protein is described along with the corresponding amino acid sequence for the protein. The protein, as expressed by R. equi, includes a signal peptide portion which is cleaved off in the process of the protein being excreted through the cell wall. Also in that process, the protein is lipid modified to provide proteins of SDS-PAGE gel estimated molecular weights of 17 through 22 kDa. A plasmid construct is provided suitable for expression in E. coli host for providing the protein. Antibodies have been raised to the protein which are useful in diagnostic and passive vaccines.
Active vaccines are provided based on the use of the protein. The protein is hydrophobic in nature when lipid modified. Hence, a process for recovery of the hydrophobic protein is provided which involves the use of a detergent.

Description

RHODOCOCCUS EQUI GENE SEQUENCE
FIFTn OF THE lNv~NllON
This invention relates to Rhodococcus equi (R. equi) bacteria, immunogenic proteins on the bacteria cell surfaces, genetic information as it relateæ to the proteins, diagnostics and vaccine components based on such proteins.
BACKGROUND OF THE l~v~llON
Fatal pneumonic infections caused by R. equi appear to be peculiar to foals and in particular foals ranging in age from one to five months. The problems associated with R. equi infection are well known to horse breeders;
hence several procedures have been developed in an attempt to minimize loss of foals to these early infections. It is generally understood that, in a foal fee~ing from their dam, the foal receives the dam's colostrum which provides passive protection in the young foal. In most situations, such immediate passive protection may be sufficient to avoid loss assuming the opportunity for bacterial challenge is kept to a minimum.
However, when the foal does not n~cessArily have the benefit of the dam's colostrum for reasons of lack of fee~ing or 1088 of the dam, or ~nc~ of bacterial infections are considered to be high, then the foal requires additional protection to avoid fatal infection.
Disease treatment has been achieved by the administration of antibiotics. However, there are strains of the bacteria which are now resistant to some antibiotics, including penicillin, ampicillin and gentomycin. A combination of the antibiotics, ethromysin and rifampin have met with some success in combating R.
equi infection.
The alternative approaches have involved a passive vaccine of hyperimmune anti-R. equi plasma, which is administered to the foals, and various types of active vaccines. None of these approaches have met with great success. The administration of antibiotics has to occur 212~g26 at an early stage of infection in order to combat the ~ e before it has fully set into the lungs. Attempts at vaccination have met with little reproducible success, except perhaps for the use of hyperimmune plasma.
However, the administration of hyperimmune serum is awkward in that the plasma has to be administered to an active foal over extended periods; for example, one hour or more. It is therefore difficult to restrain a foal for that period of time in administering the plasma serum. Also, since the administration of hyperimmune plasma is a passive immunization, the antibody titres in the foam will di~;nic~ over time.
The hyperimmune anti-R. equi plasma is obtained from horses that have been challenged by R. equi infection.
The hyperimmune anti-R. equi plasma has an immunosorbent assay value greater than 80. Commercially this plasma is available at an approximate cost of $120.00 per litre.
As already noted, the administration of a litre of plasma to a foal on a periodic basis is not only time consuming, but as well difficult to achieve on a repeated basis. In any event, it has been established that the hyperimmune plasma does offer protection in the foal to R. egui challenge. It is thought that the hyperimmune plasma contains factors other than the plasma antibodies developed by the horse to provide protection. These other factors are thought to include fibronectin, interferon, complement and cytokines.
Although some sl~cceC~ has been achieved with the hyperimmune plasma, the actual role of cellular and humoral immunity in the pathogeneses of R. equi disease remains unclear. In this respect the nature of the manner in which the foal might acquire resistance to R.
equi pneumonia is also unclear. Hence further work was suggest d by others toward identifying and characterizing the humoral comronents that imparted protection to foals and the development of improved technologies to actively 212 ~ ~12 6 stimulate or passively transfer these and other immuno protective components.
In this regard, considerable efforts were focused on two exoenzymes which are produced in large volumes during culture of R. equi. The two exoenzymes are cholesterol oxidase and phospholipase C. It was thought that in view of the large quantities of these exoenzymes they may elicit an immune response which would offer protection.
Vaccine compositions were made which contained the two eYo~n~ymes. However administration of those exoenzymes in vaccines failed to elicit an immune response which protected foals when challenged by R. equi.
There are therefore many problems associated with existing practices in developing successful vaccines for combating R. equi infection in foals. All of the background approaches appear to point to a passive style of vaccine to confer protection. It appeared that an active vaccine would not work, perhaps because the foal's immune system was not properly prepared to confer resistance during the first six months after birth. We have now determined, in accordance with this invention, that there are several aspects to the infection which are now understood and which enable us to provide a vaccine component which provides protection from R. equi fatal infection.
It is therefore a feature of the invention that the DNA sequence encoding virulent proteins is provided.
It is a further feature of the invention that the amino acid sequence is provided for the protein which may be lipid modified and which is involved in the virulent infections of R . equi. The DNA sequence information and the protein sequence information may be used in conducting assays and other related diagnostic tests to determine extent of R. equi infection and/or immune response in an animal.
The DNA sequence information may be used in accordance with another feature of this invention to ~125~2~

produce, in a suitable recombinant host, recoverable quantities of the protein. The protein may also be recovered from culture of the R. egui, because the protein appears to be the sole hydrophobic protein expressed on the surface of the bacterium.
A further feature of the invention is the provision of vaccine component(s) for use in vaccines to provide protection against R. equi challenge.
SUMMARY OF THE lNV~N l lON
According to an aspect of the invention, a substantially pure DNA molecule comprises a DNA
nucleotide sequence corresponding to a DNA nucleotide sequence of Table I (Sequence I.D. #1).
According to another aspect of the invention, an oligonucleotide comprises at least 18 consecutive nucleotides selected from the DNA sequence of the vir gene.
In accordance with another aspect of the invention, a DNA probe comprises an oligonucleotide of at least 18 consecutive nucleotides selected from the DNA sequence.
According to another aspect of the invention, a substantially pure protein comprises an amino acid residue sequence corresponding to a protein amino acid sequence of Table I (Sequence I.D. #2).
According to another aspect of the invention, a substantially pure protein sequence corresponding to amino acid residue positions 32 to 189 of Sequence I.D. #2.
According to another aspect of the invention, a recombinant cloning vector comprises a DNA molecule which may be the entire sequence of Table I or any fragment thereof.
In accordance with another aspect of the invention, the recombinant plasmid pCT-C is provided.
In accordance with another aspect of the invention, a host is transformed with the recombinant cloning vector 212542~

and when cultured in a suitable medium expresses the protein.
In accordance with another aspect of the invention, an antibody is provided which is specific for one or more antigenic determinants of the protein sequence of Table I. Such antibodies may be polyclonal or monoclonal antibodies. The antibodies may be specific for antigenic determinants in the N-terminal region of the protein sequence or the C-terminal region of the protein sequence.
In accordance with another aspect of the invention, a biologically active protein component for use in a vaccine composition is provided. The vaccine composition, upon administration to foals, is effective in developing immune resistance to challenge by R. equi.
The protein component has an amino acid sequence corresponding to the amino acid residue positions 32 to 189.
In accordance with another aspect of the invention, the biologically active protein component has a lipid molecule attached in the region of the N-terminal end of the amino acid sequence. The lipid modified protein is of varying molecular size and the molecular weight of the lipid modified protein is in the range of 17 kDa to 22 kDa.
In accordance with another aspect of the invention, a vaccine composition useful for eliciting a protective immune response in foals to resist challenge to R. equi infection is provided. The vaccine composition comprises:
i) the vaccine protein component in an amount sufficient to elicit the immune response; and ii) a pharmaceutically acceptable carrier.
In accordance with another aspect of the invention, a process is provided for recovery of the hydrophobic Vir protein from cultured cell wall by the use of a detergent.

2125~
-BRIEF DESCRIPTION OF THE DRAWINGS
Various aspects of the invention are described with reference to the accompanying drawings, wherein:
Figure 1 is Western immunoblots of recombinant and native virulence-associated protein of R. equi. Lane 1:
Immunoblot of SDS-PAGE separated E. coli XLlBlue (pBluescript) reacted with antiserum to virulence-associated protein of R. egui; Lane 2: Immunoblot of SDS-PAGE separated E. coli XLlBlue(pCT-C7) reacted with the same antiserum showing 3 immunoreactive protein bands;
Lane 3: TX-114 extracted, sonicated E. coli XLlBlue(pCT-C7) reacted with the same antiserum, showing the hydrophobicity of the two heavier bands; Lane 4: Whole cell preparation of R. equi reacted with antiserum against TX-114 extracted E. coli XLlBlue(pCT-C7) showing immunoreactivity with the 3 characteristic virulence-associated protein bands of R. equi; Lane 5: Whole cell preparation of R. equi reacted with MAb103. Molecular weight markers on the left.
Figure 2 is physical map of the plasmid pCT-C and deletion derivatives. Vector sequences are presented by the thick lines, and ~. egui sequences by the thin lines.
The 17-kDa labelled bar indicates the location of the ORF
as determined by Western immunoblots of deletion derivatives and DNA sequencing. The nucleotide sequence was determined for the regions of the gene identified by arrows, their direction indicating the direction of sequencing. E, EcoRI; B, BglII; P, PstI; N, NotI.
Figure 3 is SDS-PAGE of Rhodococcus equi strain 103 (M30). Lane 1: Coomassie blue stained whole cell preparation showing weakly staining diffuse band at 17.5-22-kDa; Lane 2: Coomassie blue stained TX-114 detergent phase extract showing heavy protein bands at 17.5- and 18-22- (diffuse) and 44-kDa (diffuse); Lane 3:
Immunoblot of whole cell preparation using murine monoclonal antibody identifying 17.5-and 18-22-kDa bands;
Lane 4: Immunoblot of TX-114 detergent phase identifying 21~5 12~

relatedness of 17.5-, 18-22-, and 44-kDa bands; Lane 5:
Autoradiograph of TX-114 detergent phase extract incllhAted with 20 ~Ci [~]-palmitic acid, showing fatty acid incorporation into the 17.5-22-kDa protein bands, sites for the prestained molecular weight markers being on left.
Figure 4 is the physical map of the pOST1 virulence plasmid of R. equi strain 103.
DET~TT,~n DESCRIPTION OF THE PREFERRED B ODIMENTS
Considerable efforts have been made in the direction of developing vaccines based on eYoen7yme, because of the major amounts of that enzyme in the culture supernatant of R. equi which, in turn, discounted any importance one might lend to the cell surface proteins which are characteristically the 15 kDa and diffuse 17 to 22 kDa proteins. We have now, however, established their role in developing significant immune responses where that immune response is protective and therefore resists challenge by R. equi infection.
In order to facilitate discussion of various aspects of the invention, the proteins of R. equi, which are virulence associated, shall be identified as Vir proteins. These proteins are understood to have SDS-PAGE
gel estimated molecular weights of approximately 15 kDa, 17.5 kDa and a diffuse band indicative of several molecular weights in the range of 18 to 22 kDa. It is understood that, in characterizing the molecular weight of the proteins by SDS-PAGE gel techniques~ there can be some variation in the molecular weights from their actual molecular weights and from gel to gel. Such variation may be due to a slight variation in gel reagents, possible impurities in the gel reagents and other similar considerations familiar to thoce skilled in the art of measuring molecular weights by SDS-PAGE gels.
Furthermore, the diffuse band having molecular weights in the range of 18 to 22 kDa is due to lipid modification of the base Vir protein having a molecular weight of 15 kDa.

It is believed that the 17.5 kDa protein is lipid modified with a lipid molecule which is of a consistent molecular weight, whereas the lipid molecule modifying the 15 kD and producing the diffuse band has a variation in molecular weight resulting in proteins having molecular weights in the range of 18 to 22 kDa.
Correspondingly, in reference to the gene sequence for ~nroA;ng the Vir proteins, the sequence is referred to as the vir gene sequence. The vir gene has the Sequence I.D. #1. The nucleotide sequence of the vir gene has been deposited in the Ge~RAnk under Acre-c-cion Number UO 5250.
Various aspects of the invention will now be described in accordance with the following headings.
Cloning and Se~ n~ing of ~e ~r gene The plasmid, which appeared to be common to the virulent strain of R. equi and therefore associated with the 17.5 kDa virulent associated protein was reported by us (19). In order to facilitate reference to various background articles, the number in brackets identifies the reference in the attached reference legend. The plasmid, which is approximately 80 kb in size, can be readily isolated from the virulent strain of R. equi in accordance with the procedure set out by us in that report, the subject matter of which is hereby incorporated by reference. We had deposited the virulent strain of R. equi with ATCC under Accession Number 33701.
That strain iB readily available from ATCC to provide a readily available source for the plasmid. We have conducted extensive studies on various virulent R. equi strains which poCcecc the subject plasmid. Further study has revealed that the plasmid of the deposited strain also exists in other strains, such as strain 103 to which we referred to here. The plasmid is of 84 kb and we identify as plasmid pOTS.

_ 212542~

The procedure used to ultimately clone and sequence the vir gene is generally described as follows, details of which are provided in the following examples. A
partial EcoRi library of the 84 kb plasmid pOTS was constructed where constructs expressing the Vir protein were detected by the use of polyclonal antibodies. The polyclonal antibodies were developed by immunizing rabbits and/or mice with the identified 17.5 kDa protein which had been previously associated with the 84 kb plasmid pOTS. The identified recombinant expressing Vir protein was subcloned to ultimately provide plasmid pCT-C7Sl having the map of Figure 2 which contained the gene sequence expressing the 17.5 kDa protein. The construct was then sequenced to provide the DNA sequence of Sequence I.D. #l which encodes for the base protein of the Vir protein and, as well, includes 5' end non-coding sequence and a 3' end non-coding sequence. These non-coding regions are most likely involved in the expression of the gene sequence encoding the mature 15 kDa protein.
The base protein has an estimated SDS-PAGE gel molecular weight of approximately 15 kDa. The increase in molecular size is due to the lipid modification of the base protein where, as apparent from Figure 1, the lipid modification can provide proteins having estimated SDS-PAGE gel molecular weight ranging from 17.5 through to 22kDa where a large variation is found in the molecular weight range of 18 to 22 kDa.
The plasmid map of Figure 2 demonstrates the inclusion of the vir gene in pCT-C to provide plasmid pCT-C7Sl. This information can also be correlated with the overall map for the plasmid as shown in Figure 4.
The plasmid map of Figure 4 is for the 84 kb plasmid which, when present in R. equi, provides a virulent strain of R. equi. The corresponding plasmid has also been isolated from the R. equi strain 103 as previously referred to. The physical map of Figure 4 for the 84 kb plasmid was constructed by analysis of single, double and a ~ a (o partial digestions with Asnl, BglII, IIEcoRI, HindIII, XbaI restriction enzymes. The digestions yielded numbered fragments 2, 5, 9, 4 and 3 respectively, with each restriction enzyme used. The small fragments of EcoRI 5, 8, and 9 were located by means of Southern blot analysis of plasmid single digestions with the respective digoxigenin-labelled EcoRI fragments. The overall size of the plasmid was determined to be 84,961 base pairs.
For ease of understAn~ing the relative location of the various enzyme restrictions sites, the enzyme restriction map has been presented in five concentric circles; each circle representing the restriction sites for the respectively identified restriction enzyme. From the inner circle to the outer circle, the enzymes are listed Asnl through EcoRI. The location of the fragments in each circle are numbered where the largest fragment begins with the number 1 and the smallest fragment ends with the number 9. The largest fragment is obviously fragment 1 which is digested by all of the listed enzymes, whereas fragment 9 has the two EcoRI digestion sites.
In relating the restriction site information of the map of Figure 4 to the plasmid construction map of Figure 2, the EcoRI sites of plasmid pCT-C are the EcoRI sites for plasmid fragment number 3 in the outer circle of Figure 4. The solid line beneath the restriction map for pCT-C is the location of the vir gene enco~;ng the Vir protein which, for convenience, is marked 17-kDa. It is apparent that the vir gene is located between the PSTl and BamHI sites. Referring to Table I, the listed DNA
sequence has indicated several restriction sites including at the 3' end, the PST1 site. In actual fact, the vir gene extends from the PSTl site towards the 5' end to the BglII site. The BglII restriction sites are shown in Figure 4, fourth outer concentric ring. The BglII restriction site between fragments 3 and 5 indicates approximately where the vir gene is located in -2125~26 fragment 3 of the restriction map of Figure 4. Had a PST1 map been made for Figure 4, the location of the vir gene in fragment 3 would be located. However in view of the sequence information provided in Table I, there is no necessity for exact location in the restriction map of Figure 4.
WIth respect to Table I, the non-coding regions of the vir gene are identified and sequenced. These non-coding regions include the BglII and PSTl sites.
Furthermore, in the sequence, the asterisks indicate the start and stop codons. As well the shine-delgarno (S.D.) and stem loop structures are indicated by underlining.
The S.D. underlined region also has the letters S.D.
above same.
The explanation for the 15 kDa protein being different from the 17.5 and 18 through 22 kDa proteins can be further realized from an analysis of the DNA
sequence of Table I. As shown in the Table, the brackets indicate the N-terminal region of the mature protein.
However, upstream of the N-terminal region of the mature protein is an amino acid sequence which is initially enco~ by the vir gene. It i6 thought that this amino acid sequence acts as a signal peptide to provide for cell wall transport, so that the protein becomes attached to the outer side of the cell wall. In that process, the signal protein is cleaved from the balance of the Vir protein. The Vir protein without the signal protein portion has a molecular weight of approximately 15 kDa.
Once the 15 kDa protein is transported through the cell wall, or during that process, a lipid modification of the protein occurs where a lipid molecule is added to the N-terminal region of 15 kDa protein to provide the characteristic lipid modified protein having a molecular weight of 17.5 kDa. In such lipid modification, there is also a range of other lipid molecule sizes which are added to the base protein to provide the range of 18 through 22 kDa lipid modified protein molecules.

21254~6 ~il. ch~r,t~-ri7~ion With the plasmid pCT-C7Sl, the protein can be expressed in E. coli and its characteristics observed.
It has been found that, in the recombinant expression of the protein in B. coli, lipid modification of the protein also occurs, the presence of the lipid being confirmed by the radiolabelling of palmitic acid or like fatty acid which is attracted to or binds to the lipids of the fatty acid as demonstrated in Figure 3, Lane 5. The characteristics of the lipid molecule are not fully understood. However, from our investigations, it is apparent that it is a lipid modified protein rather than a lipo-protein. It would appear that the lipid modification occurs after or during the excretion of the expressed protein to the cell surface. The protein, as expressed by plasmid pCT-C7Sl, includes a signal portion which is cleaved off once the Vir protein reaches the cell surface, the signal proteins simply serving as a vehicle to transport the Vir protein to the cell surface.
Once the protein is at the cell surface by appropriate mech~nicms or during this mech~nifim, the lipid molecule is added to the protein. Furthermore, based upon our investigations, the lipid molecule is added to the N-terminal end of the protein leaving the hydrophillic C-terminal end of the protein free of the molecularsurface. The antigenic portion of the Vir protein appears, however, to be primarily associated with the N-terminal region of the protein, particularly involving the lipid molecule. Hence, the preferred component for vaccines includes the lipid modified protein because it elicits the greatest immune response.

RecollLi~t E~l~ssion of the Pr~
Large quantities of the protein are required for purposes of preparing antibodies, of various types of diagnostic testing of foal serum and of preparing vaccination components. The recombinant expression of _ 13 the protein along with the discovery that, when expressed in various bacterial hosts, the desired lipid modification still occurs, provides a convenient methodology. As is appreciated in the recombinant production of the protein, changes in the sequence are permitted and sometimes intentionally used to enhance production without changing the functional properties of the protein. Furthermore, sequence changes may also occur due to non-functional differences in the sequence between various virulent strains of R. equi. The DNA
sequence, which includes the signal sequence portion and with or without the non-coding regions, may be ligated to bacterial expression vectors, such as the already-prepared pCT-C expression vector. The coding region extends from bp position 245 to 814. Other vectors include, for example, PRIT, PGX, PATH, all of which are well known and can be incorporated in E. coli cells for the production of the Vir protein. It is also appreciated that the DNA sequence can also be transferred into other cloning vehicles, such as other types of plasmids, bacteria phages, and cosmids.
It is also appreciated that the DNA sequence of Sequence I.D. #l can be manipulated by a stAn~Ard procedure such as in the use of restriction enzyme digestion, fill-in with DNA polymerase, deletion by exo-nuclease, extension by terminal deoxynucleotide transferase, ligation of synthetic or cloned DNA
sequences, site directed sequence alteration via single stranded bacterial phage intermediate or with the use of specific oligonucleotides in combination with PCR. Such modifications may be used to produce desired fragments of the protein to permit study of the function of the complete and specific portions of the protein and to perhaps determine functional uses of induced portions of the proteins, either in diagnostics or vaccine related studies.

The recombinant cloning vector contAining the DNA of this invention includes, of course, all of the n?CQccAry expression control information in the vector to ensure expression of the protein sequence when incorporated in the appropriate host. Depe~i ng upon the host, the appropriate expression control sequences may be selected from those of the LAC system, the TRP system, the TAC
system, the TRC system, major operator and promoter regions of the phage-~, the control region of FD-coke protein, the early and late promoters of SV-40, promoters derived from polyoma, adino virus, retro virus, vacsilo virus and simian virus; the promoter for C-phosphoglycerate kin~se, the promoters of yeast, acid phosphatase, the promoter of yeast a-mating factors and combination thereof. Suitable hosts include E. coli, pseudomonas, bacillus, other bacteria, yeast, fungi, insect, mouse, plants, and animals in which the protein could be expressed in milk for harvest and recovery.

Culture of R. eqlu to produce commercial qn~ntities of the Vir ~Jteill Based on our discovery that the Vir protein is the only surface protein expressed during culture of the R.
equi, which is hydrophobic, allows the commercial recovery of the protein by washing the cell surfaces with an appropriate detergent or surfactant. Washing of the cell surfaces removes the hydrophobic protein in the detergent composition. The recovered wash solution may then be treated to separate the spent detergent from the supernatant wash liquid contAinin~ the protein. The protein can then be precipitated from the wash liquid to yield a concentrated mass of the Vir protein which can be further purified and used directly in further protein studies and as a vaccine component.

. . 1 5 Antibody Prodllction In accordance with this invention, the antibodies are used as a diagnostic and as a constituent of a passive vaccine. Antibodies to the Vir protein are prepared by any one of the well known stAn~Ard teçhniques using horses, rabbits or mice in the production of polyclonal antibodies and extracted serum, or production of monoclonal antibodies based on the well known te~hniques involving mice or higher lifeforms. The passive vaccines are administered to foals to provide a sufficient in vivo quantity to combat R. equi challenge during the foals susceptible period. The use of passive vaccines involve the administration of large volumes so that they inherently have the atten~nt drawback of administration on several occasions during the sll~ceptible period of the foal. The preferred antibodies are those raised to the N-terminal end of the lipid modified proteins, since these appear to provide the greatest affinity and protection against R . egui challenge. However for diagnostic purposes and other purposes, antibodies, such as monoclonal antibodies to the C-terminal end of the Vir protein, are of value.
Active V~rrin~
The preferred commercial application of the Vir protein is in active vaccines which may be administered on a periodic basis to the foal to induce or elicit an immune response which resists subsequent challenge by R.
equi. The preferred Vir protein for use as a vaccine component is the lipid modified protein having a molecular weight in the range of 17.5 to 22. In a vaccine composition, the Vir protein may be used with or without the addition of other adjuvants and pharmaceutically acceptable carriers.
It is also understood that the Vir protein, as recovered by the detergent treatment, may be lyophilized when separated from the detergent treatment. The lyophilized material may then be stored for the subsequent purpose of making into a vaccine composition.
It is also understood that the vaccine composition may include the Vir protein as made by culture of R. equi along with Vir protein made by alternative recombinant methods.
The preparation of vaccines which contain peptide sequences as active ingredients is generally well understood in the art, as exemplified by United States patents 4,608,251; 4,601,903; 4,599,231; 4,599,230;
4,596,792 and 4,578,770, all incorporated herein by reference. Typically, such vaccines are prepared as injectables. Liquid solutions or suspension solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.
Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants which may enhance the effectiveness of the vaccine .
The vaccines are conventionally administered parenterally, for example, by injection, either subcutaneously or intramuscularly.
The proteins may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic 2125~2G
_ 17 bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. The salts are usually then solubilized in a suitable pharmaceutically acceptable carrier or excipient.
The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the foal's condition to be treated, capacity of the foal's immune system to synthesis antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered ~p~ on the judgment of the practitioner and may be peculiar to each foal. However, suitable dosage ranges are of the order of several hundred micrograms active ingredient per animal.
Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by subsequent inoculations or other administrations.
The manner of application may be varied widely. Any of the conventional methods for administration of the vaccine are applicable. These include oral application on a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection or the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size of the foal. Normally, the amount of the vaccine will be from about 1 mg to 20.0 mg per kilogram of foal, more usually from about 5 mg to 2.0 mg given subcutaneously or intramuscularly after mixing with an appropriate carrier or an adjuvant to enhance immunization with the vaccine.
Adjuvants may be included in the vaccine to enhance the immune response. Usually the adjuvants are added to enhance antigenicity to the immune system. Such adjuvants may be a suspension of minerals on which the antigen is adsorbed, or water and oil emulsions in which 212a426 antigen solution is emulsified in mineral oil (Freund's, incomplete adjuvant).
Various methods of achieving adjuvant effect for the vaccine includes use of agents such as alum, aluminum hydroxide, or phosphate, commonly used as 0.05 to 0.1 percent solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as 0.25 percent solution; aggregation of the protein in the vaccine by heat treatment with temperatures ranging between 70 to 101C for 30 second to 2 minute periods, respectively. Aggregation by reactivating with pepsin treated (Fab) antibodies to albumin, mixture with bacterial cells such as a C. parium or endotoxins or lipo-polysaccharide components of gram-negative bacteria, emulsion in physiologically acceptable oil vehicles such as mannide mono-oleate (Aracel A) or emulsion with 20 percent solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed.
Various aspects of the invention are now described in respect of detailed experiments and procedures used to clone the vir gene, identify the Vir protein, express the protein in suitable hosts or recover the protein by extractive tec~n;ques using suitable hydrophobic material extractants, the development of antibodies and the development of the vaccines.

MATERIALS AND MEl~IODS
Bacterial strains and plasmids.
Rhodococcus equi strain 103, which is similar to ATCC accession ~ 33701 and which possesses an 84 kb plasmid (pOTS), has been used although it is appreciated that similar results can be attained using the deposited strain of ATCC 33701, or other R. equi strains having the 84 Kp plasmid (pOTS). E. coli strain XLlBlue was used for cloning and transformation. The plasmid cloning vector pBluescript II (SK+) (Stratagene, La Jolla, CA) was used for cloning and transformation.

2125~26 DNA methodology.
Restriction digestions, ligations, and gel electrophoresis were done essentially as described by Sambrook and others (1). The boiling method of Holmes and Quigley (2) was used to prepare some DNA for transformation screening and restriction enzyme analysis.
Restriction enzymes and T4 DNA ligase were obtained from Boehringer MAnnheim (Laval, Quebec) and Bethesda Research Laboratories (Burlington, Ontario). Transformation of E.
coli was done by electroporation (E. coli Pulser; Bio-Rad, Mississauga, Ontario). The virulence-associated plasmid pOTS was isolated using the QIAGEN column (QIAGEN, Chatsworth, CA), modified by treating cells with 5 mg/ml of lysozyme for 3 hours at 37C. A partial EcoRI library of the plasmid was constructed using pBluescript and E. coli XLl-Blue containing recombinant molecules screened with polyclonal antibody prepared against the 17-kDa virulence-associated protein. A
recombinant expressing immunoreactive proteins was subcloned using stAn~Ard methods (1). Southern blotting by stAn~Ard methods was done to confirm the plasmid origin of the cloned fragment using digoxigenin-labelling (Boehringer M~nnheim) (1).
DNA seq~l~n~i n~.
A 1.6 kb BamHI-PstI fragment of pCT-C7 was cloned into the pBluescript sequencing vector. Unidirectional deletions used in sequencing were prepared using the Erase-a-Base system (Promega Corp., Madison, WI). The sequence of cloned R. equi DNA was determined from double-stranded plasmid templates by the dideoxy-chain termination method (3). Double stranded templates were denatured with alkali and the sequencing reactions carried out with the Seql~nA~ version 2.0 kit (United States Biochemical, Cleveland, OH). Synthetic primers designed from the DNA sequence were also used.

212~426 ~ 20 Triton X-114 phase partitioning.
R. equi was grown in nutrient broth (Difco, Detroit, MI) at 38C for about 60 hours with constant shaking at 150 rpm. Cells were harvested by centrifugation, washed with 10 mM Tris-HCl, pH 7.4, 0.15 N NaCl (TBS buffer), recentrifuged and pellets stored in microcentrifuge tubes at -70C. Extraction and phase separation with TX-114 was done essentially as described by Bordier et al (4).
Briefly, 30-50 mg wet weight of cells were added per ml 2% TX-114 (Calbiochem, San Diego, CA) in TBS with 1 mM
phenylmethylsulfonylflouride (PMSF, Sigma Chemical Co., St Louis, M0) and shaken overnight at 4C. Insoluble material was removed by centrifugation at 14,400 x g at 4C for 15 min. The supernatant was recovered, and warmed to 37C for 10 min before centrifugation at 25C
at 14,400 x g for 15 min. The upper aqueous layer was removed and re-extracted with TX-114 to make a 2~
solution. The lower TX-114 phase was washed three times with enough TBS to make a 2% TX-114 solution. The bacterial pellet was extracted with TX-114 a second time.
The detergent phase was used in parallel with the aqueous phase for subsequent electrophoresis. Protein content was determined by the BCA Protein Assay (Pierce, Rockford, IL). TX-114 extraction of E. coli XLlBlue(pCT-C7) expressing the vir gene products was done by anessentially similar process, except that the cells were sonicated before extraction. E. coli XLlBlue(pBS) was used as a control. The triton extract was precipitated with 10 vol acetone overnight at -20C, dissolved in Tris, boiled in SDS sample buffer for 10 min, and run on a 15% SDS-PAGE gel, transferred to nitrocellulose and blotted with rabbit polyclonal antiserum or mouse monoclonal antibody in accordance with the following.
Radiol~h~lli n~ of lipid-modified proteins.
R. equi 103 was labelled with [3H]-palmitic acid according to the method of Neilsen and Lampen (5). R.
equi was grown in 4 ml nutrient broth at 38C for 35 212~4~6 hours in a sh~king waterbath. 20 ~Ci ~9,10(n)-[3H]-palmitic acid (Amersham, Oakville, Ontario) was added and incubation continued for another 6 hours. The cells were then harvested, WA Sh; ng once with phosphate buffered saline (PBS), pH 7.4, and twice with 100% methanol. The methanol was removed by drying the pellet in a vacuum oven. SDS reducing buffer was added to the dried pellet and the sample was run on SDS-PAGE as described below.
The gel was fixed in isopropanol: water: acetic acid (25:65:10) for 30 min and then soaked in Amplify (Amersham) for 15 min. The gel was then dried and ~Yro~
to Kodak XOMAT AR5 X-ray film at -70C.
Gel el~Lv~oresis and immunoblotting.
For SDS-PAGE and immunoblotting, all samples were suspended in SDS reducing buffer and boiled for 10 minutes (6). Undissolved material was removed by centrifugation and samples separated in a mini-electrophoresis system (Bio-Rad) using 15% resolving and 5% stacking gels. Proteins were stained with Coomassie brilliant blue. For immunoblotting, gels were transferred to Biotrace NT (Gelman Sciences, Ann Arbor, MI) using a mini-blot electrophoretic transfer cell (Bio-Rad) (7). The membranes were blocked with 5% fish gelatin in TBS, incubated with an IgG1 murine monoclonal antibody (MablO3) for 1 h or rabbit polyclonal antibody (1:12,000 dilution) prepared against the 17-kDa protein, washed PBS-0.05% Tween 20 (PBST), and incubated for 1 h with alkaline phosphatase-conjugated goat anti-mouse IgG
F(ab)2 or anti-rabbit IgG F(ab)2 fragments (Bio/Can Scientific Inc, Mississauga, Ontario), as appropriate, then washed repeatedly. Naphthol ASBI phosphatase and Fast-Red (Sigma Chemical) in 0.01 M Tris-HCl pH 9.2 was used to visualize the blot. Molecular weights of the R.
egui virulence-associated proteins in R. equi or E. coli XLlBlue(pCT-C7) were determined using TX-114 extracts from these organisms run on 13% or 15% SDS-PAGE gels and transferred to nitrocellulose. Mouse polyclonal ~12~2~
_ 22 antiserum was used to identify the proteins of interest and the molecular weight calculated using linear regression by comparison of Rf values to low molecular weight marker st~n~rds (Bio-Rad).
Monoclonal or polyclonal antibody production.
For monoclonal antibody production, whole cell proteins of R. equi strain 103 were separated by SDS-PAGE
and electroblotted onto nitrocellulose. Horizontal strips containing the 15- and 17-kDa proteins were excised. Two strips were ground in 1.5 ml PBS and homogenized with an equal volume of Freund's incomplete adjuvant. BALB/c mice were injected with 0.5 ml antigen intraperitoneally twice at 10 day intervals and with 0.25 ml 21 days later. Serum was tested by immunoblot 14 days later for antibody production. A responder mouse was injected with 0.5 ml powdered nitrocellulose in PBS 4 days before sacrifice. Splenocytes were fused to NS-1 myeloma cells. Hybridoma supernates were tested by ELISA
using sonicated whole R. equi as antigen and by immunoblot. An IgGI-producing hybridoma (designated MablO3) was isotyped (Cedar Lane Laboratories, Hornby, Ontario) and cloned by limiting dilution. The cell culture supernatant was used in immunoblots undiluted or diluted by half with 5% gelatin in PBS. For opsonization studies and for passive immunization of mice, the IgG1 was purified using membrane affinity chromatography-protein G
capsule (Amicon, Oakville, Ontario) and quantified by protein assay (Bio-Rad).
For polyclonal antibody production against the denatured 17-kDa virulence-associated protein, the protein was recovered from whole cell proteins from bacteria grown in brain heart infusion broth at 37C for 72 hours by solubilizing in SDS reducing buffer and separated by SDS-PAGE using continuous elution electrophoresis, in a manner described with respect to mouse immunization. For polyclonal antibody production against the recombinant virulence-associated protein ~3 2 1~5~26 expressed in E. coli, the protein was extracted from E.
coli XLlBlue(pCT-C7) in TX-114 as described, precipitated with acetone at 4C overnight, and suspended in 10 mM
Tris, pH 7Ø For each of the proteins, the native R.
equi and the recombinant in E . coli, about 300 ~g was homogenized in Freund's incomplete adjuvant and injected subcutaneously twice into 2 rabbits with a two week interval, and rabbits bled for serum two weeks later.
Murine macr~r~-ge ~p~ni 7~tion assay.
IC-21 mouse macrophages (ATCC TIB 186) were cultured in RPMI contAining 10% inactivated calf serum (FCS;
Gibco, Burlington, Ontario), penicillin (100 IU/ml), and streptomycin (40 ~g/ml). Macrophages were counted, adjusted to 1 x 106 cells/ml in RPMI with 10% FCS, and added to wells of Nunc tissue culture chamber slides (Gibco). The slides were incubated for 1.5 hours at 37C
in 10% CO2 to allow adherence. R. equi strain 103 grown in nutrient broth for 72 hours at 37C was washed once in PBS, and suspended in PBS. The bacterial cells were adjusted by optical density to give a final concentration of 1 x 106/ml in hybridoma supernatant or appropriate dilution of purified MablO3. The antibody-bacterial suspensions were incubated for 15 minutes at 37C before use to replace the media in the slide chambers. After incubation at 37C in 10% CO2 for 60 minutes, the slides were washed in 3 changes of PBS for 5 minutes each with stirring. Slides were stained with Wright's stain and the presence of bacteria in 300 macrophages (10 fields of 30 cells) determined.
House immunization and challenge studies.
Female 6-8-week-old CDl mice (Charles River, Montreal) were used. For passive immunization, mice were immunized intraperitoneally with 300 ~g MablO3 one day before challenge. Immunized and nonimmunized controls were injected intravenously with 5 x 105 R. egui strain 103. Five mice in each group were sacrificed on days 1, 4, and 7 post-infection and bacterial numbers in whole lung, liver, and spleen enumerated. For active immunization, TX-114 extracted protein was precipitated with 10 vol of acetone overnight at -20C and applied to a 15% PAGE resolving gel with a 4% stacking gel in SDS
reducing buffer (6) for continuous elution electrophoresis (Electrophor Model 491 Prep Cell; Bio-Rad). Fractions were collected after the dye front and concentrated (Centrifugal Ultrafree, 10,000 Da cutoff:
Millipore Corp., Bedford, MA). These fractions were identified as the 17-kDa protein by SDS-PAGE and immunoblotting with MablO3. Fractions which were identified as the 17-kDa protein were combined, assayed for protein (BCA; Pierce) and adjusted to 400 ~g/ml. The solution was mixed with an equal volume of Freund's incomplete adjuvant and 400 ~l injected intraperitoneally into CD1 mice. The procedure was repeated 14 days later.
Control mice received adjuvant mixed with an equal volume of Tris buffered saline, administered at the same schedule as vaccinates. All mice were challenged intravenously 17 days after the second immunization with R. equi strain 103 (105 organisms/mouse). Seven mice from each group were killed on days 2 and 3 after challenge and 6 mice on days 4 and 7, and bacteria enumerated in lung, liver, and spleen.
N-terminal amino acid sequencing.
To obtain N-terminal amino acid sequence from the native 17-kDa protein, the denatured protein was recovered by continuous elution electrophoresis as described above. The elution fraction containing only the 17-kDa protein was separated on an SDS-PAGE gel, electroblotted to a polyvinylidene difluoride membrane (Bio-Rad) in 10 mM CAPS buffer and 10% methanol pH 11.0 at 14 V for 18 hours, and excised for N-terminal amino acid sequencing using automated Edman degradation.
Protein -ecQn~-~y structure and L~d~Lobicity analysis.
Predicted protein secondary structure was determined ~12542~
_ 25 using Protylze Predictor Version 3.0 software based (8, 9) -The following application of the above-described materials and methods provides the following results in respect of the various aspects of the invention.

EXAMPLE 1 Cloning and sequencing of the vir gene.
A partial EcoRI library was constructed for pOTS, of the 9 EcoRI fragments less than or equal to 10.5 kb.
Recombinants of each cloned fragment were tested by Western immunoblotting using rabbit polyclonal antibody.
A recombinant pCT-C, containing the 10.5 kb fragment, was positive on Western blot with and without IPTG induction.
Three immunoreactive bands of approximately 15-, 17.5-, and 20-kDa were identified but the diffuse band characteristic of the R. equi 18-22-kDa virulence-associated protein was not present in E. coli, as shown with reference to Figure 1, Lane 1, where molecular weight markers in kDa are in the left margin. Southern blotting confirmed the plasmid origin of the fragment.
Recombinants other than those with the 10.5 kb fragment were negative. The 10.5 kb fragment was mapped with restriction enzymes and subcloned in pBluescript.
Several deletion derivatives were constructed, as shown in Figure 2, and transformants containing the deletion derivative pCT-Cl, pCT-C6, pCT-C7, and pCT-CSl were found to produce the three immunoreactive proteins as demonstrated in Figure 1. Antiserum prepared against TX-114 extracted E. coli XLlBlue(pCT-C7), as described in the Examples, reacted with whole cell R . equi in a manner identical to ~Ab103, rPcognizing the 15-, 17.5- and diffuse 18-22-kDa protein bands of the native R. equi virulence-associated protein (Fig. 1), confirming that the gene for the virulence-associated protein had been cloned.
The 1.6 kb BamH-PstI of pCT-C7 contained the open reading frame of the virulence-associated protein, designated vir, of 570-bp which begins with a methionine start codon at nucleotide 245 of Table I and terminates with a TAG stop codon after nucleotide 811 of Table I
(Sequence I.D. #1), thus encoding a polypeptide of 189 amino acids (deduced molecular weight 19,175 Da). The open reading frame is preceded by the nucleotide se~lPncec A~r~r~AÇ which are assumed to serve as a ribosome-binAing site. What may be a rho-independent inverted repeat termination signal occurred from nucleotides 846-885 of Table I (Sequence I.D. #1).
Protein folding prediction showed the first 31 amino acids to have an alpha-helical folding, corresponding to that expected of a leader peptide (4). Protein secondary structure analy~is revealed the leader peptide to be the only significantly hydrophobic region. N-terminal amino acid analysis of the 17.5-22-kDa protein isolated from R.
equi identified the start of the mature protein at codon CGA starting at nucleotide 336 of Table I (Sequence I.D.
#1), 32 amino acids after the start codon and immediately following the predicted leader or transmembrane signal peptide (Sequence I.D. #2). N-terminal amino acid sequencing revealed also that the majority of the protein was blocked to the Edman reaction. The predicted mature polypeptide consists of 158 amino acids with a molecular mass of 16,246 Da or 16.2 kDa. The predicated molecular mass appears to be correct, because the SDS-PAGE gel estimated moleclll~r weight is in the range of 15 kDa.
Considering that there can be in the range of 10%
variation in molecular weight estimation by SDS-PAGE gel, then the 15 kDa band in Figure 1 appears to be correct and therefore corresponds to the molecular weight of the predicted mature protein sequence.
No significant nucleotide or amino acid sequence homologies were found between the vir gene nor Vir protein and other DNA and protein sequences con~; neA in the ~enR~nk, European Nolecular Biology Laboratory (EMBL), National Biomedical Rece~rch Foundation-Protein 2l2~q2~
_ 27 Identification Resource (NBRF-PIR) and Swiss-Prot data bases. The overall G+C content of the Vir protein is 60%, with usage being 59.4%, 50%, and 64.2% at codon poeitions 1, 2, and 3, respectively.
s EXAMPLE 2 Triton X-114 extraction and lipid modification of virulence-associated proteins.
Whole cell preparations of R. equi separated on SDS-PAGE and stained with Coomassie blue show an often weakly staining diffuse protein band at approximately 17.5-22-kDa (Fig. 3) and an inconsistently present band at about 15-kDa. When whole cells of R. equi were extracted with the surfactant, TX-114, three major protein bands (44-, diffuse 18-22-, and 17.5-kDa) were recovered in the detergent phase which reacted with the monoclonal antibody in a Western blot (Fig. 3). The 44-kDa band is believed to be an anomaly most likely represented by a TX-114 induced aggregation of the proteins or may be a protein-detergent micelle (10). No corresponding protein bands were observed in the aqueous phase. The 15-kDa band was not observed in TX-114 preparations (Fig. 3) but was evident in some whole cell preparations (Fig. 1). In B. coli cont~in;~g pCT-C7, but not in E. coli with pBluescript, protein bands of 17.6 and 20-kDa which reacted with rabbit polyclonal antiserum to the R . equi virulence-associated proteins were recovered in TX-114 (Fig. 1).
When the amount of ~3H]-palmitic acid was limited in R. equi, the 17.5-22-kDa proteins incorporated the radioactive fatty acid, and extensive labelling of low molecular weight lipids was observed at the bottom of the gel as per Fig. 3. Incorporation into both the diffuse 18-22- and the 17.5-kDa protein bands could be clearly disting~ ehe~ on some gels.
EXAMPLE 3 Murine macro~hage opsonization assaY.

Uptake of R. equi strain 103 by the mouse macrophage cell line was ~nhAnce~ by opsonization with the monoclonal antibody as established in Table II. The characteristic initial decline with subsequent rise compared to the controls observed was attributed to agglutination by the antibody.

EXAMPLE 4 Mouse immunization and challenge studies.
The monoclonal antibody administered intraperitoneally as a passive vaccine enhanced liver clearance at 1 but not at 4 and 7 days after intravenous challenge as set out in Table III. Clearance from liver, lung, and spleen was e~hAnce~ in mice actively immunized with the 17-kDa virulence-associated protein isolated by continuous elution electrophoresis as set out in Table IV. Antibody response in immunized mice determined by Western immunoblot against whole cell protein preparations was marked (titres >1:8000) and occurred predominantly to the virulence-associated proteins. An earlier study using a similar immunization protocol but with only 12 days from the second immunization to challenge showed 6tatiætically significantly enhanced clearance at day 3 but not at days 1, 5, and 7 following challenge.
EXAMPLE 5 Horse Immunization Studies In order to further demonstrate the highly immunogenic properties of the Vir protein, horses were vaccinated with a vaccine composition contAining the Vir protein and their antibody response was measured over time to demonstrate immune activity. The vaccine composition comprised the Vir protein component reAic~clved in Tris to about 1 mm per ml, Tris being a well known pharmaceutically acceptable carrier for the vaccine component. In addition, an adjuvant, aluminum hydroxide, was added to make up 35% by volume of the composition. The Vir protein was prepared in accordance 212~-~21~

with the aforementioned tec~n;que of detergent extraction. The detergent extraction was carried out using TRITON~ with subsequent acetone precipitation of the Vir protein from the solution separated from the detergent.
The immune response in each horse was determined by use of the well-known ELISA assay. The purpose of the ELISA test is to determine specific serum antibody response of mares and foals to Rhodococcus equi. Details of the ELISA test include plates being with 2 ~g/ml of the lipid modified protein extracted from R . equi using TRITON X-114 (antigen). Serial dilutions of horse sera are added to the plate and incubated for one hour. The plates are washed to remove excess antibody. The antigen-antibody reaction is detected by a secondary antibody to the serum horse antibody and labelled to a marker enzyme. Substrate is added followed by colorimetric measurement to determine the titre for horse immune response.
The vaccine was administered on three separate occasions. Before vaccine administration, the level of antibody titre in each horse's serum was measured by the ELISA test. Following the first vaccination dose, antibody titre was measured two weeks later at which time a further vaccination dose was administered. Four weeks later the antibody titre was measured, upon which time a further vaccination dose was administered. Also six weeks later, antibody titre was measured.
The results, as per the following Table V, clearly demonstrate a significant increase in immune response to the vaccination using the Vir protein. With antibody titers increasing after four weeks by some 5000 to 8000 fold increase, demonstrates the very high immunogenic response invoked by the Vir protein.
The above Examples in demonstrating various aspects of the invention provide valuable insight into the 212542~

characteristics of the Vir protein to now enable the use of vir gene sequence information, Vir protein sequence information in diagnostics and vaccine components.
The work described here establishes that these proteins are encoded by one plasmid-carried gene, the vir gene, and that lipid modification is responsible for the different protein forms observed. In addition, these proteins have immunoprotective properties.
The 10.5 EcoRI fragment contains the gene for the plasmid-mediated virulence-associated protein, confirming the findings of others (11). We have demonstrated we have cloned the gene for the R. equi virulence-associated proteins because:
i) the presence of three protein bands in E. coli recombinants, similar in size to the R. egui virulence-associated proteins, which reacted with rabbit polyclonal antibodie~ prepared against the ~. equi virulence-a6sociated proteins (Fig. l); the recognition by antiserum prepared against the recombinant protein of the virulence-associated protein complex in R. equi (Fig. l);

ii) the identity of the N-terminal amino acid sequence of the R. equi protein with the sequence deduced from the cloned gene;
iii) the similar hydrophobic characteristics of proteins from R. equi or E. coli demonstrated by their partition into TX-114 (Fig. 1, 3); and iv) the use of Southern blot to confirm the plasmid origin of the cloned gene. Although little is known about the genetics of rhodococci, we have now demonstrated one of the first descriptions of the efficacy of a rhodococcal promoter in E . coli ( 12).
DNA sequence analysis revealed an open reading frame corresponding to a polypeptide of molecular mass 19,175 Da. The first 31 amino acids had the characteristics of a signal sequence, starting with an N-terminal lysine followed by a alpha-helical hydrophobic region (13), and 2 12 5 ~ 2 G

terminated in a possible alanine-X-alanine signal peptidase I cleavage site (13), which immediately preceded the N-terminal amino acid sequence of the mature protein identified by amino acid sequencing. The size of the deduced mature protein of 16,246 Da which corresponded closely to the molecular weight determined from SDS-PAGE gels as the 15-kDa protein. Our fin~ing that the vir gene encodes a protein whose different sizes are due to lipid modifications thereof explains why one monoclonal antibody (NablO3) directed to the C-terminal end rPcognizes all three protein bands on immunoblotting in R. equi (Fig. 1, 3). Besides establishing the N-terminal sequence of the mature protein, N-terminal amino acid analysis of the 17.5-22-kDa protein of R. equi also showed that the majority of the protein was blocked to the Edman degradation reaction, consistent with lipid modification.
Failure of dilapidation to remove radiolabel from the site of the Vir protein showed that lipid was covalently linked to the protein. The presPnse of three forms of the protein in both R . equi and the E . col i recombinant thus demonstrates that there are probably two lipid modifications of the Vir protein, and this was confirmed by incorporation of radiolabelled palmitic acid 25 in both the 17.5- and 18-22-kDa proteins in R. equi. The two heavier bands in the E . col i recombinant also extracted into TX-114, confirming their hydrophobic nature and demonstrating their lipid modification as well.
Although the three immunoreactive proteins produced by recombinants in E. coli corresponded in size to the three forms of the protein in R. equi, the failure of the 18-22-kDa form to take its characteristically diffuse form in E . col i shows what may be an additional difference in lipid modification of the Vir protein between E . col i and R . equi . Antibody to the recombinant protein however recognized the diffuse band of R. equi.

Diffll~?nesc in SDS-PAGE gels due to lipid modifications of proteins have been described in Mycobacterium tuberculosis ( 14) and in Treponema pallidum (15). The heterogenous behaviour of the diffuse 18-22-kDa protein on SDS-PAGE may result from either variability in lipid modifications with resulting differences in molecular mass or from the binding capacity of this lipid-modified form for SDS (14).
The lipid modifications determine the hydrophobic nature of the Vir protein since the unmodified mature 15-Kda protein is predominantly hydrophillic and does not extract into TX-114 in R. equi. The ease of TX-114 extraction of the 17.5-22-kDa proteins from whole bacteria demonstrates that they are on the surface of the organism. The 15-kDa form of the protein may be largely intracellular. The site of lipid modifications is unclear but likely occurs at a site close to the N-terminal region of the mature protein. The protein lacks the consensus cleavage site of signal peptidase II and is not a lipoprotein (16). Besides lipoprotein modification, four other types of fatty acylation of proteins are recognized: palmitoylation, isoprenylation, myristoylation, and glypiation (17). The first two involve cysteine, which is absent in the Vir protein.
The absence of a glycine at the N-terminal end of the mature protein and the low molecular weight of myristic acid suggest that the Vir protein is not myristoylated.
The possible type of lipid modification of the Vir protein is therefore by glypiation, the attachment of a phosphatidylinositol-containing glycolipid (17). This suggestion is supported by the increased molecular mass of the lipid-modified proteins compared to the unmodified mature protein of 15 kDa and by the observation that the inositol fatty acid is always palmitic acid, which was readily incorporated in radiolabelling studies.
The opsonization by R. equi with MablO3 in the mouse macrophage cell line was further evidence that the 212~l2~

protein is expressed on the surface of the organism.
Opsonization in the macrophage cell line demonstrates the immunogenic potential of the protein. The temporarily enhanced clearance following passive immunization shows that the antibody plays a role in protecting immunocompetent mice from experimental infection. Active immunization of mice with denatured protein recovered by continuous elution from SDS-PAGE resulted in marked antibody response and in significant P~hAncement of tissue clearance. The increased effect of active immunization with time, the relatively poor clearance shown by MablO3, and the immunogenic effect of what is likely SDS-denatured antigen suggests that cell-mediated immune mechAni~ms produced by the 17-kDa protein is largely responsible for enhAnce~ clearance from mice.
Earlier studies of killing of R. equi by equine macrophages suggested that immunity was the result of both antibody and cellular immune mechAni~ms (18).
The additional work in mice clearly indicated the ability of the Vir protein to elicit an immune response.
We have now demonstrated that the Vir protein elicits corresponding immune response in horses, as established by the results of Example 5 and hence the importance of the Vir protein as a vaccine component for use in vaccine compositions which, when administered to foals, establishes resistance to R. equi challenge.

212~42~
_ 34 TP~BI~E I
BglII
1 CTGGGCTAGA rAAnA~A~CT TCCGCTCCGC TAATTACCGG CACTAAA~--AT AAAGr-ArGCG
SacII
61 CA.~.~..~. GGTrAr,r-ArA TCGCACCC~-A CGGGGCTCGC GGAGAGTGCC GCGGTGAGCT
121 AACGTAAGTT '~cc~AGA ~.-~.CGGGT ~cG~AAcG CTACAATCAA CTATGTCGGA
SD
181 ACTGCCC~ AAC~A~ r-~ TCCGCGAAGG CGATCGAAGG GCGACGTCCG AA~G~Ar~Ar 241 TAAGATGAAG A~. ACA AGACG~...C TP~GCr-ATC GrA4CrACAG CCGTAGCTGC
M R T L H R T V S R A I A A T A V A
301 GGCTGCGGCT ATGATTCCCG CCGGCGTCGC TAATGCG{ACC G..~..GATT CCGGTAGCAG
A A A A M I P A G V A N A T V L D S G S
361 CAGTGCGATT CTCAATAGTG GG}G QGGCAG TGGCATTGTC GGTTCTGGGA GCTATGACAG
S S A I L N S G A G S G I V G S G S Y D
421 CTCr-Ar,r-ACT TCGTTAAACC TTrA~--AAA~-A C~--AAr,CGAAC GGTcr-A4 Q A GC4ATACCGC
S S T T S L N L Q R D E P N G R A S D T
481 C~GG~AA-A4 CAGCAGTACG ACGTTCACGG AGACGTCATC AGCGCGGTCG TC~ACr~GA4 A G Q E Q Q Y D V H G D V I S A V V Y Q
541 GTTT Q CGTA TTCGCCCr~ GT CTTCGATGGC GATGr~GGGG GACTCACGCT
R F H V F G P E G R V F D G D A G G L T
EcoRII
601 .C~.GGGGCC GGCGCGTTCT GGGGC~CTCT CTTrArAAAT GACCTTCAGC G.~. ACAA
L P G A G A F W G T L F T N D L Q R L Y
661 Ar-ArArCGTC .O~..C~AGT ACAACGCCGT GGGGCCATAC CTGAACATCA A~..~..CGA
R D T V S F Q Y N A V G P Y L N I N F F
ScaI
721 TAGCTCAGGT AG~..C~.CG GCr~TA~CCA G-CC~G.aGA GTTAGTACTG GG-GGGCGT
D S S G S F L G H I Q S G G V S T V V G
781 OGoCGoCGGC TCTGGTAGCT GGCA~AACGC CTA4r-AGGcT GCACGTACTT CCGr-~ArCCC
V G G G S G S W H N A *
841 GGGTGGCr7AA AAGGGCAGGC GCGAACCGCT TCCTGCCCTT TTCGCTCAGC ~-CGG--~--AvaII
901 AG~AC~rATc GAAGATGCGC GGTCr~--AAA CATGCAGGCT GCGAGGTCAT AATAATTAAG
961 CGGGAGCAAT TTAArAr~7GcG TATCAAGGTG TGAGGTGGGT GTAr-AGGGCT GAAATTATCA
PstI
1021 CGAO.CC~.. TTCGTGGGAA TCGrAAr~0C ATTGGTGCCA ATCGCGCTGA CTGCAG

Nucl-otide sQguence of the R. cqui pOTS-derived DNA in pCT-C7S1 and the ~ ced amino acid sequencQ of the 17-kDa virulen~
as~ociated protein of R cgui The asterisks indicate the poeition of ~tart and stop codonB. Shine-Dalgarno (S D ~ and a stem-loop structure are indicated by underline { } indicateg the N-t- ; na 1 sequence of mature protein. The unique re6triction enzyme sites are indicated above the DNA sequence TABLE II
Uptake of R. 8qui by murine macrophage cell line (%
cells) in presence of monoclonal antibody to R. equi Vir protein.
% cells with R. equi Antibody dilution Al B C

No antibody 20c l5~b 25c 1:20 22c gc 23c 1:40 _ 1lbc 24c 1:80 34b 17~ 16d 1:320 54~ 18- 36b 1:1280 34b _ 45~
1:10,240 - ~ 30bc A, æupernatant; B, C, 500 ~g/ml monoclonal antibody.
Duncan's multiple range test; means with same superscript letter in same column no significant difference (P~0.05).

212~12fi _ 36 TABLE m tP~iql rl-qrtqnr~ in CDl mice immlmi7~ with monorlonql -ntihody to R.
equi Vir protein and challenged intravenously with R.
equi.

Days post Liver Spleen inf~ti-~n Tmmlmi7~d- Control ~mmuni7~ Control 1 3.69 + 0.13 4.04 ~ 0.13 3.98 + 0.12 3.87 ~ 0.16 4 4.03 ~ 0.34 3.24 ~ 1.86 4.26 ~ 0.42 3.34 ~ 1.90 7 - O.Sl ~ 1.14 2.35 i 1.39 1.93 + 1.76 300 ~ug ms~n~rlcnql antibody in~ P~ nPqlly, day-l.
b p<0.003, Duncan's ml-ltipl- range test (5 mice per group).

_ 37 TABLE IV
R~^t~riql cl~oqr~qnce in CDl m-ice imm~-ni7f~d with SDS-PAGE
eluted Vir protein and chq1l~nged iJlt~dvellously with R. equ~.

Days post Liver S~leen inf~tinn Tmmllni7~1 Control Tmmllni7~ Control 2 3.21 + 0.67 4.17 + 0.563.96 i 0.31 4.25 i 0.31 3 3.49 + 0.68- 4.41 + 0.304.01 + 0.44- 4.95 + 0.29 4 2.52 + 1.36 4.16 + 0.193.38 + 0.28- 4.44 + 0.18 7 1.60 + 1.33 2.39 i 1.102.51 + 0.35 3.03 + 0.45 p<O.OS, Duncan's mll1tiple range test (5-7 m-ice per group).

~_ 38 o o ~ o oo oo ^ ô

~ 0a4 a~ a O O O O

~; ~ o O

g3 ~ ~ o ~ 8 E~ ~ oo ^ oO oO ^ ^ ^ ^

a~ a~ a~ a~ ~ ~ a~ a~ a~ 0a4 O ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
~ ~
~ t 00 o 00 ~o ~o ~o O

2~25426 ~h~ULN~ LISTING

( 1 ) C~URRAT. INFORMATION:
(i) APPLI Q NT: Pre~cott, John F
Tan, Cuiwen (ii) TITLE OF lhvLn~ION: R~ODOCOCCUS EQUI GENE SEQUENCE
(iii) NUMBER OF XL~UL..-~S: 2 (iv) CORRESPONDENCE ADDRESS:
'A' PnDRRSSRR: Bell, Seltzer, Park ~ Gib~on B STREET: 1211 Ea~t Morehead Street, C CITY: Charlotte, D STATE: North Carolina E COUh~nY: United State~ of America ~F ZIP: 28234 (v) COMPUTER ~RAnART-R FORM:
~Al MEDIUM TYPE: Floppy di~k ~B COI~u.~n: IBM PC compatible ,C, OPERATING SYSTEM: PC-DOS/MS-DOS
~D SOFTWARE: PatentIn Relea~e #1.0, Version #1.25 (Vi) ~UnR~h~ APPLICATION DATA:
(A) APPLI Q TION NUMBER:
(B) FILING DATE:
(C) CLASSIFI Q TION:
(viii) A. ORN~/AGENT INFORMATION:
(A) NAME: Layton, Jr., Samuel G
(B) REGISTRATION NUMBER: 22,807 (ix) TRRRCnM~I Q TION INFORMATION:
(A) TELEPHONE: 704-377-1561 (B) TELEFAX: 704-334-2014 ~ 40 2 1 2 ~ q 2 6 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
A LENGTH: 1076 ba~e pair~
B TYPE: nucleic acid C, STRANDEDNESS: ~ingle D TOPOLOGY: linear ( ii ) M~T~FC~lT~ TYPE: DNA (g~n ;~
(iii) ~Y~G~A~lCAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Vir protein (B) STRAIN: P-hodococcua equi (vii) IMMEDIATE SOURCE:
(B) CLONE: pCT-C7Sl (viii) POSITION IN GENOME:
(C) UNITS: bp (xi) SEQUENCE ~SCDTPTION: SEQ ID NO:l:
CTGGGCTAGA C~ G~TCT TCCGCTCCGC TAATTACCGG CACTAAA~AT AAAGCACGCG

CAl,. ~, GGT~A~-A~A TCGC-ACCC~-A CGGGGCTCGC GGAGAGTGCC GCGGTGAGCT

AACGTAAGTT ..CCG.~AGA G~,CGGGT ,~,C~AACG CTACAATCAA CTATGTCGGA

ACTGCCC~ - aA~TP~ TCCGC~ C CGATCGAAGG GCGACGTCCG AA~C~n~C

TAAGATGAAG A~.~ACA AGACGG..~C TAAGGCGATC GCAGCCACAG CCGTAGCTGC

GGCTGCGGCT ATGATTCCCG CCGGCGTCGC TAATGCGACC ~,, GATT CCGGTAGCAG

CAGTGCGATT CTCAATAGTG GGG~AGGCAG TGGCATTGTC GG,,~,GGGA GCTATGACAG

CTC~r-ACT TCGTTAAACC TTrAa~ Cr-AA~C~-AA~ GGTCGAGCAA GCr-ATACCGC

CG~G~AAr-~G Q GCAGTACG ACGTTCACGG AGACGTCATC AGCGCGGTCG TCTAC~-Ar-Ar-GTTTCACGTA TTCGGGCCAG PA~A~GT CTTCGATGGC GATGCAGGGG GACTCACGCT

TCCTGGGGCC GGCGCGTTCT CGGCC~CTCT CTT~ACAAAT GACCTTCAGC G~C.~.ACAA

A~rA~CGTC .~..C~AGT A~AArCCCGT ~GGGC~ATAr CTGAACATCA A~ CGA

TAGCTCAGGT AG~.,C~.CG GC~ATATCCA GTCCGGTGGA GTTAGTACTG TGGTGGGCGT

2125~6 CGGCGCCGCC .~-~GG.AGCT GGr~AAr~GC CTAGrAr~GcT GCACGTACTT CCGGAAr,CCC

GGGTGGCGAA AAr~GGr~r,GC GCr-AArCGCT TCCTGCCCTT TTCGCTCAGC ~.~GG

AGT~ATC GAAGATGCGC GGTCr-Ar-AAA CATGCAGGCT GCGAGGTCAT AATAATTAAG

CGGGAGCAAT TTAACAGGCG TATCAAGGTG TGAGGTGGGT GTAr-Ar-GGCT GAAATTATCA

CGA~CC~.. ..~-~.GGGAA TCGrAAr-Ar,G ATTGGTGCCA ATCGCGCTGA CTGCAG

2~426 -(2) INFORMATION FOR SEQ ID No:2 (i) xL~uL..CE CHARACTERISTICS:
~AI LENGTH: 189 amino acids ,BI TYPE: amino acid ,C STRANDEDNESS: single ~D TOPOLOGY: linear (ii) MOLECULE TYPE: protein (iii) HY~O~n~ ICAL: YES
(iv) ANTI-SENSE: NO
(~i) ORIGINAL SOURCE:
(A) ORGANISM: Vir Protein (B) STRAIN: Rhodococcu~ equi (viii) POSITION IN GENOME:
(C) UNITS: kb (xi) SEQUENCE D~-S~PTPTION: SEQ ID NO: 2 Net Ly~ Thr Leu Hi~ Ly~ Thr Val Ser Ly~ Ala Ile Ala Ala Thr Ala Val Ala Al- Ala Ala Ala Met Ile Pro Ala Gly Val Ala A~n*Ala Thr Val Leu Asp Ser Gly Ser Ser Ser Ala Ile Leu Asn Ser Gly Ala Gly Ser Gly Ile Val Gly Ser Gly Ser Tyr Asp Ser Ser Thr Thr Ser Leu A~n Leu Gln Ly~ A~p Glu Pro AGn Gly Arg Ala Ser A~p Thr Ala Gly Gln Glu Gln Gln Tyr A~p Val Hi~ Gly Asp Val Ile Ser Ala Val Val Tyr Gln Arg Phe Hi~ Val Phe Gly Pro Glu Gly Lys Val Phe A~p Gly A~p Ala Gly Gly Leu Thr Leu Pro Gly Ala Gly Ala Phe Trp Gly Thr Leu Phe Thr A~n A~p Leu Gln Arg Leu Tyr Ly~ A~p Thr Val Ser Phe Gln Tyr A~n Ala Val Gly Pro Tyr Leu Asn Ile Acn Phe Phe Acp Ser Ser Gly Ser Phe Leu Gly Hi~ Ile Gln Ser Gly Gly Val Ser Thr Val Val Gly Val Gly Gly Gly Ser Gly Ser Trp His Asn Ala * N-te i n~l ~it- of mature prot~in 2~2~4~6 REFERENCE LEGEND
1. Sambrook, J., E. F. Fritsch, and T. ~niAtis 1989. Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory: Cold Spring Harbor, N. Y.

2. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193-197.

3. !C~n~, F., S. Nicklen, and A. R. CQ~l~on~ 1977.
DNA sequencing with chain-terminating inhibitors. Proc. Acad. Sci. USA 74:5463-5467.

4. Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114 solution. J.
Biol. Chem. 256:1604-1607.

5. Ni~l~-n, J. B. K. and J. O. Lampen. 1982.
Glyceride-cystine lipoproteins and secretion by Gram-positive bacteria. J. Bacteriol. 152:315-322.

6. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680-685.

7. Towbin, H. T., T. S~h^l ;n and J. Gordon. 1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets:
Procedures and some applications. Pro. Natl.
Acad. Sci. 76:4350-4354.

8. Chou, P. Y., and G. D. Fasman. 1978. Prediction of the secondary structure of proteins from their amino acid sequence. Adv. Enzymol. 47:45-148.

9. Ryte, J., and R. F. Doolittle. 1982. A simple method for displaying the hydrophathic character of a protein. J. Mol. Biol. 157:105-132.

10. Ratona, L. I., G. Beck, and G. S. Habicht. 1992.
Purification and immunological characterization of a major low-molecular-weight lipoprotein from Borrelia burgdorferi. Infect. Immun. 60:4995-5003.

11. Ranno, T., T. Asa~a, H. Ito, S. Takai, S. T~-lh~i, and T. ~; 7~i . 1993. Restriction map of a virulence-associated plasmid of Rhodococcus equi.
Plasmid 30:309-311.

12. Finnerty. W. R. 1992. The biology and genetics of the genus Rhodococcus Ann. Rev. Microbiol.
46:193-218.

13. Pugsley, A. P. and N. Schwartz. 1985. Export and secretion of proteins. FEMS Micro. Rev. 32:3-38.

14. Young, D. B. and T. R. Garbe. 1991. Lipoprotein antigens of Mycobacterium tuberculosis. Res.
Microbiol. 142:55-65.

15. Cr~ , L. H., R. Mout, J. n~Pr, and J. D. A.
van Embden. 1989. Characterization of lipid-modified immunogenic proteins of Treponema pallidum expressed in Escherichia coli. Microbial Pathogen. 7:175-188.

16. Wu, H. C. and ~. To~ln~g~. 1986. Biogenesis of lipoproteins in bacteria. Curr. Topics Microbiol.
Immunol. 125:127-157.

17. Field, M. C., and A. K. ~enon. 1993. Glycolipid anchoring of cell surface proteins, p. 83-134.

212~2~
In M. J. Schlesinger (ed.), Lipid modifications of proteins. CRC Press, Boca Raton, Fl.

18. Hietala, S. K., and A. A. Ardans. 1987.
Interaction of Rhodococcus equi with phagocytic cells from R. equi-exposed and non-exposed foals.
Vet. Microbiol. 14:307-320.

19. Tkachuk-saad, 0. and Prescott, J., 1991 Rhodococcus equi Plasmids: Isolation and Partial Characterization, J. Clin. Microbiol. 29:2696-2700).

Although preferred embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the apr~e~ claims.

Claims (28)

1. A substantially pure DNA molecule comprising a DNA
nucleotide sequence corresponding to a DNA nucleotide sequence of Table I (Sequence I.D. #1)

2. A substantially pure DNA molecule comprising a DNA
nucleotide sequence which encodes an amino acid sequence for a virulence-associated protein of R. equi, said protein having an amino acid sequence corresponding to the amino acid sequence of Table I (Sequence I.D. #2)

3. A synthesized oligonucleotide comprising at least 18 consecutive nucleotides selected from a DNA sequence of claim 1.

4. A DNA probe comprising said oligonucleotide of claim 3.

5. A substantially pure protein comprising an amino acid residue sequence corresponding to a protein amino acid sequence of Table I (Sequence I.D. #2)

6. A substantially pure protein comprising an amino acid residue sequence corresponding to an amino acid sequence encoded by a DNA sequence of Table I (Sequence I.D. #1)

7. A substantially pure protein sequence of claim 5 wherein said sequence corresponds to amino acid residues positions 32 to 189.

8. A recombinant cloning vector comprising a DNA
molecule of claim 1, 2, 3 or 4.

9. A recombinant plasmid pCT-C having a restriction map of Figure 2.

10. A host transformed with a recombinant cloning vector of claim 8 or 9.

11. A bacterial host transformed with a recombinant cloning vector of claim 8 or 9.

12. An E. coli host transformed with a recombinant cloning vector of claim 8 or 9.

13. An antibody specific for one or more antigenic determinants of a lipid modified protein of claim 5 having molecular weights in the range of 18 to 22 kDa.

14. An antibody of claim 13 specific for an antigenic determinant in the N-terminal region of the protein sequence.

15. An antibody of claim 13 or 14 wherein said antibody is a polyclonal antibody.

16. An antibody of claim 13 specific for an antigenic determinant in the C-terminal region of the protein sequence.

17. An antibody of claim 16 wherein said antibody is a monoclonal antibody.

18. An antibody of claim 17 wherein said monoclonal antibody is MAb103.

19. A biologically active protein component for use in a vaccine composition, the vaccine composition upon administration to foals being effective in developing immune resistance to challenge by R. equi, said protein component having an amino acid sequence corresponding to the amino acid residue positions 32 to 189 of Table I
(Sequence I.D. #2).

20. A biologically active protein component of claim 19, wherein said protein component has a lipid molecule attached in the region of the N-terminal end of said amino acid sequence.

21. A biologically active protein component of claim 19 wherein said lipid modified molecule is of varying molecular size, the molecular weight of the lipid modified protein being selected from the range of 17.5 kDa and 18 to 22 kDa.

22. A vaccine composition useful for eliciting a protective immune response in foals to resist challenge to R. equi infection, said composition comprising:
i) the vaccine protein component of claim 19 in an amount sufficient to elicit said immune response; and ii) a pharmaceutically acceptable carrier.

23. A vaccine composition of claim 22 further comprising an adjuvant.

24. A vaccine composition of claim 22 wherein said protein component is lipid modified with a lipid molecule attached in the region of the N-terminal end of said amino acid sequence.

25. A vaccine composition of claim 24 wherein said lipid modified molecule is of varying molecular size, the molecular weight of the lipid modified protein being in the range of 17 kDa to 22 kDa.

26. A process for isolating Vir protein from cultured R. equi having an expressible plasmid with the vir gene, said process comprising:

culturing said R. equi to produce on its cell wall surfaces said Vir protein which has a hydrophobic lipid modified N-terminal region;
treating said R, equi cells with a detergent solution to remove the hydrophobic Vir protein from cell wall;
separating said detergent and Vir protein from the R. equi cells;
separating said detergent from said Vir protein to provide a non-detergent solution containing said Vir protein.

27. A process of claim 26 wherein said detergent solution comprises approximately 1 to 5% by weight of detergent.

28. A process of claim 27 wherein the R. equi cells are treated with said detergent for approximately 12 to 24 hours to complete protein extraction from said cell walls.