Note: Descriptions are shown in the official language in which they were submitted.
Z125426
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.
28125426
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
12
~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
212S426
_ 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.
2125426
14
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
2125426
- 16
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
18
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
19
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
2125426
- 24
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,
2125426
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.
2125426
28
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~
- 29
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
31
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.
2125426
32
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~
33
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
2125426
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).
212S426
_ 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).
2125426
~_ 38
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oo oo ^ ô
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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
120
AACGTAAGTT ..CCG.~AGA G~,CGGGT ,~,C~AACG CTACAATCAA CTATGTCGGA
180
ACTGCCC~ - aA~TP~ TCCGC~ C CGATCGAAGG GCGACGTCCG AA~C~n~C
240
TAAGATGAAG A~.~ACA AGACGG..~C TAAGGCGATC GCAGCCACAG CCGTAGCTGC
300
GGCTGCGGCT ATGATTCCCG CCGGCGTCGC TAATGCGACC ~,, GATT CCGGTAGCAG
360
CAGTGCGATT CTCAATAGTG GGG~AGGCAG TGGCATTGTC GG,,~,GGGA GCTATGACAG
420
CTC~r-ACT TCGTTAAACC TTrAa~ Cr-AA~C~-AA~ GGTCGAGCAA GCr-ATACCGC
480
CG~G~AAr-~G Q GCAGTACG ACGTTCACGG AGACGTCATC AGCGCGGTCG TCTAC~-Ar-Ar-
540
GTTTCACGTA TTCGGGCCAG PA~A~GT CTTCGATGGC GATGCAGGGG GACTCACGCT
600
TCCTGGGGCC GGCGCGTTCT CGGCC~CTCT CTT~ACAAAT GACCTTCAGC G~C.~.ACAA
660
A~rA~CGTC .~..C~AGT A~AArCCCGT ~GGGC~ATAr CTGAACATCA A~ CGA
720
TAGCTCAGGT AG~.,C~.CG GC~ATATCCA GTCCGGTGGA GTTAGTACTG TGGTGGGCGT
780
2125~6
41
CGGCGCCGCC .~-~GG.AGCT GGr~AAr~GC CTAGrAr~GcT GCACGTACTT CCGGAAr,CCC
840
GGGTGGCGAA AAr~GGr~r,GC GCr-AArCGCT TCCTGCCCTT TTCGCTCAGC ~.~GG
900
AGT~ATC GAAGATGCGC GGTCr-Ar-AAA CATGCAGGCT GCGAGGTCAT AATAATTAAG
960
CGGGAGCAAT TTAACAGGCG TATCAAGGTG TGAGGTGGGT GTAr-Ar-GGCT GAAATTATCA
1020
CGA~CC~.. ..~-~.GGGAA TCGrAAr-Ar,G ATTGGTGCCA ATCGCGCTGA CTGCAG
1076
2~426
-
42
(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
1 5 10 15
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
100 105 110
A~p Ala Gly Gly Leu Thr Leu Pro Gly Ala Gly Ala Phe Trp Gly Thr
115 120 125
Leu Phe Thr A~n A~p Leu Gln Arg Leu Tyr Ly~ A~p Thr Val Ser Phe
130 135 140
Gln Tyr A~n Ala Val Gly Pro Tyr Leu Asn Ile Acn Phe Phe Acp Ser
145 150 155 160
Ser Gly Ser Phe Leu Gly Hi~ Ile Gln Ser Gly Gly Val Ser Thr Val
165 170 175
Val Gly Val Gly Gly Gly Ser Gly Ser Trp His Asn Ala
180 185
* N-te i n~l ~it- of mature prot~in
2~2~4~6
43
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.
44 2125426
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.
