Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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1 ~~9450
METHODS AND COMPOSITIONS FOR THE
PREPARATION OF TREPONEMA PALLIDUM
ANTIGENS, INCLUDING PURIFIED ANTIGENS
AND DNA SEQUENCES CODING THEREFOR
s
This is a divisional patent application from
application serial no. 582,451, filed on 7 November,
1988.
The present invention relates generally to compositions
for use in the preparation of Treponema pallidum antigenic
ZO proteins and peptides. In particular aspects, the invention
relates to recombinant DNA constructs for the preparation of
selected T. pallidum antigenic/immunogenic proteins and peptides.
Treponema pallidum and its various pathogenic
subspecies comprise the etiologic agents of syphilis and other
treponemal diseases. Despite the availability of effective
antibiotics, syphilis continues to result in
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worldwide morbidity and mortality. One of the principal
reasons behind the continued medical threat posed by
syphilis is the fact that this disease can go undiagnosed,
and can continue to be transmitted sexually. Moreover, by
going undetected or undiagnosed, the disease can exert
extensive damage, which may not be resolved by therapy.
This can be a particular problem in the case of cardio-
vascular syphilis, neurosyphilis, and even congenital
forms of the disease.
'
Both prevention and diagnosis are important aspects
or an overall approach to treponemal disease control.
Particularly in places where some treponemal diseases tend
to be endemic, such as Central and South America, Asia, or
Africa, preventative measures such as immunization against
the disease would likely provide the most reasonable and
successful long term approach. Immunization of individ-
uals against the disease through the use of vaccines
incorporating particular T. pallidum pathogen-specific
immunogenic proteins(s) is an approach which could be
attempted. Unfortunately, no such vaccine currently
exists.
The physician, in an attempt to make an accurate
diagnosis with no definitive historical, epidemiological,
or clinical evidence of the disease, relies totally upon
serological results for accura'_e~diagnosis. Unfortu-
nately, the severe limitations and restrictions of the
available serological procedures often preclude reliable
diagnostic conclusions (83). It is well known that the
immunologically non-specific non-treponemal screening
tests for syphilis (VDRL, RPR circle card, ART) give false
positive reactions not only among patients with acute and
chronic diseases, but among pregnant women, vaccinees, and
narcotic addicts (83-86). Furthermore, false negative
reactions occur commonly among patients with untreated
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latent and late syphilis (83,87). Reliance, then, is
placed upon the so-called "immunologically specific"
confirmatory (treponemal) tests for syphilis (FTA-ABS,
MHA-TP) to establish the presence or absence of the
disease. Yet, these procedures also produce false posi-
tive reactions due to their technical and biological
limitations, thereby creating the current diagnostic
dilemma (83).
The lack of specificity of these tests stems from a
two-fold problem. First, the preparation of either whole,
killed T. pallidum (FTA-ABS test) or ultrasonic lysates of
the organisms (MHA-TP test) requires organisms initially
cultured in rabbit testes and thus necessitates the use of
rabbit testis-contaminated T. pallidum. This contributes
to false, positive reactivity. Second, the presence of
circulating cross-reactive antibody found in the serum of
all humans (both patients and normal individuals) and
elicited in response to the common antigens of host
indigenous, non-pathogenic treponemes comprising the
"normal bacterial flora" further complicates diagnostic
accuracy. Although both the FTA-ABS and MHA-TP tests
attempt to selectively bind these non-specific antibodies
by the use of "sorbing" components prepared from one of
the non-pathogenic treponemes (T. phagedenis biotype
Reiter), their inability to effect a complete absorption
of cross-reactive antibody results in false positive
reactivities (83,88-92). Additionally, the use of
"sorbing" reagents contributes to the complexity of the
procedures.
Within the past decade, groups of investigators have
begun to characterize on a molecular level the structural
components of the Treponema pallidum, subspecies gallidum
(T. pallidum), bacterium in an attempt to provide useful
tools for both diagnosis and the prevention of syphilis
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(1). Certain of these investigators have sought to
identify and to analyze "surface-associated" or "outer
membrane" proteins of the organism in an attempt to define
important immunogens or potential virulence determinants
(1,2). For example, monoclonal antibodies directed
against a 47-ki'_odalton (kDa) antigen of T. nallidum were
reported to possess treponemicidal activity in the T.
pallidum immobilization test and in the in vitro-in vivo
neutralization test of Bishop and Miller (3,4). Moreover,
further work demonstrated that this antigen'was abundant
in T. pallidum and highly immunogenic in both human and
experimental rabbit syphilis (4-6). Even further evidence
indicated that the 47-kDa antigen had potential as a
serodiagnostic antigen and that monoclonal antibodies
directed against the 47-kDa antigen may be used for
syphilis diagnosis (5,7-13).
The 47-kDa antigen is not localized to just one
pathogenic sub~'pecies in that other pathogenic subspecies
of treponemas such as T. pallidum subsp. pertenue,
endemicum, and Treponema carateum all apparently possess
cognate 47-kDa antigens (4,10,14-17); for example, the
predominant serologic response in patients with active
pinta (T. carateum) was found to be directed against the
47-kDa antigen (18). Immunologic, physicochemical, and
genetic data support the pathogen-specificity of the 47-
kDa antigen (4,10-12,17,19,20). Additional work on the
47-kDa antigen of T. pallidum by investigators other than
the present inventor has recently been reviewed (11).
Up until the advent of the present invention, though,
there has been no economical source for 47-kDa antigen.
This is, of course, due to the requirement that T.
pallidum can be grown only in connection with a living
tissue, such as rabbit testicles. Moreover, in partic-
ular, there has been no recombinant DNA sources from which
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to prepare 47-kDa antigen proteins, peptides, or even
specific DNA segments. Accordingly, an economic source
for the provision of 47-kDa antigenic or immunogenic
protein or peptides, a source free from possible contami-
ration with animal or human viruses, would provide an
important tool to be used in the diagnosis and/or possibly
in the prevention of the disease.
Recognizing these and additional disadvantages in the
prior art, it is a general object of the invention to
provide improved methods and compositions useful in the
preparation of the 47-kDa T. pallidum membrane antigen,
antigenic/immunogenic subportions,~or biologically func-
tional equivalents thereof.
It.is a further general object of the inventions to
provide'DNA compositions useful in the preparation of the
47-kDa T. ~allidum antigen, or useful polypeptide variants
thereof . '
It is a more particular object of the invention to
provide recombinant DNA molecules which encode immunogenic
natural 47-kDa T. gallidum surface antigen, the DNA being
capable of being expressed in a variety of hosts.
The invention thus represents a realization by the
inventor that DNA encoding the 47-kDa cell surface protein
_ of T. pallidum may be successfully isolated, essentially
free of associated cellular genes, and employed as a
template in the preparation of antigenic as well as
immunogenic polypeptides reactive with anti-47-kDa poly-
clonal and monoclonal antisera. As used herein, the term
"47-kDa antigen" refers broadly to the 47-kDa T. gallidum
membrane antigen, for example, as characterized in Ref.'s
11 and 23, as well as equivalent structures, such as those
suggested by the present disclosure. Thus, in light of
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techniques known in the art and/or disclosed herein, the
term is meant to include variants of the natural sequence
protein, including allelic and functionally equivalent
variations and antigenic/immunogenic peptidyl subfragments
thereof .
In certain embodiments, the invention is thus con-
cerned :with the preparation of the natural 47-kDa
sequence, such as defined by amino acid sequences dis-
closed in Figure 3 of the present disclosure, whether
naturally derived from recombinant sources, or biological
functional equivalents thereof.
In general, as used herein, the phrase "biologically
functional equivalent" amino acids refers to the fact,that
the invention contemplates that changes may be made in
certain of the foregoing amino acid sequences) (e.a., by
natural genetic drift, strain or subspecies antigenic
variation, or by mutation of the DNA molecules hereof),
without necessarily reducing or losing their
antigenic/immunogenic identity. For example, the sequence
can be altered through considerations based on similarity
in charge (e. a., acidity or basic charges of the amino
acid side group), hydrophatic index, or amphipathic score.
In general, these broader aspects of the invention are
founded in part on the general understanding in the art
that certain amino acids may be substituted for other like
amino acids without appreciable loss of the peptide's
ability to bind to the antibodies, and thus be recognized
antigenically, or alternatively, interact with antibody
forming cells to ellicit an immune response. Exemplary
amino acid substitutions are set forth hereinbelow.
Particular embodiments of the invention include
nucleic acid molecules encoding an amino acid sequence
comprising the sequence extending from the amino acid-val
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at position 1 through the amino acid Gln' at position 434, or
through Ser at position 443 of Fig. 3, representing the full
natural sequence of the two most likely expressed 47-kDa
antigens.
However, in even more particular embodiments, the
invention comprises nucleic acid molecules encoding various
peptide sequences shown in Figure 3, for example, the peptide
sequence extending from about the amino acid Val at,position 92
through the amino acid Ser at position 119; the sequence
extending from amino acid Asp at position 132 through the amino
acid Glu at position 145; the sequence extending from the amino
acid Met at position 158 through the amino acid Asn at position
168; the sequence extending from the amino acid Val at position
181 through the amino acid Asn at position 206; the sequence
extending from the amino acid Arg at position 243 through the
amino acid Phe at position 267: the sequence extending from the
amino acid Cys at position 20 through the amino acid Tyr at
position 29; as well as nucleic acid sequences encoding
biologically functional equivalents of the foregoing peptides.
These peptidyl regions have been selected in that it has been
discovered by the present inventor that they comprise generally
hydrophilic peptidyl regions, and are thus generally preferred
for use as immunogenic/antigenic peptides in the practice of
aspects of the invention.
In certain other aspects, the invention concerns
nucleic acid molecules comprising sequences'corresponding to the
natural 47-kDa antigen gene, or selected subportions thereof,
which sequences it is contemplated will have significant utility
1 34145
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irrespective of whether they encode antigenic peptides. In such
aspects, it is contemplated, for example, that shorter or larger
nucleic acid fragments of the 47-kDa antigen gene, prepared
synthetically or otherwise, can be employed as hybridization
r.....,., t, ...... o, . ,., t, ... ". ~.1., .... ...., .. .,.... .. .a ~ i .
_ ~,... ...,..,. i ... ... a , ... .. .... ~ . .. r.. _ _
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of manners, including their use in the detection of
pathogenic T. gallidum in selected biological or clinical
samples, such as, but not limited to, lesion exudate,
cerebrospinal fluid, biopsy specimens or amniotic fluid.
By way of useful applications, as well as DNA hybridiza-
tion techniques, one may wish to refer to references such
as Ref. 78, U.S. Patent 4,358,535.
In such embodiments, the nucleic acid molecule
selected, whether DNA or RNA, will gene_ally include at
least a 10 nucleotide segment of the 47-kDa antigen
nucleic acid sequence of Figure 3, with the nucleic acid
molecule as a whole being capable of forming a detectable
stable duplex with said sequence under standard selective
nucleic acid hybridization conditions (78-80). The 10
basepair size is, selected as a general lower limit in that
at sizes smaller'than 10 bases, hybridization stabiliza-
tion during washing steps following hybridization can
become a problem, resulting in much lower signal/noise
ratios. Moreover, as the size of the probe decreases to
much below 7 to 8 bases, nonspecific hybridization may -
occur to genes having complementary sequences over short
stretches.
In more preferred embodiments, the invention contem-
plates the preparation and use of nucleic acid molecules
whose structure including sequences comprising at least a
17 nucleotide segment of the nucleic acid sequence of
Figure 3. These embodiments recognize that hybridization
probes larger than a lower limit of about 10 bases provide
more specific stable and overall more dependable hybrid.
The only disadvantage to the longer probes is that the
3~ expense of preparation can increase somewhat where the
fragment is prepared synthetically. However, with advent
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of DNA synthesizing machines and PCR technology (U. S.
Patent 4,683,202), the
expense of preparing larger DNA or RNA probes can be
obviated.
In certain additional embodiments, the inver.=ion
concerns the preparation of recombinant vectors which
incorporate one or more of the foregoing DNA molecules,
which vectors may be employed either in the preparation of
nucleic acid sequences, or for expression. of the DNA to
produce 47-kDa antigen sequences. Thus, as used herein,
the term "recombinant vector" refers to chimeric DNA
molecules which include vector DNA capable of replicating
in selected host organisms, whether prokaryotic hosts such
.as E. coli or Bacillus subtilis, or higher organisms such
as yeast, CHO or African green monkey cells.
In further.aspects, the present disclosure relates to
the 47-kDa antigen itself, as well as antigenic/immano-
genic subfragments thereof comprising polypeptides of
betcaeen about 10 and about 30 amino acids in length,
characterized by an ability to cross react immunologically
with antisera reactive against the 47-kDa T. pallidum
surface antigen.
As used herein, the phrase "having an ability to
cross react immunologically with ~antisera specific for the
47-kDa antigen", refers generally to the ability to
cross-react immunologically with polyclonal antisera of
humans, rabbits, or other animals, such as described in
Refs. 4, 10-12, 17. 19, 23, 55, or monoclonal antibodies,
as described in U.S. Patents 4,514,498 and 4,740,407.
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Thus, in preferred aspects, the invention concerns
antigenic/immunogenic 47-kDa peptide sequences, either naturally
derived from T. pallidum or recombinant E. coli, or synthetically
prepared "synthetic peptides" of Figure 3, corresponding to the
individual peptides extending from about the amino acid Val at
position 92 through the amino acid Ser at position 119: the
peptide sequence extending from amino acid Asp at position 132
through the amino acid Glu at position 145: the peptide sequence
extending from the amino acid Met at position 158 through the
amino acid Asn at position 168: the peptide sequence extending
from the amino acid Val at position 181 through the amino acid
Asn at position 206; the peptide sequence extending from the
amino acid Arg at position 243 through the amino acid Phe at
position 267; the sequence extending from the amino acid Cys at
position 20 through the amino acid Tyr at position 29; as well
as biologically functional equivalents of the foregoing peptides.
It is contemplated that such peptides will find utility both as
antigens, for example, in immunologic detection assays, or as
immunogens in the formation of vaccines.
For greatest utility in the case of vaccine or antigen
formulations, one will desire to employ peptides having a length
ranging from about 10 to about 30 amino acids in length, with
about 30 being preferred.
For the preparation of vaccine formulations suitable
for parenteral administration, the immunogens of the invention
may be formulated in sesame or peanut oil, aqueous propylene
glycol, in liposomes or Iscoms (81,82) or in sterile aqueous
solutions. Such solutions are typically suitably buffered if
1341450
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necessary and the liquid diluent first rendered isotonic with
sufficient saline or glucose. Additionally, stabilizers in the
form of, for example, sorbitol or stabilized gelatin may be
included. These particular aqueous solutions are particularly
well suited for intramuscular and subcutaneous injection, as is
1 34145D
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generally preferred for vaccination using antigenic
preparations.
However, to increase the potential immunogenicity,
and thereby improve the performance of antigen-containing
pharmaceutical preparations, one may additionally desire
to include various immunoadjuvants, such as the water-in-
oil emulsion developed by Freund. The basic ingredients
of light mineral oil (Bayol) and emulsifying agent mix-
tures such as Arlacel (A or C) are available commercially.
The antigens are emulsified in either solutions or suspen-
sions o~ the immunogen (incomplete Freund's adjuvant).
Moreover, the addition of parts or whole killed myco-
bacteria (M. butyricum, M. tuberculosis) in small amounts
to the suspension (complete Freund's adjuvant) leads to a
further enhancement of the immunogenicity of the pharma-
ceutical vaccines made in accordance with the present
invention. Recent adjuvants composed of monophosphoryl
lipid A (93,94) also may be applicable.
In still further aspects, the invention concerns
highly purified preparations of the full length 47-kDa
antigen itself, said antigen not being heretofore available
in a substantially purified form. In preferred aspects,
the invention concerns the 47-kDa antigen, derived from
recombinant sources.
FiQUre 1. Partial restriction enzyme maps and
relevant expression products of 47-kDa antigen-encoding
plasmid derivatives. Plasmid pNC81 (top) has indicated
PstI fragments A, B, and D which collectively comprise a
2.4 kb DNA fragment containing the 47-kDa antigen encoding
region. Restriction sites for pNC8l~(bottom) are desig-
nated as P (PstI), X (XhoII), K (KpnI), H (HindIII), and R
(EcoRI). The extent of the 47-kDa antigen gene sequence
in each subclone is represented by the solid line. The
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cloning vector for all pPH subclones was pUCl9. With the
exception of pPH47.7, transcription of the 47-kDa antigen
gene was opposite to that of the direction of the lac
promote:.
Figure 2. DNA sequencing strategy for the 47-k~a
antigen gene. Restriction enzyme fragments were subcloned
into bacteriophage M13 and were sequenced by the dideoxy
chain termination method. Arrows below the <restrictio::
map indicate the direction and extent of sequencing.
Either a universal 17 base primer (bar) or custom synthe-
sized oligonucleotides (*) complementary to the 47-kDa
antigen gene sequence were used for sequencing reactions.
Figure 3. DNA and corresponding amino acid
sequence of the 47-kDa immunogen of T. pallidum. The boxed
codons indicate the first and second stop codons. Arrows
above the sequence show DNA cleavage sites for the
indicated restriction enzymes. Arrows below the sequence
indicate two hydroxylamine (HA) cleavage sites for the
protein.
Figure 4. Hydrophilicity analysis of the 47-kDa
amino acid sequence. Note the prominent hydrophilic
domain a near the N-terminus of the molecule.
FicLure 5. Hydropathy plot of the 47-kDa amino acid
sequence.
Ficrure 6. Immunoaffinity chromatography of the
47-kDa antigen from E. coli pGPl-2-pNC82 cell envelopes.
Coomassie brilliant blue-stained SDS-PAGE gel (A) and
Western blot with monoclonal antibody 11E3(8). Lanes 1,
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Molecular mass (MW) standards; lanes 2, pooled, non-absorbed proteins
combined with 1 % n-octylglucoside washes; lanes 3, 0.5 M MgCl2 wash prior to
elution; lanes 4, blank; lanes 5, 47-kDa (47K) antigen eluted from the
affinity
column.
This invention concerns a variety of embodiments which relate to the
preparation and use of the 47-kDa antigen of T. pallidum. For example, the
invention discloses for the first time the preparation of the 47-kDa antigen
in a
purified state, in significant quantities, and from either natural or
recombinant DNA
sources. Moreover, the information provided by the present invention allows
the
recombinant preparation of mutant or variant protein species, through the
application of techniques such as DNA mutagenesis. Accordingly, included in
the
present disclosure is information which allows the preparation of a wide
variety of
DNA fragments having a number of potential utilities - ranging from DNA
sequences encoding relatively short immunogenic/antigenic peptidyl
subfragments
of the 47-kDa antigen, to DNA or RNA sequences useful as hybridization probes
for in vitro diagnosis, research, as well as other useful medical and
biomedical
applications.
The inventor has also identified means for the cloning and
expression of the natural 47-kDa antigen DNA sequence as it is isolated from
T.
pallidum cells. Particular embodiments include the preparation of recombinant
vectors including DNA inserts encoding the natural 47-kDa antigen gene and
expressing that DNA in microbial clones to produce the 47-kDa antigen. An
exemplary plasmid, designated pMN23, has been deposited
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with the ATCC in host E. coli cells (accession number
67.204), in order to provide a ready and accessible source
of exemplary, recombinant 47-kDa antigen and DNA to the
art.
Nucleic Acid Hybridization Embodiments
As mentioned, in cer'_ain aspects, the DNA sequ:~nce
information provided by the invention allows~for the
preparation of relatively short DNA (or RNA) sequences
having the ability to specifically hybridize to gene
sequences of the 47-kDa antigen gene. In these aspects,
nucleic acid probes of an appropriate length are prepared
based on a consideration of the natural sequence shown in
Figure 3, or derived from flanking regions of the 47-kDa
gene, such as regions downstream of the gene as found in
plasmid pMN23. The ability of such nucleic acid probes to
specifically hybridize to the 47-kDa gene sequences lend
them particular~utility in a variety of embodiments. Most
importantly, the probes can be used in a variety of
diagnostic assays for detecting the presence of pathogenic
organisms in a given sample. However, either uses are
envisioned, including the use of the sequence information
for the preparation of mutant species primers, or primers
for use in preparing other genetic constructions.
To provide certain of the advantages in accordance
with the invention, the preferred nucleic acid sequence
employed for hybridization studies or assays includes
sequences that are complementary to at least a 10 to 20,
or so, nucleotide stretch of the sequence shown in Figure
3. A size of at least 10 nucleotides in length helps to
ensure that the fragment will be.of~sufficient length to
form a duplex molecule that is both stable and selective.
Molecules having complementary sequences over stretches
greater than 10 bases in length are generally preferred,
._ 1 X41450
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though, in order to increase stability and selectivity of
the hybrid, and thereby improve the quality and degree of
specific hybrid molecules obtained. Thus, one will
generally prefer to design nucleic acid molecules having
47-kDa gene-complementary stretches of 15 to 20 nucleo-
tides, or even longer where desired. Such fragments may
be readily prepared by, for example, directly synthesizing
the fragment by chemical means, by application of nucleic
acid reproduction technology, such as the PCR technology
of U.S. Patent 4,603,102, or by introducing selected
sequences into recombinant vectors for recombinant
production.
In that the 47-kDa antigen is indicative of patho-
genie Treponema species, the present invention will find
particular utility as the basis for diagnostic hybridiza-
tion assays for detecting 47-kDa-specific RNA or DNA in
clinical samples. Exemplary clinical samples that can be
used in the diagnosis of infections are thus any samples
which could possibly include treponemal nucleic acid,
including skin lesions, lesion exudates, amniotic fluid or
the like. A variety of hybridization techniques and
systems are known which can be used in connection with the
hybridization aspects of the invention, including diag-
nostic assays such as those described in Falkow et al.,
U.S. Patent 4,358,535.
Accordingly, the nucleotide sequences of the inven-
tion are important for their ability to selectively form
duplex molecules with complementary stretches of the 47-
kDa gene. Depending on the application envisioned, one
will desire to employ varying conditions of hybridization
to achieve varying degree of selectivity of the probe
toward the target sequence. For applications requiring a
high degree of selectivity, one will typically desire to
1 X41450
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employ relatively stringent conditions to form the
hybrids, for example, ore caill select relatively low salt
and/or high temperature conditions, such as provided by
0.02M-0.15M NaCl at temperatures of 50'C to 70'C. These
conditions are particularly selective, and tolerate
little, if any, mismatch between the probe and the tem-
plate or target strand.
Of course, for some applications, for e:~ample, where
one desires to prepare mutants employing a mutant primer
strand hybridized to an underlying template, less strin-
gent hybridization conditions are called for in order to
allow formation of the heteroduplex. In these circum-
stances, one would desire to employ conditions such as
0.15M-0.9M salt, at temperatures ranging from 20'C to
55'C. In any case, it is generally appreciated that
conditions can be rendered more stringent by the addition
of increasing amounts of formamide. which serves to
destabilize the hybrid duplex in the same manner as
increased temperature. Thus, hybridization conditions can
be readily manipulated, and thus will generally be a
method of choice depending on the desired results.
In clinical diagnostic embodiments, nucleic acid
sequences of the present invention are used in combination
with an appropriate means, such as a label, for determin-
ing hybridization. A wide variety of appropriate indi-
cator means are known in the art, including radioactive,
enzymatic or other ligands, such as avidin/biotin, which
are capable of giving a detectable signal. In preferred
diagnostic embodiments, one will likely desire to employ
an enzyme tag such as urease, alkali-ne phosphatase or
peroxidase, instead of radioactive or other environmental
undesirable reagents. In the case of enzyme tags, colori-
metric indicator substrates are known which can be
employed to provide a means visible to the human eye or
134?450
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spectrophotometrically, to identify specific hybridization
with pathogen nucleic acid-containing samples.
In general, it is envisioned that the hybridization
probes described herein will be useful both as reagents in
solution hybridization as well as in embodiments employing
a solid phase. In embodiments involving a solid phase,
the test DNA (or RNA) from suspected clinical samples,
such as exudates, body fluids (e. g.. amniotic fluid
cerebrospinal fluid) or even tissues, is adsorbed or
otherwise affixed to a selected matrix or surface. ~his
fixed, single-stranded nucleic acid is then subjected to
specific hybridization with selected probes under desired
conditions. The selected conditions will depend on the
particular circumstances based on the particular criteria
required (depending, for example, on the G+C contents,
type of target nucleic acid, source of nucleic acid, size
of hybridization probe, etc.). Following washing of the
hybridized surface so as to remove nonspecifically bound
probe molecules, specific hybridization is detected, or
even quantified, by means of the label.
Purified 47-kDa Antigen
The present invention further provides various means
for both producing and isolating the 47-kDa antigen
protein, ranging from isolation of essentially pure
protein from natural sources (e. a., from T. pallidum
bacterial cells), or its isolation from recombinant DNA
sources (e. g. E. coli or microbial cells). Additionally,
by application of techniques such as DNA mutagenesis, the
present invention allows the ready preparation of so-
called "second generation" molecules having modified or
simplified protein structures. Second generation proteins
will typically share one or more properties in common with
the full-length antigen, such as a particular
1 341450
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antigenic/immunogenic epitopic core sequence. Epitopic
sequences can be provided on relatively short molecules
prepared from knowledge of the peptide, or underlying DNA
sequence information, provided by the present invention.
Such variant molecules may not only be derived from
selected immunogenic/antigenic regions of the protein
structure, but may additionally, or alternatively, include
one or more functionally equivalent amino acids selected
on the basis of similarities or even differences with
respect to the natural sequence.
Isolation of the 47-kDa antigen from either natural
or recombinant sources in accordance with the invention is
achieved preferably by detergent extraction of the protein
from recombinant or T. nallidum cells with an ionic or
non-ionic detergent, such as Sarkosyl* or Triton X-114~* in
order to first solubilize the antigen. The detergent
solution containing the solubilized antigen is then
centrifuged or otherwise filtered to remove insoluble
material, and passed over an immunoaffinity column. Other
important considerations include the significant hydro-
phobic nature of the protein, not readily apparent from
its primary amino acid sequence, that causes it to complex
or aggregate with other hydrophobic moieties, making
purification of the protein problematic. In this regard,
chromatofocussing can be used as an additional purifica-
tion aid. A preferred immunoadsorbant antibody is pro-
vided by monoclonal antibody 11E3 (ATCC HB9781) or 8G2
(ATCC HB8134). However, in general, useful antibodies :nay
be prepared as described in earlier patents (see, e.cr.
U.S. Patents 4,514,498 and 4,740,467), and those reactive
with the desired protein or peptides selected. When
detergent solubilized and immunoadsorbed as disclosed
herein, it is found that the 47-kDa antigen may be
obtained in a highly purified state. appearing as essenti-
ally a single band upon polyacrylamide gel analysis.
* Trade-mark
1341450
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Moreover, it is believed that the foregoing isolation
scheme will work equally well for isolation of
antigenic/immunogenic subfragments of the protein, requir-
ing only the generation and use of antibodies having
affinity for the desired peptidyl region.
Particular embodiments disclosed herein are directed
to the production of 4?-kDa antigen by recombinant DNA
cells in greatly improved quantities than previously
available. For example, it has been discovered that
placement of the antigen gene under the control of a T7
RNA polymerase expression system improves the expression
of the 47-kDa antigen over earlier constructs by 20-fold
in certain hosts (e.4., E. coli K38), and even 100-fold in
others (e.a., E. coli RR1). Thus it appears to be the
case that the 47-kDa gene is amenable to transcriptional
enhancement, and can be juxtaposed to heterologous pro-
moters to achieve a greatly improved production of pro-
tein. A variety of possible promoters arrangements are
disclosed in some detail below, as well as a specific
description of the preferred T7 promoter arrangement.
Enitopic Core Sequences of the 47-kDa Antis
As noted above, particular advantages of the inven-
tion may be realized through the preparation of synthetic
peptides which include epitopic/immunogenic core
sequences. These epitopic core sequences are identified
herein in particular aspects as hydrophilic regions of the
47-kDa antigen. It is proposed that these regions repre-
sent those which are most likely to promote T-cell or B-
cell stimulation, and, hence, elicit specific antibody
production. An epitopic core sequence, as used herein, is
a relatively short stretch of amino acids that is
"complementary" to, and therefore will bind, antigen
binding sites on anti-47-kDa antibodies. It will be
~3~1450
-20-
understood that in the context of the present disclosure, the
term "complementary" refers to amino acids or peptides that
exhibit an attractive force towards each other. Thus, epitope
core sequences of the present invention may be operationally
defined in terms of their ability to compete with or even
displace the binding of the 47-kDa antigen with anti-47-kDa
antisera.
In general, the size of the polypeptide antigen is not
believed to be particularly crucial, so long as it is at least
large enough to carry the identified core sequence or sequences.
The smallest core sequence of the present disclosure is on the
order of about 11 amino acids in length. Thus, this size will
generally correspond to the smallest peptide antigens prepared
in accordance with the invention. However, the size of the
particular core sequences idenitfied by the invention ranges from
11 to 28 amino acids in length. Thus, the size of the antigen
may be larger where desired, so long as it contains the basic
epitopic core sequence.
Accordingly, the inventor has identified particular
peptidyl regions of the 47-kDa antigen which are believed to
constitute epitopic core sequences comprising particular epitopes
of the protein. These epitopic core sequences are illustrated
by reference to Figure 3 as corresponding to the putative N-
terminus of the mature molecule (amino acids 20-29) and
individual peptides extending from about the amino acid Val at
position 92 through the amino acid Ser ~at position 119; the
peptide sequence extending from amino acid Asp at position 132
through the amino acid Glu at position 145: the peptide sequence
1349450
-21-
extending from the amino acid Met at position 158 through the
amino acid Asn at position 168; the peptide sequence extending
from the amino acid Val at position 181 through the amino acid
Asn at position 206; the peptide sequence extending from the
Amino acid Arg at position 243 through the amino acid Phe at
opposition 267; putative N-terminus of the mature molecule (amino
acids 20-29) and as well as biologically functional equivalents
of the foregoing peptides, as explained in more detail below.
Syntheses of the foregoing sequences, or peptides which
include the foregoing within their sequence, is readily achieved
using conventional synthetic techniques such as the solid phase
method (eTa., through the use of commerically available peptide
synthesizer such as an Applied Biosystems Model 430A Peptide
Synthesizer). Peptide antigens synthesized in this manner may
then be aliquoted in predetermined amounts and stored in
conventional manners, such as in aqueous solutions or, even more
preferably, in a powder or lyophilized state pending use.
In general, due to the relative stability of the
peptides of the invention, they may be readily stored in aqueous
solutions for fairly long periods of time if desired, eTa., up
to six months or more, in virtually any aqueous solution without
appreciable degradation or loss of antigenic activity. However,
where extended aqueous storage is contemplated it will generally
be desirable to include agents including buffers such as Tris or
phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it
may be desirable to include agents which Mill inhibit microbial
growth, such as sodium azide or Merthiolate'". For extended
storage in an aqueous state it will be desirable to store the
1341450
-21a-
solutions at 4°C, or more preferably, frozen. Of course, where
the peptides) are stored in a lyophilized or powdered state,
they may be stored virtually indefinitely, eTa., in metered
aliquots that may be rehydrated with a predetermined amount of
water (preferably distilled) or buffer prior to use.
;:
.,
_22- 1 3 4 1 4 5 0
Biological Functional Equivalent Amino Acids
As noted above, it is believed that numerous modifi-
cation and changes may be made in the structure of the
47-kDa antigen, or antigenic/immunogenic subportions
thereof, and still obtain a molecule having like or
otherwise desirable characteristics.
For example, certain amino acids may be substituted
for other amino acids in a protein structure without
appreciable loss of interactive binding capacity with
complementary structures such as antigen-binding regions
of antibodies (or, e.a.,binding sites on receptor
molecules). It is thus hypothesized by the present
inventor that various changes may be made in the sequence
of the antigenic peptides without appreciable loss of
their antibody-binding, or 47-kDa antigen competing,
activity.
The importance of the hydropathic index of amino
acids in conferring interactive biologic function on a
protein has been discussed generally by Kyte et al. (37),
wherein it is found that certain amino acids may be
substituted for other amino acids having a similar hydro-
pathic index or core and still retain a similar biological
activity. As displayed in the table below, amino acids
are assigned a hydropathic index on the basis of their
hydrophobicity and charge characteristics. It is believed
that the relative hydropathic character of the amino acid
determines the secondary structure of the resultant
protein, which in turn defines the interaction of the
protein with substrate molecules.
1 X41450
-23-
TABLE I
P.mino Acid H~dropathic Index
Isoleucine 4.5
Valine 4.2
Leucine 3.8
Phenylalanine 2.8
Cysteine/cystine 2.5
Methionine 1.9'
Alanine 1.8
Glycine -0.4
Threonine -0.7
Tryptophan -0.9
Serine -0.8
Tyrosine -1.3
Proline -1.6
Histidine -3.2
Glutarnic Acid -3.5
Glutamine -3.5
Aspartic Acid -3.5
Asparagine -3.5
Lysine -3.9 _-
Arginine -4.5
Thus, for example, isoleucine, which has a hydro-
pathic index of +4.5, can be substituted for valine (+
4.2) or leucine~(+ 3.8), and still obtain a protein having
similar biologic activity. Alternatively, at the other
end of the scale, lysine (-3.9) can be substituted for
arginine (-4.5), and so on.
Accordingly, these amino acid substitutions are
generally based on the relative similarity of R-group
substituents, for example, in terms of size, electrophilic
character, charge. and the like. In general, preferred
1 X41450
-24-
substitutions which take various of the foregoing char-
acteristics into consideration incline the following:
TABLE II
Oriqinal Residue Exemr~lary Substitutions
Ala gly; ser
Arg lys
Asn gln; his'
Asp glu
Cys ser
Gln asn
Glu asp
Gly ala
His asn; gln
Ile leu; val
Leu ile; val
Lys ~ arg; gln; glu
Met met; leu; tyr
Ser thr
Thr ser
Trp tyr
Tyr trp; phe
Val ile; leu
3 0 Immunoassa~rs
It is proposed that the 47-kDa antigen's peptides of
the invention will find utility as immunogens in connec-
tion with vaccine development, or as antigens in immuno-
assays for the detection of anti-47TkDa antigen-reactive
antibodies. Turning first to immunoassays, in their most
simple and direct sense, preferred immunoassays of the
invention include the various types of enzyme linked
-25- 1 3 4 ~ 4 5 0
immunosorbent assays (ELISAs) known to the art. However,
it will be readily appreciated that utility is not limited
to such assays, and useful e:-bodiments include RIAs and
other non-enzyme linked antibody binding assays or
procedures.
In the preferred ELISA assay, peptides incorporating
the 47-kDa antigen sequences are immobilized onto a
selected surface, preferably a surface exhibiting a
protein affinity such as the wells of a polystyrene
microtiter plate. After washing to remove incompletely
adsorbed material, one will desire to bind or coat a
nonspecific protein such as bovine serum albumin (BSA) or
casein onto the well that is known to be antigenically
neutral with regard to the test antisera. This allows for
blocking of nonspecific adsorption sites on the immobiliz-
ing surface and thus reduces the background caused by
nonspecific binding of antisera onto the surface.
After binding of antigenic material to the well,
coating with a non-reactive material to reduce background,
and washing to remove unbound material, the immobilizing
surface is contacted with the antisera or clinical or
biological extract to be tested in a manner conducive to
immune complex (antigen/antibody) formation. Such condi-
' tions preferably include diluting the antisera with
diluents such as BSA, bovine gamma globulin (BGG) and
phosphate buffered saline (PBS)/Tween ~~ These added agents
also tend to assist in the reduction of nonspecific
background. The layered antisera is then allowed to
incubate for from 2 to 4 hours, at temperatures preferably
on the order of 25~ to 27~C. Following incubation, the
antisera-contacted surface is washed so as to remove non-
immunocomplexed material. A preferred washing procedure
includes washing with a solution such as PBS/Tween*, or
borate buffer.
* Trade-mark
1341450
-26-
Following formation of specific immunocomplexes
between the test sample and the bound antigen, and subse-
quent washing, the occurrence and even amount of immuno-
complex formation may be determined by subjecting sar~~ to
a second antibody having specificity for the first. Of
course, in that the test sample will typically be of human
origin, the second antibody will preferably be an antibody
having specificity in general for human Ig. To provide a
detecting means, the second antibody will preferably have
an associated enzyme that will generate a color develop-
ment upon incubating with an appropriate chromogenic
substrate. Thus, for example, one will desire to contact
and incubate the antisera-bound surface with a urease or
peroxidase-conjugated anti-human IgG for a period of time
and under conditions which favor the development of
immunocomplex formation (e.4., incubation for 2 hours at
room temperature in a PBS-containing solution such as
PBS-Tween).
After incubation with the second enzyme-tagged
antibody, and subsequent to washing to remove unbound
material, the amount of label is quantified by incubation.
with a chromogenic substrate such as urea and bromocresol
purple or 2,2'-azino-di-(3-ethyl-benzthiazoline-6-sulfonic
acid [ABTS) and H202, in the case of peroxidase as the
enzyme label. Quantification is then achieved by measur-
ing the degree of color generation, e.cr., using a visible
spectra spectrophotometer.
Vaccine Preparation and Use
Immunogenic compositions, beliwed to be suitable for
use as an anti-treponemal vaccine, may be grepared most
readily directly from immunogenic 47-kDa proteins and/or
peptides prepared and purified in a manner disclosed
herein. Preferably the purified material is also exten-
1341450
_27_
sively dialyzed to remove undesired small molecular weight
molecules and/or lyophilization of the thus purified
material for more ready formulation into a desired
vehicle.
The preparation of vaccines which contain peptide
sequences as active ingredients is generally well under-
stood in the art, as exemplified by U.S. Patents
4,608,251; 4,601.903; 4,599,231; 4,599,230;~.4,596,792; and
a4, 578, 770. . Typi-
cally, such vaccines are prepared as injectables. Either
as liquid solutions or suspensions: solid forms suitable
for solution in, or suspension in, liquid prior to injec-
tion may also be prepared. The preparation may also be
emulsified. The active immunogenic ingredient is often
mixed with excipients which are pharmaceutically accept-
able 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 emul-
sifying agents, pH buffering agents, or adjuvants which
enhance the effectiveness of the vaccines.
The vaccines are conventionally administered
parenterally, by injection, for example, either sub-
cutaneously or intramuscularly. Additional formulations
which are suitable for other modes of administration
include suppositories and, in some cases, oral formula-
tions. For suppositories, traditional binders and car-
riers may include, for example, polyalkalene glycols or
triglycerides: such suppositories may be formed from
mixtures containing the active ingredient in the range of
0.5% to 10%. preferably 1-2%. Oral formulations include
such normally employed excipients as, for example, pharma
ceutical grades of mannitol, lactose, starch, magnesium
-- 1341450
-28_
stearate. sodium saccharine, cellulose, magnesium carbon-
ate and the like: These compositions take the form of
solutions, suspensions, tablets, pills, capsules, sus-
tained release formulations or powders and contain 10-95°s
of active ingredient, preferably 25-70$.
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 bases as
isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine, and the like.
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 subject to be treated,_~
including, e.a., the capacity of the individual's immune
system to synthesize antibodies, and the degree of protec-
tion desired. Precise amounts of active ingredient
required to be administered depend on the judgment of the
practitioner. However, suitable dosage ranges are of the
order of several hundred micrograms active ingredient per
vaccination. Suitable regimes for initial administration
and booster shots are also variable. but are typified by
an initial administration followed by subsequent inocula-
tions or other administrations.
The manner of application may be varied widely. Any
of the conventional methods for administration of a
1341450
_29.
vaccine are applicable. These are believed to 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 host.
Various methods of achieving adjuvant effect for the
vaccine includes use of agents such as aluminum hydroxide
or phosphate (alum), 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 101'C for 30 second to 2 minute periods
respectively. Aggregation by reactivating with pepsin
treated (Fab) antibodies to albumin, mixture with bac-
terial cells such as C. parvum or endotoxins or lipopoly-
saccharide components of gram-negative bacteria, emulsion
in physiologically acceptable oil vehicles such as mannide
mono-oleate (Aracel A~ or emulsion with 20 percent solu-
tion of a perfluorocarbon (Fluosol-DAB used as a block
substitute may also be employed.
In many instances, it will be desirable to have
multiple administrations of the vaccine, usually not
exceeding six vaccinations, more usually not exceeding
four vaccinations and preferably one or more, usually at
least about three vaccinations. The vaccinations will
normally be at from two to twelve week intervals, more
usually from three to five week intervals. Periodic
boosters at intervals of 1-5 years,, usually three years,
will be desirable to maintain protective levels of the
antibodies. The course of the immunization may be fol-
lowed by assays for antibodies for the supernatant anti
gens. The assays may be performed by labeling with
* Trade-mark
1341450
-30-
conventional labels, such as radionuclides, enzymes,
fluorescers, and the like. These techniques are well
known and may be found in a wide variety of patents, such
as U.S. Patent Nos. 3,791,932; 4,174;384 and 3,949,064, as
illustrative of these types of assays.
Site-Specific Mutagenesis
As noted above, site-specific mutagenesis is a
technique useful in the preparation of individual pep-
tides, or biologically functional equivalent proteins or
peptides, derived from the 47-kDa antic~n sequence,
through specific mutagenesis of the underlying DNA. The
technique further provides a ready ability to prepare and
test sequence variants, for example, incorporating one or
more of~the foregoing considerations, by introducing one
or more nucleotide sequence changes into the DNA. Site-
specific mutagenesis allows the production of mutants,
through the use of specific oligonucleotide sequences
which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent nucleotides, to
provide a primer sequence of sufficient size and sequence.
complexity to form a stable duplex on both sides of the
deletion junction being traversed. Typically, a primer of
about 17 to 25 nucleotides in length is preferred, with
about 5 to 10 residues on both sides of the junction of
the sequence being altered.
In general, the technique of site-specific muta-
genesis is well known in the art as exemplified by publi-
cations such as reference 59t.
As will be appreciated, the technique typi-.
cally employs a phage vector which exists in both a single
stranded and double stranded form. Typical vectors useful
in site-directed mutagenesis include vectors such as the
M13 phage, for example, as disclosed by reference 60.
1341450
-31-
These. phaQe are readily ,_
commercially available and their use is generally well
known to those skilled in the art.
' S In general, site-directed mutagenesis in accordance
herewith is performed by first obtaining a single-stranded
vector which includes within its sequence a DNA sequence
which encodes the 47-kDa antigen. An oligonucleotide
primer bearing the desired mutated sequencecis prepared,
generally synthetically, for example by the method of
reference 61. This primer is then annealed with the
singled-stranded vector, and subjected to DNA polymerizing
enzymes such as E. coli polymerase I Klenow fragment, in
order to complete the synthesis of the mutation-bearing
strand. Thus, a heteroduplex is formed wherein one strand
encodes;the original non-mutated sequence and the second
strand bears the desired mutation. This heteroduplex
vector is then used to transform appropriate cells such as
E. coli cells and clones are selected which include
recombinant vectors bearing the mutated seque:.ce
arrangement.
Host Cell Cultures and Vectors -
In general, of course, prokaryotes are preferred for
the initial cloning of DNA sequences and constructing the
vectors useful in the invention. For example, E. coli.
K12 strain 294 (ATCC No. 31446) is particularly useful.
Other microbial strains which may be used include E. coli
strains such as E. coli B, and E. coli X 1776 (ATCC No.
31537). These examples are, of course, intended to be
illustrative rather than limiting.
Prokaryotes may also be used for expression. The
aforementioned strains, as well as E. coli W3110 (F-,
lambda-, grototrophic. ATCC No. 273325), bacilli such as
1341450
-32-
Bacillus subtilus, or other enterobacteriacea such as
Salmonella t_yphimurium or Serratia marcesans, and various
Pseudomonas species may be used.
In ge.~.eral, plasmid vectors containing replicon and
control sequences which are derived from species compat-
ible with the host cell are used in connection with these
hosts. The vector ordinarily carries a replication site,
as well as marking sequences which are capable of provid-
ing phenotypic selection in transformed cells. For
example, E. coli is typically transformed using pBR 322, a
plasmid derived from an E. coli species (see, e.g., ref.
62). pBR 322 contains genes for ampicillin and tetra-
cycline resistance and thus provides easy means for '
identifying transformed cells. The pBR plasmid, or other
microbial plasmid or phage must also contain, or be
modified .o contain, promoters which can be used by the
microbial organism for expression of its own proteins.,
Those promoters most commonly used in recombinant DNA
construction include the B-lactamase (penicillinase) and
lactose promoter systems (63,64,65) and a tryptophan (trp)
promoter system (66,67). While these are the most com-
monly used, other microbial promoters have been discovered
and utilized, and details concerning their nucleotide
sequences have been published, enabling a skilled worker
to ligate them functionally with plasmid vectors (67).
In addition to prokaryotes, eukaryotic microbes, such
as yeast cultures may also be used. Saccharomvces
cerevisiase, or common baker's yeast is the most commonly
used among eukaryotic microorganisms, although a number of
other strains are commonly available. For expression in
Saccharomvces, the plasmid YRp7, for example, is commonly
used (69,70,71). This plasmid already contains the trill
gene which provides a selection marker for a mutant strain
-3 3- 1 ~~ 4 1 4. 5 0
of yeast lacking the ability to grow in tryptophan, for
example ATCC No. 44076 or PEP4-1 (72). The presence of
the trQl lesion as a characteristic of the yeast host cell
genome then provides an effective environment for detect-
s ing transformation by growth in the absence of tryptophan.
Suitable promoting sequences in yeast vectors include
the promoters for 3-phosphoglycerate kinase (73) or other
glycolytic enzymes (74,75), such as enolase, glycer-
aldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate
isomerase, 3-phosphoglycerate mutase, pyruvate kinase,
triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase. In constructing suitable expression plas-
mids, the termination sequences associated with these
genes are also ligated into the expression vector 3' of
the sequence desired to be expressed to provide poly-
adenylation of the mRNA and termination. Other promoters,
which have the additional advantage of transcription
controlled by growth conditions are the promoter region
for alcohol dehydrogenase 2, isocytochrome C, acid phos-
phatase, degradative enzymes associated with nitrogen
metabolism, and the aforementioned glyceraldehyde-3-
phosphate dehydrogenase, and enzymes responsible for
maltose and galactose utilization. Any plasmid vector
containing a yeast-compatible promoter, origin of replica-
tion and termination sequences is suitable.
In addition to microorganisms, cultures of cells
derived from multicellular organisms may also be used as
hosts. In principle, any such cell culture is workable,
whether from vertebrate or invertebtate culture. However,
interest has been greatest in vertebrate cells, and
propogation of vertebrate cells in culture (tissue
culture) has become a routine procedure in recent years
(76). Examples of such useful host cell lines are VERO
1341450
-34-
and HeLa cells, Chinese hamster ovary (CHO) cell lines,
and W138, BHK, COS-7 293 and MDCK cell lines. Expression
vectors for such cells ordinarily include (if necessary)
an origin of replication, a promoter located in front of
the gene to be expressed, along with any necessary ribo-
some binding sites, RNA splice sites, polyadenylation
site, and transcriptional terminator sequences.
For use in mammalian cells. the control functions on
the expression vectors are often provided by viral mate-
rial. For example, commonly used promoters are derived
from polyoma, Adenovirus 2, and most frequently Simian
Virus 40 (SV40). The early and late promoters of SV40
virus are particularly useful because both are obtained
easily from the virus as a fragment which also contains
the SV40 viral origin of replication (77). Smaller or
larger SV40 fragments may also be used, provided there is
included the approximately 250 by sequence extending from
the Hind III site toward the Bgl I site located in the
viral origin of replication. Further, it is also pos-
sible, and often desirable, to utilize promoter or control
sequences normally associated with the desired gene
sequence, provided such control sequences are compatible
with the host cell systems.
As origin of replication may be provided either by
construction of the vector to include an exogenous origin,
such as may be derived from SV40 or other viral (e. g.,
Polyoma, Adeno, VSV, BPV) source, or may be provided by
the host cell chromosomal replication mechanism. If the
vector is integrated into the host cell chromosome, the
latter is often sufficient.
1341450
-35-
EXAMPLE I
Nucleotide and corresponding Amino Acid,
Sequence of the 47-kDa Antigen Gene
The present Example illustrates steps employed by the
inventor in reducing certain aspects of the invention to
practice. In particular, the Example relates to the
structural analysis and sequencing of the cloned 47-kDa
antigen gene. The general steps which may be employed in
isolating such a gene are disclosed~.in.
PCT Application W088/02403, published 7 April,
_1988.. The present Example discloses the use of one of the
more preferred clones isolated by the foregoing proce-
dures, designated plasmid pMN23, in the sequencing of the
47-kDa antigen gene. (Deposited with the ATCC in E. coli
RR1, and expressing the 47K T. pallidum antigen, ATCC
designation 67204).
In addition to DNA sequencing studies, N-terminal
amino acid sequencing of selected trypsin and hydroxyl-
amine cleavage fragments of the native 47-kDa antigen was
employed to assist in establishing the correct reading
frame of the DNA and confirming the DNA sequence. In this
regard, 33 ~ of the entire 4?-kDa antigen amino acid
content was sequenced and found to have 100% correlation
with.the predicted amino acid sequence derived from DNA
sequencing of the cloned gene. This is the first major
treponemal antigen sequenced where DNA sequencing data has
been corroborated by determination of the amino acid
sequence for a substantial proportion of the purified
native (T. pallidum) protein.
1 ~4~450
-36-
MATERIALS AND METHODS
Bacterial strains. The virulent Nichols strain of
T. pallidum subsp. gallidum was used as the representative
pathogen in this study. It was maintained and cultivated
in the testicles of Neca Zealand White rabbits without the
use of cortisone acetate injections as described (19,55).
Ten days after inoculation, rabbits were sacrificed by
intravenous injection of T-61 Euthanasia solution
(American Hoescht Corp., Somerville, N.J.), and the testes
were asceptically removed. Treponemes were extracted on a
rotary shaker in phosphate-buffered saline (PBS) (pH 7.4),
and were isolated by differential centrifugation (55).
Treponemes were suspended in PBS for final darkfield
microscopic enumeration prior to use in antigen extrac-
tion. E, coli DH5 alpha (F endAl hsdRl7 [rk mkJ supE44
thi-1 lambda recAl avrA relAl ~80dlac Zdelta M15 delta
[lacZYA-argFJ U169) (Bethesda Research Laboratory,
Gaithersburg, MD) was used as the recipient for pUC series
plasmid derivatives (21). E. coli JM101 ([rkmk] delta
[lac pro AB] thi su,gE/F' traD3o proA + proB + lacIQ lac
Zdelta M15) (22) was used to habor M13 derivatives for DNA
sequencing analyses.
Plasmids and subcloninv into EUC19. Plasmid deriva-
tives were constructed as subclones of pNC81 (23), which
originated~from plasmid pMN23 (11). Plasmid pPH47.1
(containing PstI fragments A and B) (23) and pPH47.2
(possessing PstI fragments A, B, and D) (23) were gener-
ated by inserting the 2.3 kilobase (kb) and 2.36 kb
partial PstI fragments of pNC81 (23) into pUCl9 vector
(21). Plasmid pPH47.5 was generated by digesting pPH47.2
with Konl and recircularization (23)~. Plasmid pPH47.5 was
generated by digesting pPH47.2 with HindIII and recir-
cularization. Plasmid pPH47.6 was constructed by insert
ing the 1.35 kb EcoRI fragment of pPH47.2 into pUCl9.
-37- 1 3 4 1 4 5 0
Plasmid pPH47.7 was made by inserting the 1.1 kb XhoII-
EcoRI fragment of pPH47.2 into the BamHI-EcoRI sites of
pUCl9. In this construction, transcription of the 47-kDa
antigen mRNA is initiated from the lac promoter of pGCl9.
With the exception of pPH47.4, transcription of 47-kDa
antigen mRNA was opposita to the direction of the lac
promoter in the pUC plasmids. The 47-kDa protein deriva-
tives expressed by pPH47.5, pPH47.6, and pPH47.7 are
truncated but contain varying numbers of amino acids
(i.e.. approximately 29, 46, and 8 amino acids, respec-
tively) encoded by the plasmid vector sequence(s).
Expression of 47-kDa antigen derivatives by the various
plasmids was assessed by immunoblotting expression pro-
ducts with monoclonal antibody 11E3 (4) and rabbit anti-T.
gallidum antiserum (4,23).
Isolation of native 47-kDa antigen from T. pallidum
by Triton X-114. phase partitioning. Triton X-114 extrac-
tion and phase separation of the 47-kDa T, pallidum
protein was performed as described by Bordier (24) and as
modified by Radolf et al. (54). Briefly, whole T.
gallidum (1 x 1011) collected by differential centrifuga-
tion were extracted by rocking in a test tube end-over-end
overnight with 40 ml of PBS containing 2$ (v/v) Triton X-
114 at 4'C. The insoluble material was removed by centri-
fugation at 27,000 x g (4'C) for 20 min. The supernatant
containing soluble material was decanted and allowed to
- cloud in a 37'C waterbath for 1 min, followed by centri-
fugation at 13,000 x g (20'C) for 2 min. The aqueous
phase was removed and discarded.
At this stage, the material was processed in either
of two Ways: 1) the detergent phase (8 ml) was washed
five times by repeated dilution to 28 ml with ice-cold PBS
followed by mixing, rewarming, and centrifugation at
13,000 x g (20'C) for 2 min. The proteins in the washed
1341450
-38-
detergent phase were then precipitated overnight at -20'C
with a 10-fold volume of cold acetone; 2) alternatively,
for affinity purification prior to hydroxylamine cleavage,
the Triton X-114 extract was washed three times in 1 ml of
10 mM Tris-HC1 (pH 8.0) + 5 mM NaCl. The washed detergent
phase was diluted to 1% Triton X-114 in the 10 mM Tris-HC1
(pH 8.0) + 5 mM NaCl buffer. One ml of ReactiGel* 6X
(Pierce Chemical Co., Rockford, IL) containing 2 mg of
monoclonal antibody 11E3 (ATCC Deposit HB978<1) per ml of
resin was added batchwise to the diluted detergent phase
(23). This was incubated with end-over-end motion over-
night at 4'C. The resin was then poured into a column and
was washed with 5 bed volumes of 10 mM Tris-HC1 (pH 8.0) +
5 mM NaCl + 1% Triton X-114. Purified 47-kDa antigen was
eluted with 5 bed volumes of 3 M guanidine-HC1 (pH 5.5) +
1% Triton X-114 (flow rate of 1 drop per 8 sec).
Hydrox~rlamlne cleavacLe of the native 47-kDa antigen.
Purified 47-kDa antigen was dialyzed overnight against 18
L of distilled H20 to remove guanidine-HC1. The protein
was precipitated overnight with 10 volume of cold acetone
(-20'C). Precipitated protein was collected by centrifu-.
gation at 13,000 x g for 10 min. The pellet was suspended
in 6 M guanidine-HC1 + 2 M hydroxylamine (HA) (pH 9.0)
(25), and was incubated at 45'C for 4 hr. The reaction
mixture (1 ml) was dialyzed against 1 L of distilled H20
overnight (4'C) using 1,000 molecular weight exclusion
dialysis tubing.. The protein was lyophilized and about
100 pmoles of HA-cleaved 47-kDa antigen were subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) according to the method of Hunkapiller et al.
(26). The HA-cleaved protein was then transferred to
Millipore polyvinylidene difluoride~(PVDF) membrane
according to the method of Matsudaira (27). The three
resulting peptide bands were cut out and subjected to N-
terminal amino acid sequencing (below).
* Trade-mark
-39- 1 3 4 1 4 5 D
Amino acid sequencing' of the native 47-kDa antigen.
Approximately 100 pmoles of the 47-kDa protein were
subjected to polyacrylamide gel electrophoresis (26) and
then transferred to Whatman GF/C glass fiber filter paper
derivatized with amino propyl groups according to the
method of Aebersold et al. (28) as modified by Yuen et al.
(29). N-terminal amino acid sequencing was performed on
an Applied Biosystems Model 470A Gas Phase Sequences
coupled to an on-line Model 120A high performance liquid
chromatograph (HPLC). Attempts to sequence the N-terminus
of the intact 47-kDa protein were unsuccessful. CNBr
digestion of the apparently blocked protein immobilized on
the glass fiber sequences filter gave rise to a mixture of
peptides from which phenylthiohydantoin (PTH) amino acids
could be identified by automated Edman degradation.
Approximately 500 pmoles of the 47-kDa protein were
transferred from a 12.5% SDS-PAGE gel to nitrocellulose
paper for solid'phase tryptic digestion according to the
method of Aebersold et al. (30). Peptides were separated
by reverse-phase HPLC on an Applied Biosystems Model 130A
HPLC using a Hrownlee RP300*(2.1 x 100 mm) C8 column.
Separation was performed in 0.1% trifluoroacetic acid
using a gradient of 0 to 50% acetonitrile over a duration
of 120 minutes, at a flow rate of 50 ul/minute. Peaks
were collected manually onto 1 cm discs of Whatman*GF/C
paper. Cysteine residues were then reduced and alkylated
according to the method of Andrews and Dixon (31). Pep-
tides were sequenced directly on an Applied Biosystems
Model 470A Sequences.
DNA seguencing of the 47-kDa a~tic~en _ene. Selected
DNA fragments were ligated to M13mp18 and used to trans-
fect JM 101 cells. Recombinant phages were identified as
white plaques on LB plates containing isopropyl-beta-D-
thiogalactopyranoside (IPTG) and X-gal (5-bromo-4-chloro-
* Trade-mark
134150
-40-
3-indolyl-b-galactoside). The orientations of the inserts were
determined by restriction enzyme mapping of the replicative forms
of the phage DNA. Single-stranded phage DNAs were purified from
the culture supernatants (32). DNA sequences were determined by
the dideoxynucleotide chain termination method (33). For most
sequencing reactions, the 17 base universal primer (Bethesda
Research Laboratory, Bethesda, MD) and the Klenow fragment of DNA
polymerase I were used. Two oligonucleotides, CATGGTTGACAGCGAGG
(Fig. 3) and CCTCGCTGTCAACCATG (Fig. 3), corresponding to
nucleotide positions 471 to 489 and 489 to 471 of the 47-kDa
antigen gene, respectively, were synthesized in a Model 380B
Applied Biosystems oligonucleotide synthesizer and were used for
additional sequencing reactions. In some cases, reverse
transcriptase was used instead of the Klenow enzyme. The
reaction products were subjected to electrophoresis on standard
6% or 8% polyacrylamide sequencing gels containing 7.8 M urea or
on 4-8%, 4-10% or 6-10% polyacrylamide gradient gels with 40%
formamide to increase resolution.
Computer analyses. Beckman MicroGenie'" software
(Beckman Instruments, Palo Alto, CA) (34) was used for DNA
sequence analysis.
RESULTS
Subclones of the 47-kDa anticten q_ene. The various
subclone derivatives of the 47-kDa antigen gene and the relevant
expression products of these derivatives are shown in Figure 1.
All subclones originated from pNC81 which contains the entire 47-
kDa antigen gene and its regulatory region. The first (leftward)
PstI site of the D fragment of pNC81 (Figure 1) is located 5' to
,... 1341450
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the GC tail used in the original construction of pNC8l. Not
shown in Figure 1 is the location of an ApaI site (GGGCCC) -
1341450
-41-
that presumably was constructed fortuitously as a result
of the GC tailing method. Cleavage by ApaI leaves the GC
tail attached to the cloning vector. Plasmid construc-
tions lacking the PstI D fragment (e. g., pPH47.1) failed
to express any derivative of the 47-kDa antigen. The
addition of an active promoter at the XhoII site (upstream
from the structural gene) could restore expression of some
or all of the 47-kDa antigen (e.Q., pPH47.7). Thus, the
- basepair PstI D fragment contained a region that is
required for the expression of the 47-kDa antigen gene.
DNA secruencinq. The complete nucleotide sequence was
obtained by DNA sequencing analysis of subclones shown in
Figure 1. The DNA sequencing strategy used is outlined in
Fig. 2; virtually all of the DNA encoding the structural
gene for-the 47-kDa antigen was sequenced in both direc-
tions. Using computer analysis, an open reading frame
large enough to;represent the 47-kDa antigen (Figure 3)
was identified which was compatible with genetic expres-
lion data. The sequence contained two~stop codons (TAG and
TAA) at nucleotides 1303 and 1333, respectively. Prior
gene expression data established the direction of
transcription (11,23); the protein. Consensus sequences
for -10 Pribnow, -35 (e~Q., TTGACA), or -4 to -7 Shine-
Dalgarno regions could not be readily identified in the DNA
sequence immediately upstream from the first methionine of
the protein.
~ X41450
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The calculated molecular weight for the protein
to the first stop codon is 45,756. The molecular weight
calculated on the basis of the second stop codon is about
46,920. The protein contains 10 methionines and one
cysteine. There are 62 acidic (Asp, Glu) amino acids, 46
basic (Arg, Lys) amino acids, and the remaining amino acids
(307 are neutral; of these, 129 are hydrophobic (Phe, Trp,
Tyr, Ile, Leu, Met, Val). The overall G+C content of the
pNA was about 54% for the structural gene and consistent
with previously published G+C ratios of 52.4-53.7% for T.
pallidum (Nichols) DNA (35).
Amino acid sequencing of 47-kDa antigen ~ol~~eptide
fragments. All attempts to sequence the N-terminus of the
intact 47-kDa protein were unsuccessful; native 47-kDa
antigen preparations isolated either by gel electroelution
or by purification with Triton X-114 phase partitioning
and monoclonal antibody affinity column chromatography
therefore appeared to be "blocked" to Edman degradation.
N-germinal amino acid sequences of unfractionated cyanogen
bromide cleavage products, however, were concordant with
the predicted amino acid sequence for the 47-kDa protein.
N-terminal amino acid sequencing also was carried out on
individual trypsin and hydroxylamine cleavage fragments of
the 47-kDa antigen; Six trypsin fragments and two
hydroxylamine fragments of the 47-kDa antigen that were
analyzed by N-terminal amino acid sequencing. The trypsin
fragments were located within 16 amino acids from the N-
terminus of the molecule to within 20 amino acids of the
C-terminus of the molecule. Of the 415 amino acids
identified from the DNA sequence (Figure 3), 138 (33%) of
the amino acids contained~in the native T. pallidum 47-kDa
antigen were directly sequenced by N-terminal amino acid
sequencing. All 138 "native" amino acids sequenced had a
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100% correlation with the predicted amino acid sequence
derived from DNA sequencing of the cloned gene.
Further characterization of the 47-kDa antigen.
Hydrophilicity analysis according to the algorithm of Hopp
and Woods (36) is shown in Figure 4; a major portion of
the protein would appear to be hydrophilic by the given
parameters. In particular, one major hydrophilic domain
existed within 25 amino acids of the N-terminus of the
molecule. A hydropathy plot of Kyte and Doolittle (37)
also predicted several hydrophobic domains (Figure 5).
DISCUSSION
The structural gene for the 47-kDa antigen is local-
ized to.a 1.3 kb fragment at the most leftward (5')
portion of the 2.85 kb DNA insert of pNC8l. Therefore,
sequence analysis commenced at the 5' PstI insertion site
and was performed for approximately 2.9 kb to ensure that
t' . . .- . ~ . . __, ____ ____ ____._
30
-44- 1 3 ~ 1 4 5 0
Understanding the initiation of transcription has
been somewhat puzzling due to the significant distance
between the PstI D fragment (required for expression) and
the methionine start codon. Hansen et al. (38) reported
that expression of the 47-kDa (tmpA) and 34-kDa (tmpB)
antigens of T. ~allidum in E. coli was poor in the absence
of an expression vector, but was enhanced significantly
for tm~B when the PL promoter of bacteriophage lambda was
placed some 200 base pairs upstream of the tmpB structural
gene. The location of one tm~A promoter was less than 70
base pairs upstream of the t~A gene, but a second pro-
moter required for transcription was located between 70
and 400 base pairs upstream from the tmQA structural gene.
This situation also may be analogous to that of the
o~F and ompC genes of E. coli, both of which have fairly
long untranslated leader regions (80 base pairs for om~C-
and 110 base pairs for om F) that separate the methionine
start codon and the -10 Pribnow box (39). Upstream DNA
sequences of up to 150 nucleotides or more also have been
shown to be essential for full promoter activity in other
prokaryotic systems (40). Additionally, the 47-kDa
antigen gene may be similar to other prokaryotic genes
with unusual regulatory sequences or which are transcribed
by alternative sigma factors. Namely, certain structural
genes of enteric bacteria encoding flagellar, chemotaxis,
and motility operons appear to be under the control of
alternative sigma factors (41). The relevant regulatory
regions for these operons have not been determined, and
sequence analysis of several of these genes has failed to
1 341450
-45-
reveal plausible promoter sequences for the predominant
bacterial RNA poiymerase (41).
A particularly important feature of the 47-kDa
structural gene concerns the apparent presence of a typical
leader or signal peptide at its amino terminus (42,43).
Initially thought to be located exclusively in the outer
membrane (23,44), recent data indicate thats this molecule
may be located in both the cytoplasmic and outer membranes
of T. pallidum (59). Moreover, when synthesized at high
levels in E. coli using an expression vector system, the
cloned 47-kDa antigen partitions into both the inner and
outer membranes of that organism as well (23).
These data suggest that the mature translation
product possesses the necessary structural information for
targeting to the cytoplasmic and outer membranes of both-
T. pallidum and E. coli. Although rare, E. coli outer
membrane proteins lacking signal sequences have been
described; leader peptidase I, which can be found in both
the.cytoplasmic~and outer membrane fractions (45), repre-
sents one such protein (46). It should also be pointed
out that virtually nothing is known about the parameters
that influence protein export in T. pallidum. Neverthe-
less, Stamm et al. (47), Fiansen et al. (38) and Dallas et
al. (48) have demonstrated that post-translational proces-
sing of some T. pallidum proteins does occur. Also, a
conjectured signal peptide was noted for the t~A protein
of T. gallidum (38).
... 1 341450
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The 3' encoding region of the gene contains two stop
codons separated by nine amino acid codons. If the first
stop codon is used, the protein possesses a molecular
weight of 45,756. The discrepancy between the calculated
molecular weight (45,756) and that estimated by SDS-PAGE
(47,000) (4) is not inordinate. Therefore, the "47-kDa"
nomenclature has been retained so as not to confuse the
literature (1). While it is probable that the first TAG
stop codon represents the principal stop signal for the
termination of translation, the existence of the second
TAA stop codon, however, may explain a peculiar phenomenon
previously reported; namely, the 47-kDa antigen typically
migrates as a 47-48-kDa "doublet" on SDS-PAGE gels (23).
Occasionally, termination may fail at the first TGA stop
codon, thereby allowing the protein to be elongated an
additional 10 amino acids. The 47-kDa molecular weight
species of the 47-48-kDa doublet (i.e., to the first stop
codon) represents by far the major component. The
hypothesis therefore is consistent with the observation of
the relevant abundance of the two molecular weight deriva-
tives observed on SDS-PAGE gels.
The data disclosed by the present invention provide
the molecular basis for elucidation of the respective
roles of humoral and cell-mediated immune responses and
the immunogenicity -of the protein during infection by T.
pallidum. Hydrophilicity analysis revealed at least one
major hydrophobic domain near.the N-terminus of the
molecule. Moreover, sever.al~ 'hydrophilic domains
have been identified as likely important
immunogenic/epitopic regions. The N-terminal regions
(amino acids 92 to 119)represent a primary immunodominant
epitope. In support of this, preliminary epitope mapping
experiments showed that a vast majority of mouse mono-
clonal antibodies raised against the 47-kDa antigen react
with the N-terminal hydroxylamine cleavage fragment
1 X41450
-47-
containing this hydrophilic domain; the same was true when
human syphilitic or rabbit anti-T. pallidum sera were
examined for antibody reactivity with the hydroxylamine
cleavage fragments of the 47-kDa antigen. The protein
additionally may contain domains) that serve as poly-
clonal activators of B lymphocytes (49). This may parti-
ally explain the intense and specific fetal IgM response
to the 47-kDa antigen in congenital syphilis (7). In
addition, certain domains may serve as functional T cell
recognition epitopes (50,51) that promote the activity of
cell-mediated immunity in the clearance of T. pallidum
from early primary lesions (2) and/or during other stages
of the pathogenesis process.
There is no doubt that the 47-kDa antigen is an
integral membrane protein (23,54), but the actual basis
for the hydrophobic character of the molecule is not
readily apparent from its primary sequence. Preliminary
data derived from radiolabeling experiments with 3H
palmitate or oleate suggest that the 47-kDa antigen may be
covalently modified with.lipid. If so, this may explain
the intrinsic hydrophobic character of the 47-kDa antigen.
Its characteristic partitioning into the deter-
gent phase upon Triton X-114 extraction (20,23,59) further
substantiates its Overall hydrophobic nature: The exis-
tence of multiple hydrophilic domains also is compatible
with the notion that the 47-kDa antigen can reside in an
outer membrane, such as in the case of bacterial porins
(52). The overall structure of the 47-kDa protein,
possessing both hydrophilic and hydrophobic domains, is
consistent with its previously reported structural (hydro-
phobic characteristics) and immunogenic properties.
1341450
-48_
Monoclonal antibodies directed against the 47-kDa
antigen of T. pallidum agglutinate T. pallidum-coated
erythrocytes in the microhemagglutination assay for T.
pallidum antibodies (MHA-TP test) (4). It was proposed
that this efficient agglutination by a monoclonal antibody
was facilitated either by the presence of an abundant 47-
kDa antigen among T. pallidum and/or the presence of a
repetitive epitope within the antigen. On the basis of
sequence data provided herein, the former explanation
would appear to be correct. No significant primary repeat
epitopes were detected in the DNA and/or amino acid
sequences of the 47-kDa antigen. This finding is consis-
tent with our previous contention that the 47-kDa protein
of T. pallidum is, indeed, an abundant antigen of the
organism (4).
The availability of the entire sequence for the 47-
kDa antigen provides additional practical tools. The
entire DNA sequence or strategic constituent oligo-
nucleotide portions, including synthetic oligonucleotides,
may be used as molecular gene probes for the detection of
the organism in various tissues and/or body fluids.
Moreover, knowledge of the amino acid sequence allows the
preparation of strategic synthetic peptides for use as the
basis for improved treponemal serologic tests and/or
treponemal synthetic peptide vaccines~(53).
EXAMPLE II
Improved Production of the 4?-kDa
Antigen by Recombinant Means
E. coli derivatives containing plasmids pNC81 or
pMN23 express an amount of the 47-kDa antigen that is less
than ideal for commercial production. For this reason, a
T7 expression vector system was used in an attempt to
-49- ~ 3 ~ 1 4 5.0
increase 47-kDa antigen production by recombinant E. coli
(56). The 2.85-kb T. pallidum DNA insert from pNC81 was
ligated into pT7-6 to create pNC82, which was used to
transform E. coli strains K38 or RRl, both of which
harbored pGPl-2 (pGPl-2 is disclosed in Ref. 56, pGPl-2
encodes T7 RNA polymerase under the control of a tempera-
ture-sensitive repressor acting on the PL promoter of
bacteriophage lambda). When expression of the 47-kDa
antigen was compared between E. coli harboring pNC81 and
E. coli harboring pGPl-2-pNC82 by Western blot analysis, a
greater than 20-fold increase (cn a per cell basis) .n
antigen production was found in the er:pression vector
system for E. coli K38, or a 100-fold increase was found
for E. coli RR1. The specific procedure employed for
preparing pNC82 is shown immediately below.
Cloning into the expression vector pT7-6. A partial
PstI digestion of pNC81 was subjected to electrophoresis
on a 1~ low-melting-point agarose gel (Bethesda Research
Laboratories, Bethesda, MD.). The portion of the gel
containing the 2.85-kb PstI fragment (encoding the 47-kDa
antigen) was excised and melted at 65' C. The DNA was
harvested by column chromatography (Elutip*; Schleicher &
Schuell (57), followed by standard ethanol precipitation.
Plasmid pT7-6 was digested with PstI, treated with
alkaline phosphatase. and then ligated to the 2.85-kb
fragment of pNC81 to create pNC82. E. coli K38 or RR1
containing pGPl-2 was transformed with pNC82; trans-
formants were selected on agar plates containing ampi-
cillin and kanamycin at 50 ug/ml each, and then the clones
were tested by the radioimmunocolony blot (RICB) assay for
47-kDa antigen production (11; assay using monoclonal
antibody 11E3).
* Trade-mark
-50- 1 ~~'~450
EXAMPLE III
Purification of Recombinant
47-kDa Antigen from E. coli cells
Preparation of Cell Envelopes. As a first step in
the purification of the recombinant 47-kDa antigen, cell
envelopes were prepared from E. coli recombinant deriva-
tives containing both pGPl-2 and pNC82 (pGPl-2-pNC82), in
that a large majority of the antigen was detected in the
cell envelope fraction. Recombinant E. coli cells were
grown at 37' C in broth with the appropriate antibiotics
to maintain selective pressure on the plasmids. E. coli
cells containing pGPl-2-pNC82 were induced for 30 min at
42' C and grown for an additional 3 hrs at 37' C prior to
fractionation. Cells were harvested by centrifugation at
16,270 X g for 10 min (4' C), and the pellets were
suspended in a one-fifth volume of 10~ (wt/vol) sucrose-20
mM Tris hydroch-loride (pH 8.0)-1 mM EDTA (STE buffer) at
0' C. The cells were again harvested by using similar
centrifugation conditions and were suspended in 1/50 of
the original volume of STE. They were then frozen in
liquid nitrogen. The cells were thawed at 37' C and
lysozyme crystals were added to a final concentration of
0.2 mg/ml. After 45 min of 0' C, cells were frozen and
thawed twice to create cell envelopes.
Washed cell envelopes of E. coli were extracted with
2o Sarkosyl to produce a soluble, cytoplasmic membrane-
enriched fraction. SDS-PAGE and Western blot analysis of
the Sarkosyl-soluble and -insoluble material from pNC81
indicated that virtually all of the recombinant 47-kDa
antigen was solubilized. In contrast, similar analysis of
E. coli harboring pGPl-2-pNC82 revealed that a significant
amount of the 47-kDa protein remained in the Sarkosyl-
insoluble, outer membrane-enriched material.
~,~41450
-51-
Deterctent solubilization of the recombinant form of
the 47-kDa antigen from E. coli. To determine the optimal
solubilization conditions for the recombinant 47-kDa
antigen, cell envelopes from E. coli pGPl-2-pNC82 were
incubated with a variety of ionic and nonionic detergents
at different detergent to protein ratios.
In particular, cell envelopes containing 200 mg of
total protein in 20 ml were incubated for 1 hr at 4' C in
0.01, 0.03, 0.1, 0.3, 1.0, and 3.0% NP-40, n-octylgluco-
side, Sarkosyl, or CHAPS*. Relatively insoluble outer
membrane-enriched material was collected by centrifugation
at 110,000 Y g for 1 hr at 4~ C. The resultant super-
natants were analyzed by Western blots with monoclonal
antibody 11E3. Results from these experiments are sum-
marized;in Table 1. The 47-kDa antigen was solubilized
with concentrations of CHAPS and n-octylglucoside as low
as 0.01%. Solubilization in Sarkosyl required a detergent
concentration of 0.03%, while the 47-kDa protein was least
efficiently solubilized by NP-40.
* Trade-mark
1341450
-52-
TABLE 1
Solubilization of the recombinant 47-kDa
antigen from E. coli pGPl-2-pNC82 cell
envelopes by using ionic and nonionic detergents
Solubilization at the following
0 of detergent used to detergent
solubilize the 47-kDa antigen .
0.01 0.03 0.1 0.3 1.0 3.0
CHAPS (wt/vol) + + + + + +
n-Octylglucoside (wt/vol) + + + + + +
Sarkosyl (wt/vol) - + + + + +
NP-40 (vol/vol) - - - - + +
a Symbols: +, visible 47-kDa antigen in 50 u1 of result-
ing supernatant when Western blotted with monoclonal
antibody 11E3; -, no visible 47-kDa antigen in 50 u1 of
resulting supernatant when Western blotted with monoclonal
antibody 11E3.
Triton X-114 extraction of E. coli cell envelopes
from pGPl-2-pNC82. Localization of the recombinant 47-kDa
protein to the E.~coli cell envelope and the requirement
for detergent to solubilize the 47-kDa antigen demon-
strated the hydrophobic nature of the protein. This was
investigated further by using Triton X-114 phase parti-
tioning (58) as a means of assisting in purifying the
recombinant antigen. .
Cell envelopes from pGPl-2-pNC82-transformed E. coli
were incubated for 1 hr at 4' C in 2~ Triton X-114 at a
protein to detergent ratio of 1:5. Insoluble material was
-53- 1 ~ 4 1 4 5 0
removed by centrifugation at 14,000 X g for 15 min in a
microcentrifuge. Detergent and aqueous phases were
separated by placing tubes at 30' C for 5 min, followed by
centrifugation at 5,800 X g for 5 min (room temperature)
over 0.25 ml of a 0.25 M sucrose cushion. Detergent and
aqueous phases were analyzed by Western blotting with
anti-47-kDa monoclonal antibody, revealing that the
majority of extractable 47-kDa protein segregated into the
detergent phase. .
Purification of the 47-kDa antigen from E. coli by
deterctent extraction, immunoaffinity chromato rq ashy, and
chromatofocusin4. The foregoing studies demonstrated
that, in general, Sarkosyl or Triton X-114 detergent
extractions were the preferred methods for solubilizing
the 47-k~Da antigen from recombinant cells. Thus, the
general strategy of detergent solubilization followed by
antibody immunoaffinity chromatography was utilized to
purify the 47-kDa antigen from E. coli. Cell envelopes
from pGPl-2-pNC82 transformants (expression vector s,~stem)
were incubated in 50 mM Tris-hydrochloride (pH 8.0)+0.15 M
KCl (TK buffer) containing 2% Sarkosyl for 1 hr at 4~C.
Insoluble material was removed by centrifugation at
110,000 x g (4'C) for h hr. Prior to this, a monoclonal
antibody affinity column was prepared as follows:
Monoclonal antibody 11E3 was dialyzed in 0.1 M borate
buffer (pH 8.5). A total of 1 ml of matrix (ReactiGel 6X;
Pierce Chemical Co., Rockford, I11.) was added to 1 ml of
dialyzed monoclonal antibody (3 mg/ml) for 30 hr at 4'C.
The resulting column was washed with four bed volumes of
TK buffer containing 1% n-octylglucoside followed by a
four-bed-volume wash with 3 M guanidine hydrochloride-1%
n-octylglucoside-25 mM Tris hydrochloride (pH 8.0)-0.075 M
KC1.
1341450
-54-
A "cocktail" of monoclonal antibodies directed
against different epitopes of the 47-kDa antigen or
monospecific polyclonal antibodies also could be used to
prepare the affinity column. The detergent extract of
cell envelopes (in solution) was placed with a 1.5 ml
batch of monoclonal antibody 11E3 bound to matrix for
about 18 hr (overnight) at 4~C. The matrix was washed 3
times with four bed volumes of to n-octylglucoside in TK
buffer, followed by washing 4 times in four~bed volumes of
1% n-octylglucoside-0.5 M MgCl2 in TK buffer. The 47-kDa
antigen was eluted from the matrix by using four bed
volumes of 3 M guanidine hydrochloride (pH 8.0)-1% n-
octylglucoside-0.5 TK buffer.
The eluted material consisted of the 47-kDa antigen
purified to near homogeneity (Fig. 6A and B, lanes 5).
Silver per.iodate staining of the eluted material did,
however, reveal the presence of minor protein contaminants
with molecular masses greater than 47-kDa, but no lipo-
polysaccharide was detectable. Contaminants could be
removed by using a chromatofocusing column and collecting
the 47-kDa antigen during elution from the column with
polybuffer in fractions with a pH of 4.6 to 4.9. Approxi-
mately 34 ug of the 47-kDa antigen was recovered from a
starting quantity of about 5.5 mg of Sarkosyl-solubilized
E. coli cell envelopes.
The present invention has been described in terms of
particular embodiments found or proposed by the present
inventor to comprise preferred modes for the practice of
the invention. It will be appreciated by those of skill
in the art that, in light of the present disclosure,
numerous modifications and changes can be made in the
particular embodiments exemplified without departing from
1 X41450
-55-
the intended scope of the invention. For example, due to
codon redundency, changes can be made in the underlying
DNA sequence without affecting the protein sequence.
Moreover, due to biological functional equivalency con-
s siderations, changes can be made in protein structure
without affecting in kind or amount of the biological
action. All such modifications are intended to be
included within the scope of the appended claims.
1341450
-56-
REFERENCES
1. Norris, S.J., J.F. Alderete, N.H. Axelsen, M.J.
Bailey, S.A. Haker-Zander, J.B. Baseman, P.J. Bassford,
R.E. Baughn, A. Cockayne, P.A. Hanff, P. Hindersson, S.A.
Larsen, M.A. Lovett, S.A. Lukehart, J.N. Mil,ler, M.A.
Moskophidis, F. Muller, M.V. Norgard, C.W. Penn, L.V.
Stamm, J.D. van Embden, and K. Wicher (1987). Identity of
Treponema gallidum ssp. pallidum polypeptides: correla-
tion of sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis results from different laboratories.
Electrophoresis 8:77-92.
2. Sell, S., and S.J. Norris (1983). The biology,
pathology, and.immunology of syphilis. Int. Rev. Exp.
Pathol., 24:203-276.
3. Bishop, N.H., and J.N. Miller (1976). Fiumoral
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