Note: Descriptions are shown in the official language in which they were submitted.
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1
Description
ABUNDANT EXTRACELLULAR PRODUCTS AND
METHODS FOR THEIR PRODUCTION AND USE
Reference to Government
This invention was made with US Government support under Grant
Nos. AI-35275 and AI-31338 awarded by the Department of Health and Human
Services. The US Government has certain rights in this invention.
Field of the Invention
The present invention generally relates to immunotherapeutic agents and
vaccines against pathogenic organisms such as bacteria, protozoa, viruses and
fungus.
More specifically, unlike prior art vaccines and immunotherapeutic agents
based upon
pathogenic subunits or products which exhibit the greatest or most specific
molecular
immunogenicity, the present invention uses the most prevalent or majorly
abundant
immunogenic determinants released by a selected pathogen such as Mycobacterium
tuberculosis to stimulate an effective immune response in mammalian hosts.
Accordingly, the acquired immunity and immunotherapeutic activity produced
through
the present invention is directed to those antigenic markers which are
displayed most
often on infected host cells during the course of a pathogenic infection
without
particular regard to the relative or absolute immunogenicity of the
administered
compound.
Background of the Invention
It has long been recognized that parasitic microorganisms possess the
ability to infect animals thereby causing disease and often the death of the
host.
Pathogenic agents have been a leading cause of death throughout history and
continue
to inflict immense suffering. Though the last hundred years have seen dramatic
advances in the prevention and treatment of many infectious diseases,
complicated
host-parasite interactions still limit the universal effectiveness of
therapeutic measures.
Difficulties in countering the sophisticated invasive mechanisms displayed by
many
pathogenic vectors is evidenced by the resurgence of various diseases such as
tuberculosis, as well as the appearance of numerous drug resistant strains of
bacteria
and viruses.
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Among those pathogenic agents of major epidemiological concern,
intracellular bacteria have proven to be particularly intractable in the face
of therapeutic
or prophylactic measures. Intracellular bacteria, including the genus
Mycobacterium
and the genus Legionella, complete all or part of their life cycle within the
cells of the
infected host organism rather than extracellularly. Around the world,
intracellular
bacteria are responsible for millions of deaths each year and untold
suffering.
Tuberculosis, caused by Mycobacterium tuberculosis, is the leading cause of
death from
infectious disease worldwide, with 10 million new cases and 2.9 million deaths
every
year. In addition, intracellular bacteria are responsible for millions of
cases of leprosy.
Other debilitating diseases transmitted by intracellular agents include
cutaneous and
visceral leishmaniasis, American trypanosomiasis (Chagas disease),
listeriosis,
toxoplasmosis, histoplasmosis, trachoma, psittacosis, Q-fever, and
Legionellosis
including Legionnaires' disease. At this time, relatively little can be done
to prevent
debilitating infections in susceptible individuals exposed to these organisms.
Due to this inability to effectively protect populations from tuberculosis
and the inherent human morbidity and mortality caused by tuberculosis, this is
one of
the most important diseases confronting mankind. More specifically, human
pulmonary
tuberculosis primarily caused by M. tuberculosis is a major cause of death in
developing
countries. Capable of surviving inside macrophages and monocytes, M
tuberculosis
may produce a chronic intracellular infection. By concealing itself within the
cells
primarily responsible for the detection of foreign elements and subsequent
activation of
the immune system, M tuberculosis is relatively successful in evading the
normal
defenses of the host organism. These same pathogenic characteristics have
heretofore
prevented the development of an effective immunotherapeutic agent or vaccine
against
tubercular infections. At the same time tubercle bacilli are relatively easy
to culture and
observe under laboratory conditions. Accordingly, M tuberculosis is
particularly well
suited for demonstrating the principles and advantages of the present
invention.
Those skilled in the art will appreciate that the following exemplary
discussion of M tuberculosis is in no way intended to limit the scope of the
present
invention to the treatment of M tuberculosis. Similarly, the teachings herein
are not
limited in any way to the treatment of tubercular infections. On the contrary,
this
invention may be used to advantageously provide safe and effective vaccines
and
immunotherapeutic agents against the immunogenic determinants of any
pathogenic
agent expressing extracellular products and thereby inhibit the infectious
transmission
of those organisms.
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Currently it is believed that approximately half of the world's population
is infected by M. tuberculosis resulting in millions of cases of pulmonary
tuberculosis
annually. While this disease is a particularly acute health problem in the
developing
countries of Latin America, Africa, and Asia, it is also becoming more
prevalent in the
first world. In the United States specific populations are at increased risk,
especially
urban poor, immunocompromised individuals and immigrants from areas of high
disease prevalence. Largely due to the AIDS epidemic the incidence of
tuberculosis is
presently increasing in developed countries, often in the form of multi-drug
resistant
M. tuberculosis.
Recently, tuberculosis resistance to one or more drugs was reported in 36
of the 50 United States. In New York City, one-third of all cases tested in
1991 were
resistant to one or more major drugs. Though non-resistant tuberculosis can be
cured
with a long course of antibiotics, the outlook regarding drug resistant
strains is bleak.
Patients infected with strains resistant to two or more major antibiotics have
a fatality
rate of around 50%. Accordingly, a safe and effective vaccine against such
varieties of
M tuberculosis is sorely needed.
Initial infections of M. tuberculosis almost always occur through the
inhalation of aerosolized particles as the pathogen can remain viable for
weeks or
months in moist or dry sputum. Although the primary site of the infection is
in the
lungs, the organism can also cause infection of the bones, spleen, meninges
and skin.
Depending on the virulence of the particular strain and the resistance of the
host, the
infection and corresponding damage to the tissue may be minor or extensive. In
the
case of humans, the initial infection is controlled in the majority of
individuals exposed
to virulent strains of the bacteria. The development of acquired immunity
following the
initial challenge reduces bacterial proliferation thereby allowing lesions to
heal and
leaving the subject largely asymptomatic but possibly contagious.
When M tuberculosis is not controlled by the infected subject, it often
results in the extensive degradation of lung tissue. In susceptible
individuals lesions are
usually formed in the lung as the tubercle bacilli reproduce within alveolar
or
pulmonary macrophages. As the organisms multiply, they may spread through the
lymphatic system to distal lymph nodes and through the blood stream to the
lung
apices, bone marrow, kidney and meninges surrounding the brain. Primarily as
the
result of cell-mediated hypersensitivity responses, characteristic
granulomatous lesions
or tubercles are produced in proportion to the severity of the infection.
These lesions
consist of epithelioid cells bordered by monocytes, lymphocytes and
fibroblasts. In
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most instances a lesion or tubercle eventually becomes necrotic and undergoes
caseation.
While M. tuberculosis is a significant pathogen, other species of the
genus Mycobacterium also cause disease in animals including man and are
clearly
within the scope of the present invention. For example, M bovis is closely
related to
M tuberculosis and is responsible for tubercular infections in domestic
animals such as
cattle, pigs, sheep, horses, dogs and cats. Further, M bovis may infect humans
via the
intestinal tract, typically from the ingestion of raw milk. The localized
intestinal
infection eventually spreads to the respiratory tract and is followed shortly
by the
classic symptoms of tuberculosis. Another important pathogenic vector of the
genus
Mycobacterium is M leprae which causes millions of cases of the ancient
disease
leprosy. Other species of this genus which cause disease in animals and man
include M
kansasii, M avium intracellulare, M fortuitum, M marinum, M chelonei,
M africanum, M. ulcerans, M. microti and M scrofulaceum. The pathogenic
mycobacterial species frequently exhibit a high degree of homology in their
respective
DNA and corresponding protein sequences and some species, such as M.
tuberculosis
and M bovis are highly related.
For obvious practical and moral reasons, initial work in humans to
determine the efficacy of experimental compositions with regard to such
afflictions is
infeasible. Accordingly, in the early development of any drug or vaccine it is
standard
procedure to employ appropriate animal models for reasons of safety and
expense. The
success of implementing laboratory animal models is predicated on the
understanding
that immunodominant epitopes are frequently active in different host species.
Thus, an
immunogenic determinant in one species, for example a rodent or guinea pig,
will
generally be immunoreactive in a different species such as in humans. Only
after the
appropriate animal models are sufficiently developed will clinical trials in
humans be
carried out to further demonstrate the safety and efficacy of a vaccine in
man.
With regard to alveolar or pulmonary infections by M tuberculosis, the
guinea pig model closely resembles the human pathology of the disease in many
respects. Accordingly, it is well understood by those skilled in the art that
it is
appropriate to extrapolate the guinea pig model of this disease to humans and
other
mammals. As with humans, guinea pigs are susceptible to tubercular infection
with low
doses of the aerosolized human pathogen M. tuberculosis. Unlike humans where
the
initial infection is usually controlled, guinea pigs consistently develop
disseminated
disease upon exposure to the aerosolized pathogen, facilitating subsequent
analysis.
Further, both guinea pigs and humans display cutaneous delayed-type
hypersensitivity
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reactions characterized by the development of a dense mononuclear cell
induration or
rigid area at the skin test site. Finally, the characteristic tubercular
lesions of humans
and guinea pigs exhibit similar morphology including the presence of Langhans
giant
cells. As guinea pigs are more susceptible to initial infection and
progression of the
5 disease than humans, any protection conferred in experiments using this
animal model
provides a strong indication that the same protective immunity may be
generated in man
or other less susceptible mammals. Accordingly, for purposes of explanation
only and
not for purposes of limitation, the present invention will be primarily
demonstrated in
the exemplary context of guinea pigs as the mammalian host. Those skilled in
the art
will appreciate that the present invention may be practiced with other
mammalian hosts
including humans and domesticated animals.
Any animal or human infected with a pathogenic vector and, in
particular, an intracellular organism presents a difficult challenge to the
host immune
system. While many infectious agents may be effectively controlled by the
humoral
response and corresponding production of protective antibodies, these
mechanisms are
primarily effective only against those pathogens located in the body's
extracellular fluid.
In particular, opsonizing antibodies bind to extracellular foreign agents
thereby
rendering them susceptible to phagocytosis and subsequent intracellular
killing. Yet
this is not the case for other pathogens. For example, previous studies have
indicated
that the humoral immune response does not appear to play a significant
protective role
against infections by intracellular bacteria such as M. tuberculosis. However,
the
present invention may generate a beneficial humoral response to the target
pathogen
and, as such, its effectiveness is not limited to any specific component of
the stimulated
immune response.
More specifically, antibody mediated defenses seemingly do not prevent
the initial infection of intracellular pathogens and are ineffectual once the
bacteria are
sequestered within the cells of the host. As water soluble proteins,
antibodies can
permeate the extracellular fluid and blood, but have difficulty migrating
across the lipid
membranes of cells. Further, the production of opsonizing antibodies against
bacterial
surface structures may actually assist intracellular pathogens in entering the
host cell.
Accordingly, any effective prophylactic measure against intracellular agents,
such as
Mycobacterium, should incorporate an aggressive cell-mediated immune response
component leading to the rapid proliferation of antigen specific lymphocytes
which
activate the compromised phagocytes or cytotoxically eliminate them. However,
as will
be discussed in detail below, inducing a cell-mediated immune response does
not equal
the induction of protective immunity. Though cell-mediated immunity may be a
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prerequisite to protective immunity, the production of vaccines in accordance
with the
teachings of the present invention requires animal based challenge studies.
This cell-mediated immune response generally involves two steps. The
initial step, signaling that the cell is infected, is accomplished by special
molecules
(major histocompatibility or MHC molecules) which deliver pieces of the
pathogen to
the surface of the cell. These MHC molecules bind to small fragments of
bacterial
proteins which have been degraded within the infected cell and present them at
the
surface of the cell. Their presentation to T-cells stimulates the immune
system of the
host to eliminate the infected host cell or induces the host cell to eradicate
any bacteria
residing within.
Unlike most infectious bacteria, Mycobacterium, including
M. tuberculosis, tend to proliferate in vacuoles which are substantially
sealed off from
the rest of the cell by a membrane. Phagocytes naturally form these protective
vacuoles
making them particularly susceptible to infection by this class of pathogen.
In such
vacuoles the bacteria are effectively protected from degradation, making it
difficult for
the immune system to present integral bacterial components on the surface of
infected
cells. However, the infected cell's MHC molecules will move to the vacuole and
collect
any free (released) bacterial products or move to other sites in the host cell
to which the
foreign extracellular bacterial products have been transported for normal
presentation of
the products at the cell surface. As previously indicated, the presentation of
the foreign
bacterial products will provoke the proper response by the host immune system.
The problems intracellular pathogens pose for the immune system also
constitute a special challenge to vaccine development. Thus far, the
production of an
effective vaccine against Mycobacterium infections and, in particular, against
M. tuberculosis has eluded most researchers. At the present time the only
widely
available vaccine against intracellular pathogens is the live attenuated
vaccine BCG, an
avirulent strain of M. bovis, which is used as a prophylactic measure against
the tubercle
bacillus. Yet in 1988, extensive World Health Organization studies from India
determined that the efficacy of the best BCG vaccines was so slight as to be
unmeasurable. Despite this questionable efficacy, BCG vaccine has been
extensively
employed in high incidence areas of tuberculosis throughout the world.
Complicating
the matter even further individuals who have been vaccinated with BCG will
often
develop sensitivity to tuberculin which negates the usefulness of the most
common skin
test for tuberculosis screening and control.
Another serious problem involving the use of a live, attenuated vaccine
such as BCG is the possibility of initiating a life-threatening disease in
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immunocompromised patients. These vaccines pose a particular risk for persons
with
depressed cell-mediated immunity because of their diminished capacity to fight
a
rapidly proliferating induced infection. Such individuals include those
weakened by
malnourishment and inferior living conditions, organ transplant recipients,
and persons
infected with HIV. In the case of BCG vaccine, high risk individuals also
include those
suffering from lung disorders such as emphysema, chronic bronchitis,
pneumoconiosis,
silicosis or previous tuberculosis. Accordingly, the use of attenuated
vaccines is limited
in the very population where they have the greatest potential benefit.
The use of live attenuated vaccines may also produce other undesirable
side effects. Because live vaccines reproduce in the recipient, they provoke a
broader
range of antibodies and a less directed cell-mediated immune response than
noninfectious vaccines. Often this shotgun approach tends to occlude the
immune
response directed at the molecular structures most involved in cellular
prophylaxis.
Moreover, the use of live vaccines with an intact membrane may induce
opsonizing
antibodies which prepare a foreign body for effective phagocytosis. Thus, upon
host
exposure to virulent strains of the target organism, the presence of such
antibodies could
actually enhance the uptake of non-attenuated pathogens into host cells where
they can
survive and multiply. Further, an attenuated vaccine contains thousands of
different
molecular species and consequently is more likely to contain a molecular
species that is
toxic or able to provoke an adverse immune response in the patient. Other
problems
with live vaccines include virulence reversion, natural spread to contacts,
contaminating
viruses and viral interference, and difficulty with standardization.
Similarly, noninfectious vaccines, such as killed organisms or
conventional second generation subunit vaccines directed at strongly antigenic
membrane bound structures, are limited with respect to the inhibition of
intracellular
bacteria. Like attenuated vaccines, killed bacteria provoke an indiscriminate
response
which may inhibit the most effective prophylactic determinants. Further,
killed
vaccines still present large numbers of potentially antigenic structures to
the immune
system thereby increasing the likelihood of toxic reactions or opsonization by
the
immune system. Traditional subunit vaccines incorporating membrane bound
structures, whether synthesized or purified, can also induce a strong opsonic
effect
facilitating the entry of the intracellular pathogen into phagocytes in which
they
multiply. By increasing the rate of bacterial inclusion, killed vaccines
directed to
intracellular surface antigens may increase the relative virulence of the
pathogenic
agent. Thus, conventional attenuated or killed vaccines directed against
strongly
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antigenic bacterial surface components may be contraindicated in the case of
intracellular pathogens.
In order to circumvent the problems associated with the use of traditional
vaccines, developments have been made using extracellular proteins or their
immunogenic analogs to stimulate protective immunity against specific
intracellular
pathogens. For example, this inventor's U.S. Patent No. 5,108,745, issued
April 28,
1992 discloses vaccines and methods of producing protective immunity against
Legionella pneumophila and M tuberculosis as well as other intracellular
pathogens.
These prior art vaccines are broadly based on extracellular products
originally derived
from proteinaceous compounds released extracellularly by the pathogenic
bacteria into
broth culture in vitro and released extracellularly by bacteria within
infected host cells
in vivo. As disclosed therein, these vaccines are selectively based on the
identification
of extracellular products or their analogs which stimulate a strong immune
response
against the target pathogen in a mammalian host.
More specifically, these prior art candidate extracellular proteins were
screened by determining their ability to provoke either a strong lymphocyte
proliferative response or a cutaneous delayed-type hypersensitivity response
in
mammals which were immune to the pathogen of interest. Though this disclosed
method and associated vaccines avoid many of the drawbacks inherent in the use
of
traditional vaccines, conflicting immunoresponsive results due to cross-
reactivity and
host variation may complicate the selection of effective immunizing agents.
Thus,
while molecular immunogenicity is one indication of an effective vaccine,
other factors
may complicate its use in eliciting an effective immune response in vivo.
More importantly, it surprisingly was discovered that, particularly with
respect to M tuberculosis, conventional prior art methods for identifying
effective
protective immunity inducing vaccines were cumbersome and potentially
ineffective.
For example, SDS-PAGE analysis of bulk M tuberculosis extracellular protein
followed by conventional Western blot techniques aimed at identifying the most
immunogenic of these extracellular components produced inconsistent results.
Repeated testing failed to identify which extracellular product would produce
the
strongest immunogenic response and, consistent with prior art thinking,
thereby
function as the most effective vaccine. Many of the extracellular products of
M tuberculosis are well known in the art, having been identified and, in some
cases,
sequenced. Further, like any foreign protein, it can be shown that these known
compounds induce an immune response. However, nothing in the art directly
indicates
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that any of these known compounds will induce protective immunity as
traditionally
identified.
Accordingly, it is a principal object of the present invention to provide
vaccines or immunotherapeutic agents and methods for their production and use
in
mounting an effective immune response against infectious bacterial pathogens
which do
not rely upon traditional vaccine considerations and selection techniques
based upon
highly specific, strongly immunogenic operability.
It is another object of the present invention to provide vaccines or
immunotherapeutic agents and methods for their use to impart acquired immunity
in a
mammalian host against intracellular pathogens including M tuberculosis, M
bovis, M
kansasii, M avium-intracellulare, M. fortuitum, M chelonei, M. marinum, M.
scrofulaceum, M leprae, M africanum, M ulcerans and M. microti.
It is an additional object of the present invention to provide easily
produced vaccines and immunotherapeutic agents exhibiting reduced toxicity
relative to
killed or attenuated vaccines.
Summary of the Invention
The present invention accomplishes the above-described and other
objects by providing compounds for use as vaccines and/or immunotherapeutic
agents
and methods for their production and use to generate protective or therapeutic
immune
responses in mammalian hosts against infection by pathogens. In a broad
aspect, the
invention provides the means to induce a protective or therapeutic immune
response
against infectious vectors producing extracellular compounds. While the
compounds of
the present invention are particularly effective against pathogenic bacteria,
they may be
used to generate a protective or therapeutic immune response to any pathogen
producing majorly abundant extracellular products.
For purposes of the present invention, the term "majorly abundant"
should be understood as a relative term identifying those extracellular
products released
in the greatest quantity by the pathogen of interest. For example, with
respect to
M. tuberculosis grown under various conditions of culture to an optical
density of
approximately 0.5, one skilled in the art should expect to obtain on the order
of 10 g/L
or more of a majorly abundant extracellular product. Thus, out of the total
exemplary 4
mg/L total output of extracellular product for M tuberculosis grown under
normal or
heat shock conditions, approximately fifteen to twenty (alone or in
combination) of the
one hundred or so known extracellular products will constitute approximately
ninety
percent of the total quantity. These are the majorly abundant extracellular
products
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contemplated as being within the scope of the present invention and are
readily
identifiable as the broad bands appearing in SDS/PAGE gels. In addition, the
extracellular products of interest may further be characterized and
differentiated by
amino acid sequencing. The remaining extracellular products are minor. Those
skilled
5 in the art will also appreciate that the relative quantitative abundance of
specific major
extracellular products may vary depending upon conditions of culture. However,
in
most cases, the identification of an individual majorly abundant extracellular
product
will not change.
Accordingly, the present invention may be used to protect a mammalian
10 host against infection by viral, bacterial, fungal or protozoan pathogens.
It should be
noted that in some cases, such as in viral infections, the majorly abundant
extracellular
products may be generated by the infected host cell. While active against all
microorganisms releasing majorly abundant extracellular products, the vaccines
and
methods of the present invention are particularly effective in generating
protective
immunity against intracellular pathogens, including various species and
serogroups of
the genus Mycobacterium. The vaccines of the present invention are also
effective as
immunotherapeutic agents for the treatment of existing disease conditions.
Surprisingly, it has been found by this inventor that immunization with
the most or majorly abundant products released extracellularly by bacterial
pathogens or
their immunogenic analogs can provoke an effective immune response
irrespective of
the absolute immunogenicity of the administered compound. Due to their release
from
the organism and hence their availability to host molecules involved in
antigen
processing and presentation and due to their naturally high concentration in
tissue
during infection, the majorly abundant extracellular products of a pathogenic
agent are
processed and presented to the host immune system more often than other
bacterial
components. In the case of intracellular pathogens, the majorly abundant
extracellular
products are the principal immunogenic determinants presented on the surface
of the
infected host cells and therefore exhibit a greater presence in the
surrounding
environment. Accordingly, acquired immunity against the majorly abundant
extracellular products of a pathogenic organism allows the host defense system
to
swiftly detect pathogens sequestered inside host cells and effectively inhibit
them.
More particularly, the principal or majorly abundant products released by
pathogenic bacteria appear to be processed by phagocytes and other host immune
system mechanisms at a greater rate than less prevalent or membrane bound
pathogenic
components regardless of their respective immunogenic activity or specificity.
This
immunoprocessing disparity is particularly significant when the pathogenic
agent is an
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intracellular bacteria sequestered from normal immune activity. By virtue of
their
profuse and continual presentation to the infected host's immune system, the
most
prevalent bacterial extracellular products or their immunogenic analogs
provoke a
vigorous immune response largely irrespective of their individual molecular
immunogenic characteristics.
Majorly abundant extracellular products are the principal constituents of
proteins and other molecular entities which are released by the target
pathogen into the
surrounding environment. Current research indicates that in some instances a
single
majorly abundant extracellular product may comprise up to 40% by weight of the
products released by a microorganism. More often, individual majorly abundant
extracellular products account for between from about 0.5% to about 25% of the
total
products released by the infectious pathogen. Moreover, the top five or six
majorly
abundant extracellular products may be found to comprise between 60% to 70% of
the
total mass released by a microorganism. Of course those skilled in the art
will
appreciate that the relative levels of extracellular products may fluctuate
over time as
can the absolute or relative quantity of products released. For example, pH,
oxidants,
osmolality, heat and other conditions of stress on the organism, stage of life
cycle,
reproduction status and the composition of the surrounding environment may
alter the
composition and quantity of products released. Further, the absolute and
relative levels
of extracellular products may differ greatly from species to species and even
between
strains within a species.
In the case of intracellular pathogens, extracellular products appear to
expand the population of specifically immune lymphocytes capable of detecting
and
exerting an antimicrobial effect against macrophages containing live bacteria.
Further,
by virtue of their repeated display on the surface of infected cells, the
majorly abundant
or principal extracellular products function as effective antigenic markers.
Accordingly,
pursuant to the teachings of the present invention, vaccination and the
inducement of
protective immunity directed to the majorly abundant extracellular products of
a
pathogenic bacteria or their immunogenically equivalent determinants, prompts
the host
immune system to mount a rapid and efficient immune response with a strong
cell-
mediated component when subsequently infected by the target pathogen.
In direct contrast to prior art immunization activities which have
primarily been focused on the production of vaccines and the stimulation of
immune
responses based upon the highly specific molecular antigenicity of individual
screened
pathogen components, the present invention advantageously exploits the
relative
abundance of bacterial extracellular products or their immunogenic analogs
(rather than
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their immunogenic specificities) to establish or induce protective immunity
with
compounds which may actually exhibit lower immunogenic specificity than less
prevalent extracellular products. For the purposes of this disclosure an
immunogenic
analog is any molecule or compound sufficiently analogous to at least one
majorly
abundant extracellular product expressed by the target pathogen, or any
fraction thereof,
to have the capacity to stimulate a protective immune response in a vaccinated
mammalian host upon subsequent infection by the target pathogen. In short, the
vaccines of the present invention are identified or produced by selecting the
majorly
abundant product or products released extracellularly by a specific pathogen
(or
molecular analogs capable of stimulating a substantially equivalent immune
response)
and isolating them in a relatively pure form or subsequently sequencing the
DNA or
RNA responsible for their production to enable their synthetic or endogenous
production. The desired prophylactic immune response to the target pathogen
may then
be elicited by formulating one or more of the isolated immunoreactive products
or the
encoding genetic material using techniques well known in the art and
immunizing a
mammalian host prior to infection by the target pathogen.
It is anticipated that the present invention will consist of at least one, two
or, possibly even several well defined immunogenic determinants. As a result,
the
present invention produces consistent, standardized vaccines which may be
developed,
tested and administered with relative ease and speed. Further, the use of a
few well
defined molecules corresponding to the majorly abundant secretory or
extracellular
products greatly reduces the risk of adverse side effects associated with
conventional
vaccines and eliminates the possible occlusion of effective immunogenic
markers.
Similarly, because the present invention is not an attenuated or a killed
vaccine the risk
of infection during production, purification or upon administration is
effectively
eliminated. As such, the vaccines of the present invention may be administered
safely
to immunocompromised individuals, including asymptomatic tuberculosis patients
and
those infected with HIV. Moreover, as the humoral immune response is directed
exclusively to products released by the target pathogen, there is little
chance of
generating a detrimental opsonic immune component. Accordingly, the present
invention allows the stimulated humoral response to assist in the elimination
of the
target pathogen from antibody susceptible areas.
Another beneficial aspect of the present invention is the ease by which
the vaccines may be harvested or produced and subsequently purified and
sequenced.
For example, the predominantly abundant extracellular products may be obtained
from
cultures of the target pathogen, including M. tuberculosis or M bovis, with
little effort.
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As the desired compounds are released into the media during growth, they can
readily
be separated from the intrabacterial and membrane-bound components of the
target
pathogen utilizing conventional techniques. More preferably, the desired
immunoreactive constituents of the vaccines of the present invention may be
produced
and purified from genetically engineered organisms into which the genes
expressing the
specific extracellular products of M tuberculosis, M. bovis, M leprae or any
other
pathogen of interest have been cloned. As known in the art, such engineered
organisms
can be modified to produce higher levels of the selected extracellular
products or
modified immunogenic analogs. Alternatively, the immunoprotective products,
portions thereof or analogs thereof, can be chemically synthesized using
techniques well
known in the art or directly expressed in host cells injected with naked genes
encoding
therefor. Whatever production source is employed, the immunogenic components
of
the predominant or majorly abundant extracellular products may be separated
and
subsequently formulated into deliverable vaccines using common biochemical
procedures such as fractionation, chromatography or other purification
methodology
and conventional formulation techniques or directly expressed in host cells
containing
directly introduced genetic constructs encoding therefor.
For example, in an exemplary embodiment of the present invention the
target pathogen is M tuberculosis and the majorly abundant products released
extracellularly by M. tuberculosis into broth culture are separated from other
bacterial
components and used to elicit an immune response in mammalian hosts.
Individual
proteins or groups of proteins are then utilized in animal based challenge
experiments to
identify those which induce protective immunity making them suitable for use
as
vaccines in accordance with the teachings of the present invention. More
specifically,
following the growth and harvesting of the bacteria, by virtue of their
physical
abundance the principal extracellular products are separated from
intrabacterial and
other components through centrifugation and filtration. If desired, the
resultant bulk
filtrate is then subjected to fractionation using ammonium sulfate
precipitation with
subsequent dialysis to give a mixture of extracellular products, commonly
termed EP.
Solubilized extracellular products in the dialyzed fractions are then purified
to
substantial homogeneity using suitable chromatographic techniques as known in
the art
and as described more fully below.
These exemplary procedures result in the production of fourteen
individual proteinaceous major extracellular products of M. tuberculosis
having
molecular weights ranging from 110 kilo Daltons (KD) to 12 KD. Following
purification each individual majorly abundant extracellular product exhibits
one band
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14
corresponding to its respective molecular weight when subjected to
polyacrylamide gel
electrophoresis thereby allowing individual products or groups of products
corresponding to the majorly abundant extracellular products to be identified
and
prepared for use as vaccines in accordance with the teachings of the present
invention.
The purified majorly abundant extracellular products may further be
characterized and
distinguished by determining all or part of their respective amino acid
sequences using
techniques common in the art. Sequencing may also provide information
regarding
possible structural relationships between the majorly abundant extracellular
products.
Subsequently, immunization and the stimulation of acquired immunity in
a mammalian host system may be accomplished through the teachings of the
present
invention utilizing a series of subcutaneous or intradermal injections of
these purified
extracellular products over a course of time. For example, injection with a
purified
majorly abundant bacterial extracellular product or products in incomplete
Freund's
adjuvant followed by a second injection in the same adjuvant approximately
three
weeks later can be used to elicit a protective response upon subsequent
challenge with
the virulent pathogen. Other exemplary immunization protocols within the scope
and
teachings of the present invention may include a series of three or four
injections of
purified extracellular product or products or their analogs in Syntex Adjuvant
Formulation (SAF) over a period of time. While a series of injections may
generally
prove more efficacious, the single administration of a selected majorly
abundant
extracellular product or its immunogenic subunits or analogs can impart the
desired
immune response and is contemplated as being within the scope of the present
invention
as well.
Such exemplary protocols can be demonstrated using art accepted
laboratory models such as guinea pigs. For example, as will be discussed in
detail,
immunization of several guinea pigs with a combination of five majorly
abundant
extracellular products (purified from M tuberculosis as previously discussed)
was
accomplished with an immunization series of three injections of the bacterial
products
in SAY adjuvant with corresponding sham-immunization of control animals.
Exemplary dosages of each protein ranged from 100 pg to 2 g. Following the
last
vaccination all of the animals were simultaneously exposed to an infectious
and
potentially lethal dose of aerosolized M tuberculosis and monitored for an
extended
period of time. The control animals showed a significant loss in weight when
compared
with the animals immunized with the combination of the majorly abundant
extracellular
products of M tuberculosis. Moreover, half of the control animals died during
the
observation period while none of the immunized animals succumbed to
tuberculosis.
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Autopsies conducted after this experiment revealed that the non-immunized
control
animals had significantly more colony forming units (CFU) and corresponding
damage
in their lungs and spleens than the protected animals. Seventeen additional
combinations of purified majorly abundant extracellular products provided
5 immunoprophylaxis when tested, thereby demonstrating the scope of the
present
invention and broad range of vaccines which may be formulated in accordance
with the
teachings thereof.
However, it should be emphasized that the present invention is not
restricted to combinations of secretory or extracellular products. For
example, several
10 alternative experimental protocols demonstrate the capacity of a single
abundant
extracellular product to induce mammalian protective immunity in accordance
with the
teachings of the present invention. In each experiment guinea pigs were
immunized
with a single majorly abundant extracellular product purified from M
tuberculosis EP
using the chromatography protocols detailed herein. In one example the animals
were
15 vaccinated in multiple experiments with an adjuvant composition containing
a purified
abundant secretory product having a molecular weight corresponding to 30 KD.
In
another example of the present invention, different guinea pigs were
vaccinated with an
adjuvant composition containing an abundant extracellular product isolated
from
M tuberculosis having a molecular weight corresponding to 71 KD. Following
their
respective immunizations both sets of animals and the appropriate controls
were
exposed to lethal doses of aerosolized M tuberculosis to determine vaccine
effectiveness.
More particularly, in one experiment six guinea pigs were immunized
with 100 .tg of 30 KD protein in SAF on three occasions spread over a period
of six
weeks. Control animals were simultaneously vaccinated with corresponding
amounts
of a bulk preparation of extracellular proteins (EP) or buffer. Three weeks
after the
final vaccination, the animals were challenged with an aerosolized lethal dose
of
M tuberculosis and monitored for a period of 14 weeks. The 30 KD immunized
guinea
pigs and those immunized with the bulk extracellular preparation had survival
rates of
67% and 50% respectively (illustrating the unexpectedly superior performance
of the
majorly abundant extracellular product versus EP), while the sham-immunized
animals
had a survival rate of only 17%. Upon termination of the experiment the
animals were
sacrificed and examined for viable tubercle bacilli. Unsurprisingly, the non-
immunized
animal showed markedly higher concentrations of M tuberculosis in the lungs
and
spleen.
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16
Similar experiments were performed on those animals vaccinated with
71 KD protein. In one experiment six guinea pigs were vaccinated with an SAF
adjuvant composition containing 100 tg purified 71 KD protein two times over a
period
of three weeks. Other animals were similarly immunized with a bulk preparation
of
unpurified extracellular proteins or EP for use as a positive control and with
buffer for
use as a negative control. Following exposure to lethal doses of aerosolized
tubercle
bacilli the weight of the guinea pigs was monitored for a period of 6 months.
Once
again the animals immunized with the purified form of the abundant
extracellular
product developed protective immunity with respect to the virulent M
tuberculosis. By
the end of that period the buffer immunized animals showed a significant loss
in weight
when compared with the immunized animals. Further, while the positive controls
and
71 KD immunized animals had survival rates of 63% and 50% respectively, the
non-
immunized animals all died before the end of the observation period.
It is important to note that the formulation of the vaccine is not critical to
the present invention and may be optimized to facilitate administration.
Solutions of
the purified immunogenic determinants derived from the majorly abundant
pathogenic
extracellular products may be administered alone or in combination in any
manner
designed to generate a protective immune response. The purified protein
solutions may
be delivered alone, or formulated with an adjuvant before being administered.
Specific
exemplary adjuvants used in the instant invention to enhance the activity of
the selected
immunogenic determinants are SAF, adjuvants containing Monophosphoryl Lipid A
(MPL), Freund's incomplete adjuvant, Freund's complete adjuvant containing
killed
bacteria, gamma interferons (Radford et al., American Society of Hepatology
2008-
2015, 1991; Watanabe et al., PNAS 86:9456-9460, 1989; Gansbacher et al.,
Cancer
Research 50:7820-7825, 1990; Maio et al., Can. Immunol. Immunother. 30:34-42,
1989; U.S. Patent Nos. 4,762,791 and 4,727,138), MF59, MF59 plus MTP, MF59
plus
IL-12, MPL plus TDM (Trehalose (Dimycolate), QS-21, QS-21 plus IL-12, IL-2
(American Type Culture Collection Nos. 39405, 39452 and 39516; see also U.S.
Patent
No. 4,518,584), IL-12, IL-15 (Grabstein et al., Science 264:965-968, 1994),
dimethyldioctadecyl ammonium (ddA), ddA plus dextran, alum, Quil A, ISCOMS,
(Immunostimulatory Complexes), Liposomes, Lipid Carriers, Protein Carriers,
and
Microencapsulation techniques. Additional adjuvants that may be useful in the
present
invention are water-in-oil emulsions, mineral salts (for example, alum),
nucleic acids,
block polymer surfactants, and microbial cell walls (peptido glycolipids).
While not
limiting the scope of the invention it is believed that adjuvants may magnify
immune
responses due to the slow release of antigens from the site of injection.
CA 02222000 2008-03-07
17
Alternatively, genetic material encoding the genes for one or more of the
immunogenic determinants derived from the majorly abundant pathogenic
extracellular
products may be coupled with eucaryotic promoter and/or secretion sequences
and
injected directly into a mammalian host to induce and endogenous expression of
the
immunogenic determinants and subsequent protective immunity.
According to an aspect of the present invention, there is provided a
vaccinating agent for use in immunizing a mammalian host susceptible to
disease caused
by a pathogen from the genus Mycobacterium, consisting essentially of a
recombinant
purified Mycobacterium 32A kD extracellular protein, and an adjuvant and one
additional purified Mycobacterial extracellular protein selected from the
group consisting
of purified Mycobacterial extracellular 110 kD protein, purified Mycobacterial
extracellular 80 kD protein, purified Mycobacterial extracellular 45 kD
protein, purified
Mycobacterial extracellular 23.5 kD protein, purified Mycobacterial
extracellular 14 kD
protein; with the proviso that said vaccinating agent does not contain an
immunologically protective amount of unpurified Mycobacterium extracellular
protein
and wherein said vaccinating agent induces a protective immune response in
said
mammalian host.
According to another aspect of the present invention, there is provided a
vaccinating agent for use in immunizing a mammalian host susceptible to
disease caused
by a pathogen from the genus Mycobacterium, consisting essentially of a
recombinant
purified Mycobacterium 32A kD extracellular protein, a purified 23.5 kD
Mycobacterium protein, a purified Mycobacterium 16 kD protein and an adjuvant,
with
the proviso that said vaccinating agent does not contain an immunologically
protective
amount of unpurified Mycobacterium extracellular protein and wherein said
vaccinating
agent induces a protective immune response in said mammalian host.
According to still another aspect of the present invention, there is
provided a vaccinating agent for use in immunizing a mammalian host
susceptible to
disease caused by a pathogen from the genus Mycobacterium, consisting
essentially of a
recombinant purified Mycobacterium 32A kD extracellular protein, and an
adjuvant,
with the proviso that said vaccinating agent does not contain an
immunologically
protective amount of unpurified Mycobacterium extracellular protein and
wherein said
vaccinating agent induces a protective immune response in said mammalian host.
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17a
In accordance with an aspect of the present invention, there is provided a
vaccinating agent for use in immunizing a mammalian host susceptible to
disease caused by
a pathogen from the genus Mycobacterium, consisting of a recombinant purified
Mycobacterium 32A kD extracellular protein (having the N-terminal amino acid
sequence of
SEQ ID NO. 28), and an adjuvant and one additional purified Mycobacterial
extracellular
protein selected from the group consisting of purified Mycobacterial
extracellular 110 kD
protein (having the N-terminal amino acid sequence of SEQ ID NO. 34), purified
Mycobacterial extracellular 80 kD protein (having the N-terminal amino acid
sequence of
SEQ ID NO. 33), purified Mycobacterial extracellular 45 kD protein (having the
N-terminal
amino acid sequence of SEQ ID NO. 30), purified Mycobacterial extracellular
23.5 kD
protein (having the N-terminal amino acid sequence of SEQ ID NO. 24), purified
Mycobacterial extracellular 14 kD protein (having the N-terminal amino acid
sequence of
SEQ ID NOs. 19 or 20); with the proviso that said vaccinating agent does not
contain an
immunologically protective amount of unpurified Mycobacterium extracellular
protein and
wherein said vaccinating agent induces a protective immune response in said
mammalian
host.
In accordance with another aspect of the present invention, there is provided
a vaccinating agent for use in immunizing a mammalian host susceptibly to
disease caused
by a pathogen from the genus Mycobacterium, consisting of a recombinant
purified
Mycobacterium 32A kD extracellular protein (having the N-terminal amino acid
sequence of
SEQ ID NO. 28), a purified 23.5 kD Mycobacterium protein (having the N-
terminal amino
acid sequence of SEQ ID NO. 24), a purified Mycobacterium 16 kD protein
(having the N-
terminal amino acid sequence of SEQ ID NOs. 21 or 22) and an adjuvant, with
the proviso
that said vaccinating agent does not contain an immunologically protective
amount of
unpurified Mycobacterium extracellular protein and wherein said vaccinating
agent induces a
protective immune response in said mammalian host.
In accordance with another aspect of the present invention, there is provided
a vaccinating agent for use in immunizing a mammalian host susceptible to
disease caused
by a pathogen from the genus Mycobacterium, consisting of a recombinant
purified
Mycobacterium 32A kD extracellular protein (having the N-terminal amino acid
sequence of
SEQ ID NO. 28), and an adjuvant, with the proviso that said vaccinating agent
does not
contain an immunologically protective amount of unpurified Mycobacterium
extracellular
protein and wherein said vaccinating agent induces a protective immune
response in said
CA 02222000 2010-09-13
l7b
mammalian host.
Other objects, features and advantages of the present invention will be
apparent to those skilled in the art from a consideration of the following
detailed description
of preferred exemplary embodiments thereof taken in conjunction with the
figures which will
first be described briefly.
Brief Description of the Drawings
Figure 1 is a representation of 4 Coomassie blue stained gels, labeled ]a to
Id, illustrating the purification of exemplary majorly abundant extracellular
products of M.
tuberculosis as identified by sodium dodecyl sulfate polyacrylamide gel
electrophoresis
(SDS-PAGE).
Figure 2 is a tabular representation identifying the five N-terminal amino
acids of fourteen exemplary majorly abundant extracellular products of M.
tuberculosis
(Sequence ID Nos. 1-14) and the apparent molecular weight for such products.
Figure 3 is a tabular representation of the extended N-terminal amino acid
sequence of three exemplary majorly abundant secretory products of M.
tuberculosis
(Sequence JD Nos. 15-17) which were not distinguished by the five N-terminal
amino acids
shown in Figure 2.
Figure 4 is a graphical comparison of the survival rate of guinea pigs
immunized with exemplary purified majorly abundant 30 KD secretory product of
M
tuberculosis versus positive controls immunized with a prior art bulk
preparation of
extracellular proteins and non-immunized negative controls following exposure
to an
aerosolized lethal dose of M tuberculosis.
Figure 5 is a graphical comparison of mean guinea pig body weight of
animals immunized with purified majorly abundant 71 KD extracellular product
versus
positive controls immunized with a prior art bulk preparation of extracellular
proteins from
M. tuberculosis and non-immunized negative controls following exposure to an
aerosolized
lethal dose of M. tuberculosis.
Figure 6 is a graphical comparison of the survival rate of guinea pigs
immunized in Figure 5 with exemplary majorly abundant purified 71 KD
extracellular
product of M tuberculosis versus positive controls immunized with a prior art
bulk
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18
preparation of extracellular proteins from M tuberculosis and non-immunized
negative
controls following exposure to an aerosolized lethal dose of M tuberculosis.
Figure 7 is a graphical comparison of mean guinea pig body weight of
animals immunized with exemplary purified majorly abundant 71 KD extracellular
product and non-immunized negative controls following exposure to an
aerosolized
lethal dose of M. tuberculosis in a second, separate experiment.
Figures 8a and 8b are graphical comparisons of lymphocyte proliferative
responses to exemplary purified majorly abundant 71 KD extracellular product
in PPD+
(indicative of infection with M. tuberculosis) and PPD- human subjects. Figure
8a is a
graph of the values measured at 2 days after incubation of lymphocytes with
this
antigen while Figure 8b is a graph of the values measured at 4 days after
incubation.
Figure 9 is a graphical comparison of mean guinea pig body weight of
animals immunized with vaccine comprising a combination of extracellular
products
produced according to the teachings of the present invention and non-immunized
controls following exposure to an aerosolized lethal dose of M tuberculosis.
Figure 10 is a graphical comparison of mean guinea pig body weight of
animals immunized with three different dosages of a vaccine comprising a
combination
of extracellular products produced according to the teachings of the present
invention
and non-immunized controls following exposure to an aerosolized lethal dose of
M. tuberculosis.
Figure 11 is a graphical comparison of mean guinea pig body weight of
animals immunized with vaccines comprising six different combinations of
extracellular products produced according to the teachings of the present
invention and
non-immunized controls following exposure to an aerosolized lethal dose of
M. tuberculosis.
Figures 12a and b are graphical illustrations of the mapping of the
immunodominant epitopes of the 30 KD protein of M. tuberculosis. Figure 12a
illustrates the percentage of 24 guinea pigs immunized with the 30 KD protein
responding to overlapping peptides (15-mer) covering the entire 30 KD protein
sequence. Figure 12b illustrates a corresponding set of data for a group of 19
sham
immunized guinea pigs. The response of each group of animals to native 30 KD
protein, purified protein derivative (PPD) and concanavalin A (con A) appears
at the
right of each graph.
Figure 13 provides a diagrammatic representation of the constructs used
for the expression of recombinant 30 kDa protein. The diagram depicts the
pET22b
vectors used for the expression of recombinant 30 kDa protein. The vectors
express the
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19
mature 30 kDa protein fused to its own leader (30W-pET22b) or the plasmid
encoded
pe1B leader (30M-pET22b). Abbreviations used: On, ColE1 type origin of
replication;
Fl ori, phage Fl origin of replication; Amp, ampicillin resistance gene;
30W/M, full-
length (30W)) or mature (30M) 30 kDa protein; lacI, lac repressor gene; PT 7,
phage T7
RNA polymerase specific promoter; NdeI and NcoI, restriction enzyme sites at
vector/insert junctions.
Figure 14 shows electrophoresis test results and a Western blot analysis
which confirm the expression of full-length and mature 30 kDa protein in E.
coli
BL21(DE3)pLysS.
Figure 15 is a diagrammatic representation of an alternate construct
system used to express the 30 kDa protein.
Figure 16 shows electrophoresis test results which confirm the
expression of the M. tuberculosis 30 kDa protein in M. smegmatis.
Figure 17 depicts the results of a Western blot analysis, confirming the
expression of the M. tuberculosis 30 kDa protein in M. smegmatis.
Detailed Description
The present invention is directed to compounds and methods for their
production and use against pathogenic organisms as vaccines and
immunotherapeutic
agents. More specifically, the present invention is directed to the production
and use of
majorly abundant extracellular products released by pathogenic organisms,
their
immunogenic analogs or the associated genetic material encoding therefor as
vaccines
or immunotherapeutic agents and to associated methods for generating
protective
immunity in mammalian hosts against infection. These compounds will be
referred to
as vaccines throughout this application for purposes of simplicity.
In exemplary embodiments, illustrative of the teachings of the present
invention, the majorly abundant extracellular products of M. tuberculosis were
distinguished and subsequently purified. Guinea pigs were immunized with
purified
forms of these majorly prevalent extracellular products with no determination
of the
individual product's specific molecular immunogenicity. Further, the exemplary
immunizations were carried out using the purified extracellular products alone
or in
combination and with various dosages and routes of administration. Those
skilled in
the art will recognize that the foregoing strategy can be utilized with any
pathogenic
organism or bacteria to practice the method of the present invention and,
accordingly,
the present invention is not specifically limited to vaccines and methods
directed
against M. tuberculosis.
CA 02222000 2008-03-07
In these exemplary embodiments, the majorly abundant extracellular
products of M. tuberculosis were separated and purified using column
chromatography.
Determination of the relative abundance and purification of the extracellular
products was
accomplished using polyacrylamide gel electrophoresis. Following purification
of the
5 vaccine components, guinea pigs were vaccinated with the majorly abundant
extracellular
products alone or in combination and subsequently challenged with M.
tuberculosis. As
will be discussed in detail, in addition to developing the expected measurable
responses to
these extracellular products following immunization, the vaccines of the
present invention
unexpectedly conferred an effective immunity in these laboratory animals
against
10 subsequent lethal doses of aerosolized M tuberculosis.
While these exemplary embodiments used purified forms of the
extracellular products, those skilled in the art will appreciate that the
present invention
may easily be practiced using immunogenic analogs which are produced through
recombinant means or other forms of chemical synthesis using techniques well
known in
15 the art. Further, immunogenic analogs, homologs or selected segments of the
majorly
abundant extracellular products may be employed in lieu of the naturally
occurring
products within the scope and teaching of the present invention.
A further understanding of the present invention will be provided to those
skilled in the art from the following non-limiting examples which illustrate
exemplary
20 protocols for the identification, isolation, production and use of majorly
abundant
extracellular products (alone and in combination) as vaccines.
EXAMPLES
EXAMPLE 1
ISOLATION AND PRODUCTION OF BULK EXTRACELLULAR
PROTEINS (EP) FROM MYCOBACTERIUM TUBERCULOSIS
M. tuberculosis Erdman strain (ATCC 35801) was obtained from the
American Tissue Culture Collection (Rockville, Md.). The lyophilized bacteria
were
reconstituted in Middlebrook 7H9 culture medium (Difco Laboratories, Detroit,
Mich.)
and maintained on MiddlebrookTM 7H11 agar. 7H11 agar was prepared using Bacto
Middlebrook 7H10 agar (Difco), OADC Enrichment Medium (Difco), 0.1% casein
enzymatic hydrolysate (Sigma), and glycerol as previously described by Cohn
(Cohn,
M.1., Am. Rev. Respir. Dis. 98:295-296). Following sterilization by
autoclaving, the agar
was dispensed into bacteriologic PetriTM dishes (100 by 15 mm) and allowed to
cool.
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21
M tuberculosis was then plated using sterile techniques and grown at
37 C in 5% C02-95% air, 100% humidity. After culture on 7H11 for 7 days, the
colonies were scraped from the plates, suspended in 7119 broth to 108 CFU/ml
and
aliquoted into 1.8-m1 Nunc cryotubes (Roskilde, Denmark). Each liter of the
broth was
prepared by rehydrating 4.7 g of Bacto Middlebrook 7H9 powder with 998 ml of
distilled water, and 2 ml of glycerol (Sigma Chemical Co., St. Louis, Mo.)
before
adjusting the mixture to a pH value of 6.75 and autoclaving the broth for 15
min at
1211C. The aliquoted cells were then slowly frozen and stored at -70 C. Cells
stored
under these conditions remained viable indefinitely and were used as needed.
Bulk extracellular protein (EP) preparations were obtained from cultures
of M. tuberculosis grown in the Middlebrook 7H9 broth made as above. Following
reconstitution, 150 ml aliquots of the broth were autoclaved for 15 min at
121'C and
dispensed into vented Co-star 225 cm2 tissue culture flasks. M. tuberculosis
cells stored
at -70 C as described in the previous paragraph were thawed and used to
inoculate
7H 11 agar plates. After culture for 7 days, the colonies were scraped from
the plates,
suspended in a few ml of 7H9 broth, and sonicated in a water bath to form a
single cell
suspension. The M. tuberculosis cells were suspended in the sterile 150 ml
aliquots at
an initial optical density of 0.05, as determined by a Perkin-Elmer Junior
model 35
spectrophotometer (Norwalk, Conn). The cells were then incubated at 37 C in 5%
C02-95% air for 3 weeks until the suspension showed an optical density of 0.4
to 0.5.
These cultures were used as stock bottles for subsequent cultures also in 7H9
broth.
The stock bottles were sonicated in a water bath to form a single cell
suspension. The
M tuberculosis cells were then diluted in 7H9 broth to an initial optical
density of 0.05
and incubated at 37 C in 5% CO2-95% air for 2V2 to 3 weeks until the
suspension
showed an optical density of 0.4 to 0.5. Culture supernatant was then decanted
and
filter sterilized sequentially through 0.8 m and 0.2 m low-protein-binding
filters
(Gelman Sciences Inc., Ann Arbor, Mich.). The filtrate was then concentrated
approximately 35 fold in a Filtron Minisette with an Omega membrane having a
10 KD
cutoff and stored at 4 C. Analysis of the bulk extracellular protein
preparation by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed
a
protein composition with multiple bands. Bulk extracellular protein mixture
(EP) was
prepared by obtaining a 40%-95% ammonium sulfate cut of the culture filtrate.
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22
EXAMPLE 2
PURIFICATION OF PRINCIPAL MAJORLY ABUNDANT
EXTRACELLULAR PRODUCTS OF MYCOBACTERIUM TUBERCULOSISS
Ammonium sulfate (grade I, Sigma) was added to the sterile culture
filtrate of Example 1 in concentrations ranging from 10% to 95% at 0 C and
gently
stirred to fractionate the proteins. The suspension was then transferred to
plastic
bottles and centrifuged in a swinging bucket rotor at 3,000 rpm on a RC3B
SorvallTM
Centrifuge to pellet the resulting precipitate. The supernatant fluid was
decanted and,
depending on the product of interest, the supernatant fluid or pellet was
subjected to
further purification. When the product of interest was contained in the
supernatant
fluid a second ammonium sulfate cut was executed by increasing the salt
concentration above that of the first cut. After a period of gentle stirring
the solution
was then centrifuged as previously described to precipitate the desired
product and the
second supernatant fluid was subjected to further purification.
Following centrifugation, the precipitated proteins were resolubilized
in the appropriate cold buffer and dialyzed extensively in a SpectraporTM
dialysis
membrane (Spectrum Medical Industries, Los Angeles, California) with a 6,000
to
8,000 molecular weight cut-off to remove the salt. Extracellular protein
concentration
was determined by a bicinchoninic acid protein assay (Pierce Chemical Co.,
Rockford, Illinois) and fraction components were determined using SDS-PAGE.
The
fractions were then applied to chromatography columns for further
purification.
Using the general scheme outlined immediately above fourteen
extracellular products were purified from the bulk extracellular protein
filtrate
obtained by the process detailed in Example 1. The exact ammonium sulfate
precipitation procedure and chromatography protocol is detailed below for each
extracellular product isolated.
A. 110 KD Extracellular Product
1. A 50%-100% ammonium sulfate precipitate was obtained as
discussed above.
2. The resolubilized precipitate was dialyzed and applied to a
DEAE SepharoseTM CL-6B or QAE Sepharose ion exchange column in column buffer
consisting of 10% sorbitol, 10 mM potassium phosphate, pH 7, 5 mM 2-
mercaptoethanol, and 0.2mM EDTA and eluted with a sodium chloride
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23
gradient. Fractions containing 110 KD protein elute at approximately 550 mM
salt and
were collected.
3. Collected fractions were applied to S200 Sepharose size
fractionation column in PBS (phosphate buffered saline) buffer. The protein
eluted as a
homogeneous 110 KD protein.
B. 80 KD Extracellular Product
1. The 0-25% ammonium sulfate cut (1 hour at 0 C) was discarded
and the 25%-60% ammonium sulfate cut (overnight at 0 C) was retained as
discussed
above.
2. A DEAE CL-6B column (Pharmacia) was charged with 25 mM
Tris, pH 8.7 containing 1M NaCl and equilibrated with 25 mM Tris, pH 8.7, 10
mM
NaCl and the protein sample was dialyzed against 25 mM Tris, pH 8.7, 10 mM
NaCl
and applied to the column. The column was washed overnight with the same
buffer. A
first salt gradient of 10 mM to 200 mM NaCl in 25 mM Tris, pH 8.7 was run
through
the column to elute other proteins. A second salt gradient (200 to 300 mM
NaCI) was
run through the column and the 80 KD protein eluted at approximately 275 mM
NaCl.
3. A Q-Sepharose HP column was charged with 25 mM Tris, pH
8.7, 1M NaCl and re-equilibrated to 25 mM Tris, pH 8.7, 10 mM NaCl. The
protein
sample was dialyzed against 25 mM Tris, pH 8.7, 10 mM NaCl and applied to the
column. The column was washed in the same buffer and then eluted with 200-300
mM
NaCl in 25 mM Tris, pH 8.7.
4. Fractions containing the 80 KD protein were collected and
dialyzed against 25 mM Tris, pH 8.7, 10 mM NaCl, and then concentrated in a
Speed-
Vac concentrator to 1-2 ml. The protein sample was applied to a Superdex 75
column
and eluted with 25 mM Tris, pH 8.7, 150 mM NaCl. The 80 KD protein eluted as a
homogenous protein.
C. 71 KD Extracellular Product
1. A 40%-95% ammonium sulfate precipitate was obtained as
discussed above with the exception that the 71 KD product was cultured in 7H9
broth at
pH 7.4 and at 0% CO2 and heat-shocked at 42 C for 3h once per week. The
precipitate
was dialyzed against Initial Buffer (20 mM HEPES, 2 mM MgAc, 25 mM KCI, 10 mM
(NH4)2SO4, 0.8 mM DL-Dithiothreitol, pH 7.0).
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2. The resolubilized precipitate was applied to an ATP Agarose
column equilibrated with Initial Buffer. Effluent was collected and reapplied
to the
ATP Agarose column. The 71 KD protein bound to the column.
3. Subsequently the ATP Agarose column was washed, first with
Initial Buffer, then 1 M KCI, then Initial Buffer.
4. Homogeneous 71 KD protein was eluted from the column with
mM ATP and dialyzed against phosphate buffer.
D. 58 KD Extracellular Product
10 1. A 25%-50% ammonium sulfate precipitate was obtained as
discussed above.
2. The resolubilized precipitate was dialyzed and applied to a
DEAE-Sepharose CL-6B or QAE-Sepharose column and eluted with NaCl. Collected
fractions containing the 58 KD Protein eluted at approximately 400 mM NaCl.
3. Collected fractions were then applied to a Sepharose CL-6B size
fractionation column. The protein eluted at approximately 670-700,000 Daltons.
4. The eluted protein was applied to a thiopropyl-sepharose column.
The homogeneous 58 KD protein eluted at approximately 250-350 MM
2-mercaptoethanol. The eluted protein was monitored using SDS-PAGE and
exhibited
the single band shown in Figure 1 A, co 1. 2.
E. 45 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
2.5 mM Tris, pH 8.7 containing 1 M NaCl and equilibrated with 25 mM Tris, 10
mM
NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to column. The column was then washed overnight
with the same buffer.
c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 45 KD protein eluted at
approximately
40 mM NaCl.
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3. a. A Q-Sepharose HP (Pharmacia) column was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and re-equilibrated with 25 mM Tris, 10
mM
NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
5 10 mM NaCl, pH 8.7 and applied to column with subsequent washing using the
same
buffer.
c. The column was eluted with 10-150 mM NaCl in 25 mM
Tris, pH 8.7.
4. a. Fractions containing the 45 KD product were collected,
10 pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentration to
1 ml in a Speed Vac concentrator.
b. Concentrate was Applied to Superdex 75 column
equilibrated with 25 mM Tris 150 mM NaCl, pH 8.7. The product eluted as a
homogeneous protein. The eluted protein was monitored using SDS-PAGE and
15 resulted in the single band shown in Figure 1 B, co 1. 2.
F. 32 KD Extracellular Product (A)
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
20 b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
25 b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing overnight
with same buffer.
c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 32 KD protein eluted at
approximately
70 mM NaCl.
3. a. Fractions containing the 32 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then Applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with this
buffer. The 32 KD product eluted as homogeneous protein.
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4. a. A Q-Sepharose HP column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl, and re-equilibrated with 25 mM Tris,
10 mM
NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing in the
same
buffer.
c. The column was eluted with a 100-300 mM NaCl
gradient. Labeled 32A, the homogeneous protein elutes at approximately 120 mM
NaCl and is shown as a single band in Figure 1B, co 1.4.
G. 32 KD Extracellular Product (B)
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
20 10 mM NaCl, pH 8.7 and applied to the column with subsequent washing
overnight
with same buffer.
c. A preliminary salt gradient of 10 mM to 200 mM NaCl in
25 mM Tris, pH 8.7 was run, eluting various proteins. Following column
equilibration,
a second salt gradient (200 to 300 mM NaCI) was run. The 32 KD protein eluted
at
25 approximately 225 mM NaCl.
3. a. A Q-Sepharose HP column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl, and re-equilibrated with 25 mM Tris,
10 mM
NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing in the
same
buffer.
c. The column was eluted with a 200-300 mM NaCl
gradient in the same buffer.
4. a. Fractions containing the 32 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
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b. The concentrate was then applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with the
same
buffer. The 32 KD product, labeled 32B to distinguish it from the protein of
32 KD
separated using protocol H, eluted as homogeneous protein and is shown as a
single
band on Figure 1 B, co 1. 3.
H. 30 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing overnight
with same buffer.
c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 30 KD protein eluted at
approximately
140 mM NaCl.
3. a. Fractions containing the 30 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then Applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with this
buffer. The 30 KD product eluted as homogeneous protein and is shown as a
single
band on Figure 1 B, co 1. 5.
1. 24 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
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b. The protein sample was dialyzed against 25 mM Tris,
mM NaCl, pH 8.7 and applied to the column with subsequent washing overnight
with same buffer.
c. A preliminary salt gradient of 10 mM to 200 mM NaCl in
5 25 mM Tris, pH 8.7 was run, eluting various proteins. Following column
equilibration
a second salt gradient (200 to 300 mM NaCI) was run. The 24 KD elutes at
approximately 250 mM NaCl.
3. a. A Q-Sepharose HP column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl, and re-equilibrated with 25 mM Tris,
10 mM
10 NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing in the
same
buffer.
c. The column was eluted with a 200-300 mM NaCl
gradient in the same buffer.
4. a. Fractions containing the 24 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with the
same
buffer. The 24 KD product eluted as homogeneous protein and is shown as a
single
band on Figure 1B, column 7.
J. 23.5 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25%-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column prior to subsequent washing
overnight
with same buffer.
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c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 23.5 KD protein eluted at
approximately
80 mM NaCl.
3. a. A Q-Sepharose HP column was charged with 25 mM
Tris, pH 8.7 containing 1 M NaCl, and re-equilibrated with 25 mM Tris, 10 mM
NaCl,
pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
mM NaCl, pH 8.7 and applied to the column with subsequent washing in the same
buffer.
10 c. The column was eluted with 100-300 mM NaCl in
25 mM Tris, pH 8.7.
d. Steps 3a to 3c were repeated.
4. a. Fractions containing 23.5 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with the
same
buffer. The 23.5 KD product eluted as homogeneous protein. The eluted protein
was
monitored using SDS-PAGE and resulted in the single band shown in Figure 1B,
column 6.
K. 23 KD Extracellular Product
1. a. Ammonium sulfate cuts of 0-25% (lh at 0 C) and 25%-
60% (overnight at 0 C) were discarded.
b. A 60%-95% ammonium sulfate cut was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
50 mM Bis-Tris pH 7.0 containing 1 M NaCl and equilibrated with 50 mM Bis-
Tris,
100 mM NaCl, pH 7Ø
b. The protein sample was dialyzed against 50 mM Bis-Tris,
pH 7.0, 100 mM NaCl buffer and applied to the column before washing the column
overnight with the same buffer.
c. The column was eluted with a 100 to 300 mM NaCl linear
gradient in 50 mM Bis-Tris pH 7Ø
d. Fractions were collected containing the 23 KD protein
which eluted at approximately 100-150 mM NaCl.
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3. a. The protein fractions were dialyzed against 25 mM Tris,
pH 8.7, 10 mM NaCl and concentrated to 1-2 ml on a Savant Speed Vac
Concentrator.
b. The concentrate was applied to a Superdex 75 column
equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7. The product elutes as a
5 homogeneous protein as is shown in Figure 1B col. 8.
L. 16 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
10 b. The 25-60% ammonium sulfate cut (overnight at 0 C)
was retained.
2. a. A DEAE CL-6B column (Pharmacia) was charged with
2.5 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
10 mM NaCl, pH 8.7.
15 b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing overnight
in
the same buffer.
c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 16 KD protein eluted at
approximately
20 50 mM NaCl.
3. a. Fractions containing 16 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then applied to a Superdex 75
25 column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with
the same
buffer. A 16 KD product eluted as homogeneous protein. The eluted protein was
monitored using SDS-PAGE and resulted in the single band shown in Figure I B,
col. 9.
30 M. 14 KD Extracellular Product
1. a. A 0-25% ammonium sulfate cut (1 hour at 0 C) was
discarded.
b. The 25-60% ammonium sulfate cut (overnight at 0 C)
was retained.
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2. a. A DEAE CL-6B column (Pharmacia) was charged with
25 mM Tris, pH 8.7 containing 1 M NaCl and then equilibrated with 25 mM Tris,
mM NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
5 10 mM NaCl, pH 8.7 and applied to the column with subsequent washing
overnight in
the same buffer.
c. The column was eluted with a salt gradient (10 mM to
200 mM) in 25 mM Tris, pH 8.7 buffer. The 14 KD protein eluted at
approximately
60 mM NaCl.
10 3. a. A Q-Sepharose HP column was charged with 25 mM
Tris, pH 8.7 containing 1 M NaCl, and re-equilibrated with 25 mM NaCl, pH 8.7.
b. The protein sample was dialyzed against 25 mM Tris,
10 mM NaCl, pH 8.7 and applied to the column with subsequent washing in the
same
buffer.
c. The column was eluted with 10-150 mM NaCl in 25 mM
Tris, pH 8.7.
d. Steps 3a through 3c were repeated.
4. a. Fractions containing 14 KD product were collected,
pooled and dialyzed against 25 mM Tris, 10 mM NaCl, pH 8.7, before
concentrating the
protein sample to 1 ml in a Speed-Vac Concentrator.
b. The concentrate was then applied to a Superdex 75
column equilibrated with 25 mM Tris, 150 mM NaCl, pH 8.7 and eluted with this
buffer. The 14 KD product eluted as homogeneous protein. The eluted protein
was
monitored using SDS-PAGE and resulted in the single band shown in Figure 1 C,
column 2.
N. 12 KD Extracellular Products
1. A 0-10% ammonium sulfate precipitate was obtained (overnight
at 4 C).
2. The resolubilized precipitate was applied to a S200 Sephacryl
size fractionation column eluting the protein as a 12 KD molecule.
3. The protein fractions were applied to a DEAE-Sepharose CL-6B
or QAE-Sepharose ion exchange column and eluted with an NaCl gradient as
previously described. Fractions containing two homogeneous proteins having
molecular weights of approximately 12 KD eluted at approximately 300-350 mM
NaCl
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and were collected. The proteins were labeled 12A and 12B and purified as a
doublet
shown in Figure 1 D, co 1. 2.
As illustrated in the SDS-PAGE profile of Figure 1, the principal or
majorly abundant extracellular proteins of M tuberculosis were purified to
homogeneity through the use of the protocols detailed in Examples 2A - 2N
above.
More particularly, Figure 1 illustrates four exemplary 12.5% acrylamide gels
developed
using SDS-PAGE and labeled 1 A, I B, IC, and ID. The standard in lane 1 of
gels IA-
1 C has proteins with molecular weights of 66, 45, 36, 29, 24, 20, and 14 KD.
In gel 1 D
the standard in lane 1 contains proteins with molecular weights of 68, 45, 31,
29, 20,
and 14 KD. The lanes containing the respective purified extracellular products
show
essentially one band at the reported molecular weight of the individual
protein. It
should be noted that in gel 1 D the 12 KD protein runs as a doublet visible in
lane 2.
Sequence analysis shows that the lower 12 KD (or 12B KD band) is equivalent to
the
upper 12 KD (or 12A KD) band except that it lacks the first 3 N-terminal amino
acids.
Further analysis of these individual exemplary majorly abundant
extracellular products is provided in Figure 2. More particularly Figure 2 is
a tabular
compilation of N-terminal sequence data obtained from these purified
extracellular
products showing that the majority of the isolated products are indeed
distinct
(Sequence ID Nos. 1-14). Proteins 32A, 32B and 30 all had the same 5 N-
terminal
amino acids therefore further sequencing was necessary to fully characterize
and
differentiate them. Figure 3 shows the extended N-terminal amino acid
sequences for
these three purified secretory products (Sequence ID Nos. 15-17). Different
amino
acids at positions 16 (Sequence ID No. 17), 31 (Sequence ID No. 16) and 36
(Sequence
ID No. 16) demonstrate that these isolated proteins are distinct from one
another despite
their similarity in molecular weight.
In addition to proteins 30, 32A and 32B, extended N-terminal amino acid
sequences of other majorly abundant extracellular products were determined to
provide
primary structural data and to uncover possible relationships between the
proteins.
Sequencing was performed on the extracellular products purified according to
Example
2 using techniques well known in the art. Varying lengths of the N-terminal
amino acid
sequence, determined for each individual extracellular product, are shown
below
identified by the apparent molecular weight of the intact protein, and
represented using
standard one letter abbreviations for the naturally occurring amino acids. In
keeping
with established rules of notation, the N-terminal sequences are written left
to right in
the direction of the amino terminus to the carboxy terminus. Those positions
where the
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identity of the determined amino acid is less than certain are underlined.
Where the
amino acid at a particular position is unknown or ambiguous, the position in
the
sequence is represented by a dash. Finally, where two amino acids are
separated by a
slash, the correct constituent has not been explicitly identified and either
one may
occupy the position in that sequence.
PROTEIN N-TERMINAL AMINO ACID SEQUENCE
5 10 15 20 25 30 35
12 KD FDTRL MRLED EMKEG RYEVR AELPG VDPDK DVDIM
40 45
VRDGQ LTIKA ERT
(Sequence ID No. 18)
5 10 15 20 25 30
14 KD ADPRL QFTAT TLSGA PFDGA S/NLQGK PAVLW
(Sequence ID Nos. 19 and 20)
5 10 15 20 25 30
16 KD AYPIT GKLGS ELTMT DTVGQ VVLGW KVSDL
40 45
30 F/YKSTA VIPGY TV-EQ QI
(Sequence ID Nos. 21 and 22)
5 10 15 20
23 KD AETYL PDLDW DYGAL EPHIS GQ
(Sequence ID No. 23)
5 10
23.5 KD APKTY -EELK GTD
(Sequence ID No. 24)
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10 15 20 25 30 35
24 KD APYEN LMVPS PSMGR DIPVA FLAGG PHAVY LLDAF
40 45 50 55 60
5 NAGPD VSNWV TAGNA MMTLA -KGIC/S
(Sequence ID Nos. 25 and 26)
5 10 15 20 25 30 35
30 KD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG NNSPA
15 VYLLD
(Sequence ID No. 27)
5 10 15 20 25 30 35 40
32A KD FSRPG LPVEY LQVPS PSMGR DIKVQ FQSGG ANSP- LYLLD
(Sequence ID No. 28)
5 10 15 20
32B KD FSRPG LPVEY LQVPS A-MGR DI
(Sequence ID No. 29)
5 10 15 20 25 30
KD DPEPA PPVPD DAASP PDDAA APPAP ADPP-
(Sequence ID No. 30)
5 10 15 20
58 KD TEKTP DDVFK LAKDE KVLYL
(Sequence ID No. 31)
5
71 KD ARAVG I
(Sequence ID No. 32)
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80 KD TDRVS VGN
5 (Sequence ID No. 33)
5 10 15 20
10 110 KD NSKSV NSFGA HDTLK V-ERK RQ
(Sequence ID No. 34)
DNA sequencing was performed on the 30, 32A, 16, 58, 23.5, and 24
KD proteins using techniques well known in the art. These DNA sequences, and
the
corresponding amino acids, including upstream and downstream sequences, are
shown
below identified by the apparent molecular weight of the intact protein and
represented
using standard abbreviations and rules of notation.
KD DNA SEQUENCE
1/1 31/11
ATG ACA GAC GTG AGC CGA AAG ATT CGA GCT TGG GGA CGC CGA
25 met thr asp val ser arg lys ile arg ala trp gly arg arg
61/21
TTG ATG ATC GGC ACG GCA GCG GCT GTA GTC CTT CCG GGC CTG
leu met ile gly thr ala ala ala val val leu pro gly leu
91/31
30 GTG GGG CTT GCC GGC GGA GCG GCA ACC GCG GGC GCG
val gly leu ala gly gly ala ala thr ala gly ala
121/41 151/51
TTC TCC CGG CCG GGG CTG CCG GTC GAG TAC CTG CAG GTG CCG
phe ser arg pro gly leu pro val glu tyr leu gln val pro
181/61
TCG CCG TCG ATG GGC CGC GAC ATC AAG GTT CAG TTC CAG AGC
ser pro ser met gly arg asp ile lys val gln phe gln ser
211/71 241/81
GGT GGG AAC AAC TCA CCT GCG GTT TAT CTG CTC GAC GGC CTG
giy gly asn asn ser pro ala val tyr leu leu asp gly leu
271/91
CGC GCC CAA GAC GAC TAC AAC GGC TGG GAT ATC AAC ACC CCG
arg ala gin asp asp tyr asn gly trp asp ile asn thr pro
301/101
GCG TTC GAG TGG TAC TAC CAG TCG GGA CTG TCG ATA GTC ATG
ala phe glu trp tyr tyr gin ser gly leu ser ile val met
331/111 361/121
CCG GTC GGC GGG CAG TCC AGC TTC TAC AGC GAC TGG TAC AGC
pro val gly gly gin ser ser phe tyr ser asp trp tyr ser
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391/131
CCG GCC TGC GGT AAG GCT GGC TGC CAG ACT TAC AAG TGG GAA
pro ala cys gly lys ala gly cys gln thr tyr lys trp glu
421/141 451/151
ACC TTC CTG ACC AGC GAG CTG CCG CAA TGG TTG TCC GCC AAC
thr phe leu thr ser glu leu pro gln trp leu ser ala asn
481/161
AGG GCC GTG AAG CCC ACC GGC AGC GCT GCA ATC GGC TTG TCG
arg ala val lys pro thr gly ser ala ala ile gly leu ser
511/171
ATG GCC GGC TCG TCG GCA ATG ATC TTG GCC GCC TAC CAC CCC
met ala gly ser ser ala met ile leu ala ala tyr his pro
541/181 571/191
CAG CAG TTC ATC TAC GCC GGC TCG CTG TCG GCC CTG CTG GAC
gln gin phe ile tyr ala gly ser leu ser ala leu leu asp
601/201
CCC TCT CAG GGG ATG GGG CCT AGC CTG ATC GGC CTC GCG ATG
pro ser gln gly met gly pro ser leu ile gly leu ala met
631/211 661/221
GGT GAC GCC GGC GGT TAC AAG GCC GCA GAC ATG TGG GGT CCC
gly asp ala gly gly tyr lys ala ala asp met trp gly pro
691/231
TCG AGT GAC CCG GCA TGG GAG CGC AAC GAC CCT ACG CAG CAG
ser ser asp pro alatrp glu arg asn asp pro thr gln gln
21/241
ATC CCC AAG CTG GTC GCA AAC AAC ACC CGG CTA TGG GTT TAT
ile pro lys leu val ala asn asn thr arg leu trp val tyr
751/251 781/261
TGC GGG AAC GGC ACC CCG AAC GAG TTG GGC GGT GCC AAC ATA
cys gly asn gly thr pro asn glu leu gly gly ala asn ile
811/271
CCC GCC GAG TTC TTG GAG AAC TTC GTT CGT AGC AGC AAC CTG
pro ala glu phe leu glu asn phe val arg ser ser asn leu
841/281 871/291
AAG TTC CAG GAT GCG TZC AAC GCC GCG GGC GGG CAC AAC GCC
lys phe gln asp ala tyr asn ala ala gly gly his asn ala
901/301
GTG TTC AAC TTC CCG CCC AAC GGC ACG CAC AGC TGG GAG TAC
val phe asn phe pro pro asn gly thr his ser trp glu tyr
931/311
TGG GGC GCT CAG CTC AAC GCC ATG AAG GGT GAC CTG CAG AGT
trp/g3l2y ala gin leu asn ala met lys gly asp leu gin ser
TCG TTA GGC GCC GGC TGA
ser leu gly ala gly OPA (Sequence ID No. 35)
32A KD DNA SEQUENCE
1/1 31/11
ATG CAG CTT GTT GAC AGG GTT CGT GGC GCC GTC ACG GGT ATG
met gln leu val asp arg val arg gly ala val thr gly met
61/21
TCG CGT CGA CTC GTG GTC GGG CCC CTC CCC CCG GCC CTA CTG
ser arg arg leu val val gly ala val gly ala ala leu val
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91/31 121/41
TCC GGT CTG GTC GGC GCC GTC GGT GGC ACG GCG ACC GCG GGG
ser gly leu val gly ala val gly gly thr ala thr ala gly
151/51
GCA TTT TCC CGG CCG GGC TTG CCG GTG GAG TAC CTG CAG GTG
ala phe ser arg pro gly leu pro val glu tyr leu gln val
181/61
CCG TCG CCG TCG ATG GGC CGT GAC ATC AAG GTC CAA TTC CAA
pro ser pro ser met gly arg asp ile lys val gln phe gln
211/71 241/81
AGT GGT GGT GCC AAC TCG CCC GCC CTG TAC CTG CTC GAC GGC
ser gly gly ala asn ser pro ala leu tyr leu leu asp gly
271/91
CTG CGC GCG CAG GAC GAC TTC AGC GGC TGG GAC ATC AAC ACC
leu arg ala gln asp asp phe ser gly trp asp ile asn thr
301/101 331/111
CCG GCG TTC GAG TCC TAC GAC CAG TCG GGC CTG TCG GTG GTC
pro ala phe glu trp tyr asp gln ser gly leu ser val val
361/121
ATG CCG GTG GGT GGC CAG TCA AGC TTC TAC TCC GAC TGG TAC
met pro val gly gly gin ser ser phe tyr ser asp trp tyr
391/131
CAG CCC GCC TGC GGC AAG GCC GGT TGC CAG ACT TAC AAG TGG
gln pro ala cys gly lys ala gly cys gin thr tyr lys trp
421/141 451/151
GAG ACC TTC CTG ACC ACC CAC CTC CCC GGG TGG CTC CAC CCC
glu thr phe leu thr ser glu leu pro gly trp leu gln ala
481/161
AAC AGG CAC GTC AAG CCC ACC GGA AGC GCC GTC TGC GGT CTT
asn arg his val lys pro thr gly ser ala val val gly leu
511/171 541/181
TCG ATG GCT GCT TCT TCG GCG CTG ACG CTG GCG ATC TAT CAC
ser met ala ala ser ser ala leu thr leu ala ile tyr his
571/191
CCC CAG CAG TTC GTC TAC GCG GGA GCG ATG TCG GGC CTG TTG
pro gln gln phe val tyr ala gly ala met ser gly leu leu
601/201
GAC CCC TCC CAG GCG ATG GGT CCC ACC CTG ATC GGC CTG GCG
asp pro ser gln ala met gly pro thr leu ile gly leu ala
631/211 661/221
ATG GGT GAC GCT GGC GGC TAC AAG GCC TCC GAC ATG TGG GGC
met gly asp ala gly gly tyr lys ala ser asp met trp gly
691/231
CCG AAG GAG GAC CCG GCG TGG CAG CGC AAC GAC CCG CTG TTG
pro lys glu asp pro ala trp gln arg asn asp pro leu leu
721/241 751/251
AAC GTC GGG AAG CTG ATC GCC AAC AAC ACC CGC GTC TGG GTG
asn val gly lys leu ile ala asn asn thr arg val trp val
781/261
TAC TGC GGC AAC GGC AAG CCG TCG GAT CTG GGT GGC AAC AAC
tyr cys gly asn gly lys pro ser asp leu gly gly asn asn
811/271
CTG CCG GCC AAG TTC CTC GAG GGC TTC GTG CGG ACC AGC AAC
leu pro ala lys phe leu glu gly phe val arg thr ser asn
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38
841/281 871/291
ATC AAG TTC CAA GAC GCC TAC AAC GCC GGT GGC GGC CAC AAC
ile lys phe gln asp ala tyr asn ala gly gly gly his asn
901/301
GGC GTG TTC GAC TTC CCG GAC AGC GGT ACG CAC AGC TGG GAG
gly val phe asp phe pro asp ser gly thr his ser trp glu
931/311 961/321
TAC TGG GGC GCG CAG CTC AAC GCT ATG AAG CCC GAC CTG CAA
tyr trp gly ala gln leu asn ala met lys pro asp leu gln
991/331
CGG GCA CTG GGT GCC ACG CCC AAC ACC GGG CCC GCG CCC CAG
arg ala leu gly ala thr pro asn thr gly pro ala pro gln
GGC GCC TAG
gly ala AMB (Sequence ID No. 36)
16 KD DNA SEQUENCE
1/1 31/11
atg AAG CTC ACC ACA ATG ATC AAG ACG GCA GTA GCG GTC GTG GCC atg GCG GCC ATC
GCG
Met lys leu thr thr met ile lys thr ala val ala val val ala met ala ala ile
ala
61/21 91/31
ACC TTT GCG GCA CCG GTC GCG TTG GCT GCC TAT CCC ATC ACC GGA AAA CTT GGC AGT
GAG
thr phe ala ala pro val ala leu ala ala tyr pro ile thr gly lys leu gly ser
glu
121/41 151/51
CTA ACG ATG ACC GAC ACC GTT GGC CAA GTC GTG CTC GGC TGG AAG GTC AGT GAT CTC
AAA
leu thr met thr asp thr val gly gln val val leu gly trp lys val ser asp leu
lys
181/61 . 211/71
TCC AGC ACG GCA GTC ATC CCC GGC TAT CCG GTG GCC GGC CAG GTC TGG GAG GCC ACT
GCC
ser ser thr ala val ile pro gly tyr pro val ala gly gln val trp glu ala thr
ala
241/81 271/91
ACG GTC AAT GCG ATT CGC GGC AGC GTC ACG CCC GCG GTC TCG CAG TTC AAT GCC CGC
ACC
thr val asn ala ile arg gly ser val thr pro ala val ser gln phe asn ala arg
thr
301/101 331/111
GCC GAC GGC ATC AAC TAC CGG GTG CTG TGG CAA GCC GCG GGC CCC GAC ACC ATT AGC
GGA
ala asp gly ile asn tyr arg val leu trp gln ala ala gly pro asp thr ile ser
gly
361/121 391/131
GCC ACT ATC CCC CAA GGC GAA CAA TCG ACC GGC AAA ATC TAC TTC GAT GTC ACC GGC
CCA
ala thr ile pro gln gly glu gln ser thr gly lys ile tyr phe asp val thr gly
pro
421/141 451/151
TCG CCA ACC ATC GTC GCG ATG AAC AAC GGC ATG GAG GAT CTG CTG ATT TGG GAG CCG
TAG
ser pro thr ile val ala met asn asn gly met glu asp leu leu ile trp glu pro
AMB
(Sequence ID No. 92)
CA 02222000 1997-11-24
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39
58 KD DNA SEQUENCE
1/1 31/11
gtg ACG GAA AAG ACG CCC GAC GAC GTC TTC AAA CTT GCC AAG GAC GAG AAG GTC GAA
TAT
val thr glu lys thr pro asp asp val phe lys leu ala lys asp glu lys val glu
tyr
61/21 91/31
GTC GAC GTC CGG TTC TGT GAC CTG CCT GGC ATC ATG CAG CAC TTC ACG ATT CCG GCT
TCG
val asp val arg phe cys asp leu pro gly ile met gln his phe thr ile pro ala
ser
121/41 151/51
GCC TTT GAC AAG AGC GTG TTT GAC GAC GGC TTG GCC TTT GAC GGC TCG TCG ATT CGC
GGG
ala phe asp lys ser val phe asp asp gly leu ala phe asp giy ser ser ile arg
gly
181/61 211/71
TTC CAG TCG ATC CAC GAA TCC GAC ATG TTG CTT CTT CCC GAT CCC GAG ACG GCG CGC
ATC
phe gln ser ile his glu ser asp met leu leu leu pro asp pro glu thr ala arg
ile
241/81 271/91
GAC CCG TTC CGC GCG GCC AAG ACG CTG AAT ATC AAC TTC TTT GTG CAC GAC CCG TTC
ACC
asp pro phe arg ala ala lys thr leu asn ile asn phe phe val his asp pro phe
thr
301/101 331/111
CTG GAG CCG TAC TCC CGC GAC CCG CGC AAC ATC GCC CGC AAG GCC GAG AAC TAC CTG
ATC
leu glu pro tyr ser arg asp pro arg asn ile ala arg lys ala glu asn tyr leu
ile
361/121 391/131
AGC ACT GGC ATC GCC GAC ACC GCA TAC TTC GGC GCC GAG GCC GAG TTC TAC ATT TTC
GAT
ser thr gly ile ala asp thr ala tyr phe gly ala glu ala glu phe tyr ile phe
asp
421/141 451/151
TCG GTG AGC TTC GAC TCG CGC GCC AAC GGC TCC TTC TAC GAG GTG GAC GCC ATC TCG
GGG
ser val ser phe asp ser arg ala asn gly ser phe tyr glu val asp ala ile ser
gly
481/161 511/171
TGG TGG AAC ACC GGC GCG GCG ACC GAG GCC GAC GGC AGT CCC AAC CGG GGC TAC AAG
GTC
trp trp asn thr gly ala ala thr glu ala asp gly ser pro asn arg gly tyr lys
val
541/181 571/191
CGC CAC AAG GGC GGG TAT TTC CCA GTG GCC CCC AAC GAC CAA TAC GTC GAC CTG CGC
GAC
arg his lys gly gly tyr phe pro val ala pro asn asp gln tyr val asp leu arg
asp
601/201 631/211
AAG ATG CTG ACC AAC CTG ATC AAC TCC GGC TTC ATC CTG GAG AAG GGC CAC CAC GAG
GTG
iys met leu thr asn leu ile asn ser gly pheli e leu glu lys gly his his glu
val
691/231
GGC AGC GGC GGA CAG GCC GAG ATC AAC TAC CAG TTC AAT TCG CTG CTG CAC GCC GCC
GAC
gly ser gly gly gln ala glu ile asn tyr gln phe asn ser leu leu his ala ala
asp
721/241 751/251
GAC ATG CAG TTG TAC AAG TAC ATC ATC AAG AAC ACC GCC TGG CAG AAC GGC AAA ACG
GTC
asp met gln leu tyr lys tyr ile ile lys asn thr ala trp gln asn gly lys thr
val
781/261 811/271
ACG TTC ATG CCC AAG CCG CTG TTC GGC GAC AAC GGG TCC GGC ATG CAC TGT CAT CAG
TCG
thr phe met pro lys pro leu phe gly asp asn gly ser gly met his cys his gln
ser
841/281 871/291
CTG TGG AAG GAC GGG GCC CCG CTG ATG TAC GAC GAG ACG GGT TAT GCC GGT CTG TCG
GAC
leu trp lys asp gly ala pro leu met tyr asp glu thr gly tyr ala gly leu ser
asp
901/301 931/311
ACG GCC CGT CAT TAC ATC GGC GGC CTG TTA CAC CAC GCG CCG TCG CTG CTG GCC TTC
ACC
thr ala arg his tyr ile gly gly leu leu his his ala pro ser leu leu ala phe
thr
961/321 991/331
AAC CCG ACG GTG AAC TCC TAC AAG CGG CTG GTT CCC GGT TAC GAG GCC CCG ATC AAC
CTG
asn pro thr val asn ser tyr lys arg leu val pro gly tyr glu ala pro ile asn
leu
1021/341 1051/351
GTC TAT AGC CAG CGC AAC CGG TCG GCA TGC GTG CGC ATC CCG ATC ACC GGC AGC AAC
CCG
val tyr ser gln arg asn arg ser ala cys val arg ile pro ile thr gly ser asn
pro
1081/361 1111/371
AAG GCC AAG CGG CTG GAG TTC CGA AGC CCC GAC TCG TCG GGC AAC CCG TAT CTG GCG
TTC
lys ala lys arg leu glu phe arg ser pro asp ser ser gly asn pro tyr leu ala
phe
1141/381 1171/391
TCG GCC ATG CTG ATG GCA GGC CTG GAC GGT ATC AAG AAC AAG ATC GAG CCG CAG GCG
CCC
ser ala met leu met ala gly leu asp gly ile lys asn lys ile glu pro gln ala
pro
1201/401 1231/411
GTC GAC AAG GAT CTC TAC GAG CTG CCG CCG GAA GAG GCC GCG AGT ATC CCG CAG ACT
CCG
val asp lys asp leu tyr glu leu pro pro glu glu ala ala ser ile pro gln thr
pro
1261/921 1291/431
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ACC CAG CTG TCA GAT GTG ATC GAC CGT CTC GAG GCC GAC CAC GAA TAC CTC ACC GAA
GGA
thr gln leu ser asp val ile asp arg leu glu ala asp his glu tyr leu thr glu
gly
1321/441 1351/451
GGG GTG TTC ACA AAC GAC CTG ATC GAG ACG TGG ATC AGT TTC AAG CGC GAA AAC GAG
ATC
5 gly val phe thr asn asp leu ile glu thr trp ile ser phe lys arg glu asn glu
ile
1381/461 1411/471
GAG CCG GTC AAC ATC CGG CCG CAT CCC TAC GAA TTC GCG CTG TAC TAC GAC GTT taa
glu pro val asn ile arg pro his pro tyr glu phe ala leu tyr tyr asp val OCH
10 (Sequence ID No. 93)
23.5 KD DNA SEQUENCE
1/1 31/11
gtg CGC ATC AAG ATC TTC ATG CTG GTC ACG GCT GTC GTT TTG CTC TGT TGT TCG GST
GIG
15 val arg ile lys ile phe met leu val thr ala val val leu leu cys cys ser gly
val
61/21 91/31
GCC ACG GCC GCG CCC AAG ACC TAC TGC GAG GAG TTG AAA GGC ACC GAT ACC GGC CAG
GCG
ala thr ala ala pro lys thr tyr cys glu glu leu lys gly thr asp thr gly gln
ala
121/41 151/51
20 TGC CAG ATT CAA ATG TCC GAC CCG GCC TAC AAC ATC AAC ATC AGC CTG CCC AGT TAC
TAC
cys gln ile gln met ser asp pro ala tyr asn lie asn ile ser leu pro ser tyr
tyr
181/61 211/71
CCC GAC CAG AAG TCG CTG GAA AAT TAC ATC GCC CAG ACG CGC GAC AAG TTC CTC AGC
GCG
pro asp gln lys ser leu glu asn tyr ile ala gln thr arg asp lys phe leu ser
ala
25 241/81 271/91
GCC ACA TCG TCC ACT CCA CGC GAA GCC CCC TAC GAA TTG AAT ATC ACC TCG GCC ACA
TAC
ala thr ser ser thr pro arg glu ala pro tyr glu leu asn ile thr ser ala thr
tyr
301/101 331/111
CAG TCC GCG ATA CCG CCG CGT GGT ACG CAG GCC GTG GTG CTC AAG GTC TAC CAG AAC
GCC
30 gln ser ala ile pro pro arg gly thr gln ala val val leu lys val tyr gln asn
ala
361/121 391/131
GGC GGC ACG CAC CCA ACG ACC ACG TAC AAG GCC TTC GAT TGG GAC CAG GCC TAT CGC
AAG
gly gly thr his pro thr thr thr tyr lys ala phe asp trp asp gln ala tyr arg
lys
421/141 451/151
35 CCA ATC ACC TAT GAC ACG CTG TCG CAG GCT GAC ACC GAT CCG CTG CCA GTC GTC TTC
CCC
pro ile thr tyr asp thr leu trp gln ala asp thr asp pro leu pro val val phe
pro
481/161 511/171
ATT GTG CAA GGT GAA CTG AGC AAG CAG ACC GGA CAA CAG GTA TCG ATA GCG CCG AAT
GCC
40 ile val gln gly giu leu ser lys gin thr 571/gin n gin val ser ile ala pro
asn ala
541/181 GGC TTG GAC CCG GTG AAT TAT CAG AAC TTC GCA GTC ACG AAC GAC GGG GTG
ATT TTC TTC
gly leu asp pro val asn tyr gln asn phe ala val thr asn asp gly val ile phe
phe
601/201 631/211
TTC AAC CCG GGG GAG TTG CTG CCC GAA GCA GCC GGC CCA ACC CAG GTA TTG GTC CCA
CGT
phe asn pro gly glu leu leu pro glu ala ala gly pro thr gln val leu val pro
arg
661/221
TCC GCG ATC GAC TCG ATG CTG GCC tag
ser ala ile asp ser met leu ala AMB
(Sequence ID No. 94)
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41
24 KD DNA SEQUENCE
1/l 31/11
ATG AAG GGT CGG TCG GCG CTG CTG CGG GCG CTC TGG ATT GCC GCA CTG TCA TTC GGG
TTG
Met lys gly arg ser ala leu leu arg ala leu trp ile ala ala leu ser phe gly
leu
61/21 91/31
GGC GGT GTC GCG GTA GCC GCG GAA CCC ACC GCC AAG GCC GCC CCA TAC GAG AAC CTG
ATG
gly gly val ala val ala ala glu pro thr ala lys ala ala pro tyr glu asn leu
met
121/41 151/51
GTG CCG TCG CCC TCG ATG GGC CGG GAC ATC CCG GTG GCC TTC CTA GCC GGT GGG CCG
CAC
val pro ser pro ser met gly arg asp ile pro val ala phe leu ala gly gly pro
his
181/61 211/71
GCG GTG TAT CTG CTG GAC GCC TTC AAC GCC GGC CCG GAT GTC AGT AAC TGG GTC ACC
GCG
ala val tyr leu leu asp ala phe asn ala gly pro asp val ser asn trp val thr
ala
241/81 271/91
GGT AAC GCG ATG AAC ACG TTG GCG GGC AAG GGG ATT TCG GTG GTG GCA CCG GCC GGT
GGT
gly asn ala met asn thr leu ala gly lys gly ile ser val val ala pro ala gly
gly
301/101 331/111
GCG TAC AGC ATG TAC ACC AAC TGG GAG CAG GAT GGC AGC AAG CAG TGG GAC ACC TTC
TTG
ala tyr ser met tyr thr asn trp glu gin asp gly ser lys gin trp asp thr phe
leu
361/121 391/131
TCC GCT GAG CTG CCC GAC TGG CTG GCC GCT AAC CGG GGC TTG GCC CCC GGT GGC CAT
GCG
ser ala glu leu pro asp trp leu ala ala asn arg gly leu ala pro gly gly his
ala
421/141 451/151
GCC GTT GGC GCC GCT CAG GGC GGT TAC GGG GCG ATG GCG CTG GCG GCC TTC CAC CCC
GAC
ala val gly ala ala gln gly gly tyr gly ala met ala leu ala ala phe his pro
asp
481/161 511/171
CGC TTC GGC TTC GCT GGC TCG ATG TCG GGC TTT TTG TAC CCG TCG AAC ACC ACC ACC
AAC
arg phe gly phe ala gly ser met ser gly phe leu tyr pro ser asn thr thr thr
asn
541/181 571/191
GGT GCG ATC GCG GCG GGC ATG CAG CAA TTC GGC GGT GTG GAC ACC AAC GGA ATG TGG
GGA
gly ala ile ala ala gly met gln gln phe gly gly val asp thr asn gly met trp
gly
601/201 631/211
GCA CCA CAG CTG GGT CGG TGG AAG TGG CAC GAC CCG TGG GTG CAT GCC AGC CTG CTG
GCG
ala pro gln leu gly arg trp lys trp his asp pro trp val his ala ser leu leu
ala
661/221 691/231
CAA AAC AAC ACC CGG GTG TGG GTG TGG AGC CCG ACC AAC CCG GGA GCC AGC GAT CCC
GCC
gln asn asn thr arg val trp val trp ser pro thr asn pro gly ala ser asp pro
ala
721/241 751/251
GCC ATG ATC GGC CAA GCC GCC GAG GCG ATG GGT AAC AGC CGC ATG TTC TAC AAC CAG
TAT
ala mer ile gly gin ala ala glu ala met gly asn ser arg met phe tyr asn gin
tyr
781/261 811/271
CGC AGC GTC GGC GGG CAC AAC GGA CAC TTC GAC TTC CCA GCC AGC GGT GAC AAC GGC
TGG
arg ser val gly gly his asn gly his phe asp phe pro ala ser gly asp asn gly
trp
841/281 871/291
GGC TCG TGG GCG CCC CAG CTG GGC GCT ATG TCG GGC GAT ATC GTC GGT GCG ATC CGC
TAA
gly ser trp ala pro gin leu gly ala met ser gly asp ile val gly ala ile arg
OCH
(Sequence ID No. 95)
This sequence data, combined with the physical properties ascertained
using SDS-PAGE, allow these representative majorly abundant extracellular
products of
the present invention to be characterized and distinguished. The analysis
described
indicates that these proteins constitute the majority of the extracellular
products of
M tuberculosis, with the 71 KD, 30 KD, 32A KD, 23 KD and 16 KD products
comprising approximately 60% by weight of the total available extracellular
product. It
is further estimated that the 30 KD protein may constitute up to 25% by weight
of the
total products released by M tuberculosis. Thus, individual exemplary majorly
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42
abundant extracellular products of M tuberculosis useful in the practice of
the present
invention may range anywhere from approximately 0.5% up to approximately 25%
of
the total weight of the extracellular products.
As previously discussed, following the inability of traditional Western
blot analysis to consistently identify the most immunogenically specific
extracellular
products, the present inventor decided to analyze the immunogenicity of the
majorly
abundant extracellular products based upon their abundance and consequent ease
of
identification and isolation. Surprisingly, it was found that these majorly
abundant
extracellular products induce unexpectedly effective immune responses leading
this
inventor to conclude that they may function as vaccines. This surprising
discovery led
to the development of the non-limiting functional theory of this invention
discussed
above.
To demonstrate the efficacy of the present invention, additional
experiments were conducted using individual majorly abundant extracellular
products
and combinations thereof at various exemplary dosages to induce protective
immunity
in art accepted laboratory models. More specifically, purified individual
majorly
abundant extracellular products were used to induce protective immunity in
guinea pigs
which were then challenged with M tuberculosis. Upon showing that these
proteins
were capable of inducing protective immunity, combinations of five purified
majorly
abundant extracellular products was similarly tested using differing routes of
administration. In particular the 30 KD abundant extracellular product was
used to
induce protective immunity in the accepted animal model as was the purified
form of
the 71 KD extracellular product. As with the individual exemplary majorly
abundant
extracellular products the combination vaccines of five majorly abundant
extracellular
products conferred protection against challenge with lethal doses of M.
tuberculosis as
well. Results of the various studies of these exemplary vaccines of the
present
invention follow.
Specific pathogen-free male Hartley strain guinea pigs (Charles River
Breeding Laboratories, North Wilmington, Massachusetts) were used in all
experiments
involving immunogenic or aerosol challenges with M tuberculosis. The animals
were
housed two or three to a stainless steel cage and allowed free access to
standard guinea
pig chow and water. After arrival at the animal facility, the guinea pigs were
observed
for at least one week prior to the start of each experiment to ensure that
they were
healthy.
Initial experiments were conducted using individual majorly abundant
extracellular products believed to comprise between 3% to 25% of the total
extracellular
CA 02222000 1997-11-24
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43
proteins normally present. These experiments demonstrate that majorly abundant
extracellular products elicit an effective immune response. More particularly,
isolated
30 KD and 71 KD extracellular products were shown to be individually capable
of
generating a cell-mediated immune response that protected guinea pigs upon
exposure
to lethal doses of M tuberculosis as follows.
EXAMPLE 3
PURIFIED 30 KD PROTEIN SKIN TESTING FOR
CELL-MEDIATED IMMUNITY OF 30 KD IMMUNIZED GUINEA PIGS
To illustrate that a measurable immune response can be induced by
purified forms of abundant extracellular products, a cutaneous
hypersensitivity assay
was performed. Guinea pigs were immunized with the exemplary majorly abundant
M. tuberculosis 30 KD secretory product purified according to Example 2 and
believed
to comprise approximately 25% of the total extracellular product of M.
tuberculosis. In
three independent experiments, guinea pigs were immunized three times three
weeks
apart with 100 g of substantially purified 30 KD protein in SAF adjuvant.
Control
animals were similarly injected with buffer in SAF. Three weeks after the last
immunization the guinea pigs were challenged with the exemplary 30 KD protein
in a
cutaneous hypersensitivity assay.
Guinea pigs were shaved over the back and injections of 0.1, 1 and 10 g
of 30 KD protein were administered intradermally with resulting erythema
(redness of
the skin) and induration measured after 24 hours as shown in Table A below.
Data are
reported in terms of mean measurement values for the group standard error
(SE) as
determined using traditional methods. ND indicates that this particular aspect
of the
invention was not done.
TABLE A
Erythema (mm) to 71 KD (Mean SE)
Guinea Pig
Status n 0.1 ag 1.0 tg 10.0 ua
Ex w.
Immunized 6 1.2 0.5 3.9 0.8 6.9 1.0
Controls 5 ND ND 3.0 0.9
Expt. 2
Immunized 6 0.5 0.5 5.4 0.7 8.1 0.6
Controls 3 0 0 2.5 0 1.7 0.8
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44
Erythema (mm) to 71 KD (Mean SE)
Guinea Pig
Status n 0.1 ua 1.0 ug 10.0 mg
Ext)t. 3
Immunized 6 ND 1.7 1.1 6.2 0.3
Controls 3 ND ND 2.0 0.0
Induration (mm) to 30 KD (Mean SE)
Guinea Pig
Status n 0.1 ug 1.0 ug 10.0 ug
Expt. 1
Immunized 6 0 0 3.3 0.3 5.6 0.9
Controls 5 ND ND 1.6 1.0
x t.2
Immunized 6 0 0 3.8 0.7 4.9 1.2
Controls 3 0 f 0 0.8 f 0.8 1.7 0.8
Expt. 3
Immunized 6 ND 1.1 1.1 4.7 0.4
Controls 3 ND 0 0 0 0
As shown in Table A, guinea pigs immunized with the exemplary 30 KD
secretory product exhibited a strong cell-mediated immune response as
evidenced by
marked erythema and induration. In contrast, the control animals exhibited
minimal
response.
To confirm the immunoreactivity of the 30 KD secretory product and
show its applicability to infectious tuberculosis, non-immunized guinea pigs
were
infected with M tuberculosis and challenged with this protein as follows.
EXAMPLE 4
PURIFIED 30 KD PROTEIN TESTING FOR CELL-MEDIATED
IMMUNE RESPONSES OF GUINEA PIGS
INFECTED WITH M. TUBERCULOSIS
To obtain bacteria for use in experiments requiring the infection of
guinea pigs, M tuberculosis was first cultured on 7H11 agar and passaged once
through
a guinea pig lung to insure that they were virulent. For this purpose, guinea
pigs were
challenged by aerosol with a 10 ml suspension of bacteria in 7H9 broth
containing
approximately 5 x 104 bacteria/ml. After the guinea pigs became ill, the
animals were
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sacrificed and the lungs, containing prominent M tuberculosis lesions, were
removed.
Each lung was ground up and cultured on 7H11 agar for 7 days to 10 days. The
bacteria
were scraped from the plates, diluted in 7H9 broth containing 10% glycerol,
sonicated
in a water bath to obtain a single cell suspension, and frozen slowly at -70 C
at a
5 concentration of approximately 2 x 10' viable bacteria/ml. Viability of the
frozen cells
was measured by thawing the bacterial suspension and culturing serial
dilutions of the
suspension on 7H11 agar. Just before a challenge, a vial of bacterial cells
was thawed
and diluted to the desired concentration in 7H9 broth.
The guinea pigs were exposed to aerosols of the viable M tuberculosis
10 in a specially designed lucite aerosol chamber. The aerosol chamber
measured 14 by 13
by 24 in. and contained two 6 inch diameter portals on opposite sides for
introducing or
removing guinea pigs. The aerosol inlet was located at the center of the
chamber
ceiling. A vacuum pump (Gast Mfg. Co., Benton Harbor, Michigan) delivered air
at 30
lb/in2 to a nebulizer-venturi unit (Mes Inc., Burbank, California), and an
aerosol was
15 generated from a 10-m1 suspension of bacilli. A 0.2 m breathing circuit
filter unit
(Pall Biomedical Inc., Fajardo, Puerto Rico) was located at one end of the
chamber to
equilibrate the pressure inside and outside of the assembly. Due to safety
considerations, the aerosol challenges were conducted with the chamber placed
completely within a laminar flow hood.
20 The animals were exposed to pathogenic aerosol for 30 minutes during
which time the suspension of bacilli in the nebulizer was completely
exhausted. Each
aerosol was generated from the 10 ml suspension containing approximately 5.0 x
104
bacterial particles per ml. Previous studies have shown that guinea pig
exposure to this
concentration of bacteria consistently produces infections in non-protected
animals.
25 Following aerosol infection, the guinea pigs were housed in stainless steel
cages
contained within a laminar flow biohazard safety enclosure (Airo Clean
Engineering
Inc., Edgemont, Pennsylvania) and observed for signs of illness. The animals
were
allowed free access to standard guinea pig chow and water throughout the
experiment.
In this experiment, the infected guinea pigs were sacrificed and splenic
30 lymphocyte proliferation was measured in response to various concentrations
of the 30
KD protein. More specifically, splenic lymphocytes were obtained and purified
as
described by Brieman and Horwitz (J. Exp. Med. 164:799-811) which is
incorporated
herein by reference. The lymphocytes were adjusted to a final concentration of
10'/ml
in RPMI 1640 (GIBCO Laboratories, Grand Island, New York) containing
penicillin
35 (100 U/ml), streptomycin (100 g/ml), and 10% fetal calf serum (GIBCO) and
incubated with various concentrations of purified 30 KD secretory product in a
total
CA 02222000 2008-03-07
46
volume of 100 l in microtest wells (96-well round-bottom tissue culture
plate;
FalconTM Labware, Oxnard, California) for 2 days at 37 C in 5% C02-95% air and
100% humidity. Noninfected animals were used as negative controls. At the end
of the
incubation period, 0.25 gCi of [3H]thymidine (New England Nuclear, Boston,
Massachusetts) was added to each well and the cells were further incubated for
2 hours
at 37 C in 5% C02-95% air at 100% humidity. A multisample automated cell
harvester
(Skatron Inc., Sterling, Virginia) was used to wash each well, and the
effluent was
passed through a filtermat (Skatron). Filtermat sections representing separate
microtest
wells were placed in scintillation vials, and 2 ml of Ecoscint H liquid
scintillation
cocktail (National Diagnostics, Manville, New Jersey) was added. Beta particle
emission was measured in a beta scintillation counter (Beckman Instruments
Inc.,
Fullerton, California).
Tissue samples from the infected and noninfected guinea pigs were
assayed against 1 and 10 gg/ml of isolated 30 KD secretory protein. Samples
were then
monitored for their ability to incorporate [3H]thymidine. The results of these
assays
were tabulated and presented in Table B below.
Data are reported as a stimulation index which, for the purposes of this
disclosure, is defined as:
mean [3H]thymidine incorporation of lymphocytes incubated with
antigen / mean [3H]thymidine incorporation of lymphocytes incubated without
antigen.
TABLE B
Stimulation Indices to 30KD (Mean+ SE)
Guinea Pig
Status n 1.0 gg/ml 10.0 gg/ml
Infected 6 2.2 0.2 9.7 4.6
Controls 6 1.5 0.3 2.0 0.8
As shown in Table B, the cells of the infected animals exhibited a strong
response to the exemplary 30 KD protein as manifested by dose dependent
splenic
lymphocyte proliferation in response to exposure to this majorly abundant
secretory
product. Conversely, the uninfected control animals showed little lymphocyte
proliferation. Accordingly, the 30 KD secretory product clearly induces a cell-
mediated
immune response in mammals infected with M. tuberculosis.
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To illustrate the protective aspects of the vaccines of the present
invention, guinea pigs were immunized with purified 30 KD protein and exposed
to
M. tuberculosis as follows.
EXAMPLE 5
CHALLENGE OF 30 KD IMMUNIZED GUINEA PIG
WITH AEROSOLIZED M TUBERCULOSIS
As before, the animals were immunized three times at three week
intervals with 100 pg of the exemplary 30 KD secretory protein in SAF. Control
guinea
pigs were immunized with 120 p.g of bulk EP in SAF or sham-immunized with
buffer
in the same adjuvant. Three weeks after the last immunization, the animals
were
challenged with aerosolized M tuberculosis as described in Example 4. The
survival
rates for the three groups of animals were monitored and are graphically
presented in
Figure 4. Absolute mortality was determined 14 weeks after challenge as
presented in
Table C below.
TABLE C
Status of Survivors/ Percent
Guinea Piss Challenged Survival
30 KD Immunized 4/6 67%
EP Immunized 3/6 50%
Sham Immunized 1/6 17%
As shown in Figure 4 guinea pigs immunized three times with the
exemplary 30 KD protein were protected against death. Approximately 67% of the
guinea pigs immunized with the 30 KD protein survived whereas only 17% of the
control sham-immunized guinea pigs survived.
Weight retention of the immunized animals was also monitored (data not
shown) and further illustrates the prophylactic capacity of vaccines
incorporating
majorly abundant extracellular products produced by pathogenic bacteria as
taught by
the present invention. While the immunized animals appeared to maintain their
weight,
the high mortality rate of the sham-immunized animals precluded the graphical
comparison between the immunized animals and the control animals.
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Following conclusion of the weight monitoring study, the surviving
animals were sacrificed and the right lung and spleen of each animal was
assayed for
viable M tuberculosis. The animals were soaked in 2% amphyl solution (National
Laboratories, Montvale, New Jersey), and the lungs and spleen were removed
aseptically. The number of macroscopic primary surface lesions in the lungs
were
enumerated by visual inspection. Colony forming units (CFU) of M. tuberculosis
in the
right lung and spleen were determined by homogenizing each organ in 10 ml of
7H9
with a mortar and pestle and 90-mesh Norton Alundum (Fisher), serially
diluting the
tissue homogenate in 7H9, and culturing the dilutions on duplicate plates of
7Hll agar
by using drops of 0.1 ml/drop. All plates were kept in modular incubator
chambers and
incubated 12 to 14 days at 37 C in 5% CO2, 95% air at 100% humidity. The assay
was
conducted using this protocol and the results of the counts are presented in
Table D
below in terms of mean colony forming units (CFU) standard error (SE).
TABLE D
Mean CFU + SE
Guinea Pig
Status n Right Lung Spleen
30 KD Immunized 4 3.4 1.7 x 107 7.7 3.9 x 106
Sham-immunized 1 1.8 x 10$ 8.5 x 107
Log-Difference 0.73 1.04
As shown in Table D, immunization with the exemplary 30 KD
secretory protein limited the growth of M tuberculosis in the lung and the
spleen.
Although only data from the one surviving sham-immunized animal was available
for
comparative purposes, the four surviving 30 KD immunized animals had 0.7 log
fewer
CFU in their lungs and 1 log fewer CFU in their spleen than the surviving sham-
immunized animal. Based on previous demonstrations of a high correlation
between
CFU counts and mortality, the surviving animal likely had fewer CFU in the
lungs and
spleen than the animals who died before a CFU analysis could be performed.
Again
this reduction of CFU in the lungs and spleens of the immunized animals
conclusively
demonstrates the scope and operability of the present invention.
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The immunoprotective potential of another majorly abundant
extracellular product from M tuberculosis, the 71 KD extracellular product,
was tested
in its isolated form to demonstrate its immunoprotective capacity.
EXAMPLE 6
PURIFIED 71 KD PROTEIN SKIN TEST OF GUINEA PIGS
IMMUNIZED WITH A BULK PREPARATION OF EP
To demonstrate the potential of 71 KD protein to provoke an effective
immune response in animals, this isolated majorly abundant extracellular
product was
used to skin test guinea pigs immunized with a bulk preparation of M.
tuberculosis
extracellular proteins (EP) in a cutaneous hypersensitivity assay. As
discussed above,
bulk EP will impart acquired immunity against infection by M. tuberculosis but
to a
lesser extent than the vaccines of the present invention.
Guinea pigs were immunized on two occasions spaced three weeks apart,
with 120 g of a bulk preparation of EP prepared as detailed in Example 1. The
vaccination was prepared in incomplete Freund's adjuvant with sham-immunized
animals receiving buffer in place of EP. Three weeks after the last
vaccination the
guinea pigs from each group were shaved over the back and skin tested with an
intradermal injection of 0.1, 1.0 and 10 gg of 71 KD protein. 10.0 g of
buffer was
used as a control and all injections were performed using a total volume of
0.1 ml. The
diameters of erythema and induration were measured after 24 hours with the
results as
shown in Table E below. Data are reported in terms of mean measurement values
for
the group standard error (SE) as determined using traditional methods.
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TABLE E
Erythema (mm) to 71 KD (Mean SE)
Guinea Pig
Status n 0.1 ug 1.0 ug 10.0 ug
Immunized 4 6.5 0.7 11.9 1.4 18.9 2.2
Controls 3 2.5 1.4 5.0 2.9 11.8 2.1
Induration (mm) to 71 KD (Mean + SE)
Guinea Pig
Status n 0.1 jig 1.0 ug 10.0 Lug
Immunized 4 3.6 1.1 6.8 1.1 11.6 0.8
Controls 3 0.7 0.7 3.7 0.9 7.8 1.0
The responses of the immunized animals were almost twice the response
of the guinea pigs challenged with buffer alone and were comparable to those
5 challenged with bulk EP identical to that used to immunize the animals (data
not
shown).
To further confirm that the purified exemplary 71 KD majorly abundant
extracellular product elicits cell-mediated immune responses, the bulk EP
immunized
guinea pigs were sacrificed and splenic lymphocyte proliferation was measured
in
10 response to various concentrations of the 71 KD protein. Nonimmunized
animals were
used as controls. Following the protocol of Example 4, the lymphocytes were
incubated
with and without 71 KD protein for 2 days and then assayed for their capacity
to
incorporate [3H]thymidine.
Data is reported in terms of stimulation indices calculated as in Example
15 4. The results of this 71 KD challenge are shown in Table F below.
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TABLE F
Stimulation Indices to 71 KD (Mean SE)
Guinea Pig
Status n 0.01 jig/ml 0.1 jig/ml 1.0 u.g/ml
Immunized 4 1.5 0.1 2.3 0.5 8.1 2.2
Controls 2 1.7 0.6 1.6 0.4 2.5 0.6
Stimulation Indices to EP (Mean + SE)
Guinea Pig
Status n 0.01 ml 0.1 g/ml 1.0 g/ml
Immunized 4 1.5 0.1 2.2 0.3 5.3 1.4
Controls 2 1.4 0.2 1.5 0.2 1.2 0.1
As shown in Table F, stimulation indices for the lymphocyte
proliferation assay were comparable to the results obtained in the cutaneous
hypersensitivity assay. Both the 71 KD and bulk EP tested samples showed
responses
between two and three times higher than those obtained with the controls
indicating that
isolated exemplary 71 KD majorly abundant extracellular product is capable of
provoking a cell-mediated immune response in animals immunized with
M tuberculosis extracts. However, it should again be emphasized that the
purified
majorly abundant or principal extracellular product is free of the problems
associated
with prior art or bulk compositions and is more readily adaptable to synthetic
and
commercial production making the vaccines of the present invention superior to
the
prior art.
More particularly the bulk preparation cannot be manufactured easily on
a large scale through modem biomolecular techniques. Any commercial production
of
these unrefined bulk preparations containing all extracellular products would
involve
culturing vast amounts of the target pathogen or a closely related species and
harvesting
the resultant supernatant fluid. Such production methodology is highly
susceptible to
contamination by the target pathogen, toxic byproducts or other parasitic
agents.
Further, the large number of immunogenic determinants in such a preparation is
far
more likely to provoke a toxic immune reaction in a susceptible segment of the
immunized population. Using these unrefined bulk preparations also negates the
use of
the most popular skin tests currently used for tuberculosis screening and
control.
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In direct contrast, the vaccines of the present invention can be mass-
produced in relative safety using high yield transformed hosts. Similarly, the
vaccines
of the present invention can be produced in identical, easy to standardize
batches as
opposed to the wider variable production of bulk extracellular products.
Moreover, as
the number of immunogenic determinants presented to the host immune system is
relatively small, toxic reactions and the chance of invalidating popular
screening tests
are greatly reduced.
EXAMPLE 7
PURIFIED 71 KD PROTEIN SKIN TEST OF
71 KD IMMUNIZED GUINEA PIGS
Following demonstration that the isolated exemplary 71 KD majorly
abundant extracellular product generates a cell-mediated immune response in
bulk EP
immunized animals, it was shown that the purified form of this majorly
abundant
product was able to induce a cell-mediated immune response in animals
immunized
with 71 KD.
Guinea pigs were twice vaccinated with 100 g of purified 71 KD
protein in SAF three weeks apart. Control animals were sham-immunized with
buffer
in SAF on the same schedule. Three weeks after the last immunization both sets
of
animals were intradermally challenged with I and 10 g of isolated 71 KD
protein. The
resulting erythema and indurations were measured after 24 hours with the
results shown
in Table G below.
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TABLE G
E hema (mm) to 71 KD (Mean SE)
Guinea Pig
Status n L R9 1.0 jig 10.0 jig
Immunized 3 0 0 6.5 1.5 15.0 1.5
Controls 3 0 0 2.7 1.3 6.7 1.3
Induration (mm) to 71 KD (Mean + SE)
Guinea Pig
Status n 0 ua 1.0 jig 10.0 jig
Immunized 3 0 0 3.0 1.0 9.3 0.3
Controls 3 0 0 0 0 1.3 1.3
The extent of induration and erythema was much greater in the
immunized animals than in the non-immunized control animals demonstrating that
a
strong cell-mediated immune response to 71 KD protein had been initiated by
the
vaccination protocol of the present invention.
To further confirm the capacity of this abundant extracellular product to
induce an effective immune response on its own in accordance with the
teachings of the
present invention, lymphocyte proliferation assays were performed. Animals
immunized as in Table G were sacrificed and splenic lymphocyte proliferative
assays
were run using the protocol established in Example 4. The tissue samples from
the 71
KD immunized guinea pigs and those from the control guinea pigs were
challenged
with 0.1, 1 and 10 gg/ml of isolated 71 KD protein and monitored for their
ability to
incorporate [3H]thymidine. Stimulation indices were calculated as previously
described. The results of these assays are presented in Table H below.
TABLE H
Stimulation Indices to 71 KD (Mean + SE)
Guinea Pig
Status n 0.1 ml 1.0 g/ml 10.0 jig/ml
Immunized 3 4.0 1.3 5.6 2.5 12.2 5.1
Controls 3 1.3 0.3 1.3 0.3 3.2 f 1.5
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As with the cutaneous hypersensitivity assay, the 71 KD immunized
animals showed a much higher response to purified 71 KD than did the sham-
immunized controls. Though expected of a foreign protein, such results clearly
show
that a majorly abundant extracellular product has the capacity to induce an
cell-
mediated immune response.
After establishing that an isolated majorly abundant extracellular protein
will induce an effective cell-mediated immune response, further experiments
were
conducted to confirm that any such response is cross-reactive against tubercle
bacilli as
follows.
EXAMPLE 8
PURIFIED 71 KD PROTEIN CHALLENGE OF GUINEA PIGS
INFECTED WITH M TUBERCULOSIS
Non-immunized guinea pigs were infected with aerosolized
M tuberculosis as reported in Example 4. Purified protein derivative (PPD-
CT68;
Connaught Laboratories Ltd.) was employed as the positive control to ensure
that the
infected animals were demonstrating a cell-mediated immune response indicative
of
M tuberculosis. Widely used in the Mantoux test for tuberculosis exposure, PPD
is
generally prepared by ammonium sulfate fractionation and comprises a mixture
of small
proteins having an average molecular weight of approximately 10 KD. Immune
responses to PPD are substantially analogous to those provoked by the bulk EP
fractions isolated in Example 1.
Three weeks after infection the guinea pigs were challenged
intradermally with 0.1, 1 and 10 g of the exemplary purified majorly abundant
71 KD
extracellular protein. Uninfected animals used as controls were similarly
challenged
with the isolated protein. The extent of erythema and induration were measured
24
hours later with the results reported in Table I below.
TABLEI
Erythema (mm) to 71 KD (Mean SE)
Guinea Pig
Status n 0.1 ua 1.0 g 10.0112
Infected 7 9.5 1.7 13.4 1.3 19.7 1.3
Controls 6 2.3 2.3 3.5 2.2 7.8 1.9
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Induration (mm) to 71 KD (Mean + SE)
Guinea Pig
Status n 0.1 up- 1.0 ug 10.0 ug
Infected 7 5.3 1.8 8.7 1.6 13.4 1.1
Controls 6 0 0 0.8 0.8 0 0
As shown in Table I, strong immune responses are present in the infected
animals challenged with the exemplary purified majorly abundant extracellular
protein
of the present invention. These responses are on the order of three to four
times greater
5 for erythema and more than 10 times greater for induration than those of the
uninfected
animals, confirming that the prominent 71 KD extracellular protein induces a
strong
cell-mediated immune response in M tuberculosis-infected animals.
To further corroborate these results the infected animals and uninfected
animals
were sacrificed and subjected to a lymphocyte proliferative assay according to
the
10 protocol of Example 4. The tissue samples from both sets of guinea pigs
were assayed
against 0.1, 1 and 10 gg/ml of isolated 71 KD protein and PPD. The samples
were then
monitored for their ability to incorporate [3H]thymidine as previously
described with the
results of these assays presented in Table J below.
15 TABLE J
Stimulation Indices to 71 KD (Mean SE)
Guinea Pig
Status n 0.1 g/ml 1.0 g/ml 10.0 g/ml
Infected 3 2.4 0.5 6.2 1.8 29.1 16.2
Controls 3 1.1 0.1 2.6 0.8 18.2 6.1
Stimulation Indices to PPD (Mean SE)
Guinea Pig
Status n 0.1 g/ml 1.0 Ug/ml 10.0 jig/ml
Infected 3 1.0 0.1 4.0 1.5 11.4 3.4
Controls 3 0.9 0.2 0.9 0.03 1.5 0.3
As with the results of the cutaneous sensitivity assay, Table J shows that
the stimulation indices were much higher for the infected tissue than for the
uninfected
samples. More specifically, the mean peak stimulation index of infected
animals was 2-
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fold higher to the exemplary 71 KD protein and 3-fold higher to PPD than it
was to
uninfected controls confirming that a strong cell-mediated immune response is
induced
in animals infected with M tuberculosis by the exemplary majorly abundant
extracellular protein vaccines of the present invention.
Following this demonstration of cross-reactivity between the exemplary
purified 71 KD majorly abundant protein and M tuberculosis, additional
experiments
were performed to demonstrate that an effective immune response could be
stimulated
by these exemplary purified samples of the majorly abundant extracellular
products as
disclosed by the present invention.
EXAMPLE 9
CHALLENGE OF 71 KD IMMUNIZED GUINEA PIGS
WITH AEROSOLIZED M. TUBERCULOSIS
To demonstrate the immunoprotective capacity of exemplary majorly
abundant or principal extracellular protein vaccines, guinea pigs were
immunized twice,
3 weeks apart, with 100 g of the exemplary majorly abundant 71 KD protein
purified
according to Example 2. Control animals were immunized with 120 jig bulk EP
from
Example 1 or buffer. All animals were immunized using the adjuvant SAF. Three
weeks after the last immunization, guinea pigs immunized with the exemplary 71
KD
protein were skin-tested with 10 g of the material to evaluate whether a cell-
mediated
immune response had developed. The control animals and 71 KD immunized guinea
pigs were then infected with aerosolized M. tuberculosis as detailed in
Example 4.
Following infection the animals were monitored and weighed for six months.
The graph of Figure 5 contrasts the weight loss experienced by the sham-
immunized group to the relatively normal weight gain shown by the 71 KD and
bulk EP
immunized animals. Data are the mean weights SE for each group. Mortality
curves
for the same animals are shown in the graph of Figure 6. The absolute
mortality rates
for the study are reported in Table K below.
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TABLE K
Status of Survivors/ Percent
Guinea Pigs Challenged Survival
71 KD Immunized 3/6 50%
EP Immunized 5/8 62.5%0
Sham Immunized 0/6 0%
Both the weight loss curves and the mortality rates clearly show that the
majorly abundant extracellular proteins of the present invention confer a
prophylactic
immune response. This is emphasized by the fact that 100% of the non-immunized
animals died before the end of the monitoring period.
EXAMPLE 10
CHALLENGE OF 71 KD IMMUNIZED GUINEA PIGS
WITH AEROSOLIZED M. TUBERCULOSIS
A similar experiment was conducted to verify the results of the previous
Example and show that the administration of an exemplary principal
extracellular
protein can confer a protective immune response in animals. In this
experiment, guinea
pigs were again immunized three times, 3 weeks apart, with 100 g of the 71 KD
extracellular protein in SAF. Control guinea pigs were sham-immunized with
buffer in
SAF. Three weeks after the last immunization, the animals were challenged with
aerosolized M. tuberculosis and weighed weekly for 13 weeks. Mean weights SE
for
each group of 6 guinea pigs were calculated and are graphically represented in
Figure 7.
This curve shows that the sham-immunized animals lost a considerable amount of
weight over the monitoring period while the immunized animals maintained a
fairly
consistent body weight. As loss of body mass or "consumption" is one of the
classical
side effects of tuberculosis, these results indicate that the growth and
proliferation of
tubercle bacilli in the immunized animals was inhibited by the exemplary
vaccine of the
present invention.
Protective immunity having been developed in guinea pigs through
vaccination with an abundant extracellular product in an isolated form,
experiments
were run to demonstrate the inter-species immunoreactivity of the vaccines of
the
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present invention and to further confirm the validity and applicability of the
guinea pig
model.
EXAMPLE 11
TESTING CELL-MEDIATED IMMUNITY OF PPD POSITIVE HUMANS
WITH PURIFIED 71 KD PROTEIN
To assess the cell-mediated component of a human immune response to
the exemplary 71 KD majorly abundant protein, the proliferation of peripheral
blood
lymphocytes from PPD-positive and PPD-negative individuals to the protein were
studied in the standard lymphocyte proliferation assay as reported in Example
4 above.
A positive PPD, or tuberculin, response is well known in the art as being
indicative of
previous exposure to M. tuberculosis. The proliferative response and
corresponding
incorporation of [3H]thymidine were measured at two and four days. Data for
these
studies is shown in Figures 8A and 8B. Figure 8A shows the response to various
levels
of 71 KD after two days while Figure 8B shows the same responses at four days.
As illustrated in Figures 8A and 8B, the mean peak stimulation index of
PPD-positive individuals was twofold higher to the 71 KD protein and threefold
higher
to PPD than that of PPD negative individuals. Among PPD-positive individuals,
there
was a linear correlation between the peak stimulation indices to the exemplary
71 KD
protein and to PPD demonstrating that a strong cell-mediated response is
stimulated by
the most prominent or majorly abundant extracellular products of M.
tuberculosis in
humans previously exposed to M tuberculosis. This data corresponds to the
reactivity
profile seen in guinea pigs and confirms the applicability of the guinea pig
model to
other mammals subject to infection.
Thus, as with the previously discussed 30 KD exemplary protein, the
development of a strong immune response to the majorly abundant 71 KD
extracellular
product demonstrates the broad scope of the present invention as evidenced by
the fact
that the 71 KD product is also effective at stimulating cell-mediated immunity
in
humans.
Again, it should be emphasized that the present invention is not limited
to the extracellular products of M. tuberculosis or to the use of the
exemplary 71 KD
protein. Rather the teachings of the present invention are applicable to any
majorly
abundant extracellular product as demonstrated in the examples.
Additional studies were performed in order to ascertain whether
combinations of majorly abundant extracellular products of M. tuberculosis
would
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provide protective immunity as well. In general, these studies utilized guinea
pigs
which were immunized either intradermally or subcutaneously with various
dosages of
vaccines comprising combinations of 5 purified extracellular proteins of M.
tuberculosis
in SAF three times, 3 or 4 weeks apart.
The first protein combination used for the immunization procedure,
labeled Combination I, was comprised of 71 KD, 32A KD, 30 KD, 23 KD, and 16 KD
proteins purified according to the protocols described in Example 2. This
combination
is believed to comprise up to 60% of the total extracellular protein normally
present in
M. tuberculosis culture supernatants. These proteins selected for use in
Combination I,
are identified with an asterisk in Figure 2. Combination I vaccine containing
100 g,
g, or 2 g of each protein was administered intradermally with the adjuvant
SAF.
Combination I vaccine containing 20 g of each protein was also administered
subcutaneously in similar experiments. Negative control guinea pigs were sham-
immunized with equivalent volumes of SAF and buffer on the same schedule while
15 positive controls were immunized using 120 gg of the bulk extracellular
protein
preparation from Example 1 in SAF. All injection volumes were standardized
using
buffer.
EXAMPLE 12
20 RESPONSE OF COMBINATION I IMMUNIZED GUINEA PIGS TO A
CHALLENGE WITH COMBINATION I VACCINE
To determine if the animals had developed a measurable immune
response following vaccination with the Combination I mixture of principal
extracellular products, a cutaneous hypersensitivity assay was performed.
Guinea pigs
were shaved over the back and injected intradermally with 1.0 g and 10.0 g
of the
same combination of the five purified extracellular proteins. 10.0 g of
buffer was used
as a control and all injections were performed using a total volume of 0.1 ml.
The
diameters of erythema and induration at skin tests sites were measured at 24
hours after
injection.
The results of the measurements are presented in Table L below. Data
are again reported in terms of mean measurement values for the group
standard error
(SE) as determined using traditional methods. ND indicates that this
particular aspect
of the experiment was not done.
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TABLE L
Guinea Pig Erythema (mm) (Mean + SE)
Status n PD 1.0 ug 10.0 jig
Immunized 6 0 11.4 4.6 17.4 2.6
Controls 6 0 ND 6.0 0.5
Induration (mm) (Mean + SE)
PD 1.0ua 10.0ua
Immunized 6 0 7.3 0.8 11.6 1.2
Controls 6 0 ND 4.2 0.3
The data clearly demonstrate that a strong cell-mediated immune
response to the Combination I extracellular proteins was generated by the
vaccinated
5 animals. The immunized guinea pigs show erythema and induration measurements
almost three times greater than the control animals.
EXAMPLE 13
IMMUNOPROTECTIVE ANALYSIS OF COMBINATION I VACCINE
10 AGAINST AEROSOLIZED M TUBERCULOSIS
Three weeks after the last immunization, the guinea pigs used for the
preceding hypersensitivity assay were challenged with aerosolized M.
tuberculosis,
Erdman strain and weighed weekly for 10 weeks. This aerosol challenge was
15 performed using the protocol of Example 4. Six animals immunized with 100
g of the
principal extracellular products of Combination I, along with equal sized
groups of
positive and negative controls, were challenged simultaneously with
aerosolized
M tuberculosis. Positive controls were immunized three times with 120 g EP in
SAF.
Guinea pigs that died before the end of the observation period were
20 autopsied and examined for evidence of gross tuberculosis lesions. Such
lesions were
found in all animals which expired during the study.
Differences between immunized and control animals in mean weight
profiles after aerosol challenge were analyzed by repeated measures analysis
of variance
(ANOVA). Differences between immunized and control guinea pigs in survival
after
25 challenge were analyzed by the two-tailed Fisher exact test. Data are the
mean weights
t standard error (SE) for each group of six guinea pigs.
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Results of the weekly weight determinations following challenge are
shown in Figure 9. Compared with guinea pigs immunized with the combination of
extracellular products, sham-immunized animals lost 15.9% of their total body
weight.
Weights of the positive controls were similar to those of animals immunized
with the
combination of five purified extracellular proteins. Body weights were
normalized
immediately before challenge. The difference between animals immunized with
Combination I and sham-immunized controls was highly significant with p
<.0000001
by repeated measures ANOVA.
Mortality was determined ten and one-half weeks after challenge. All
three of the sham-immunized animals died within three days of each other
between ten
and ten and one-half weeks after challenge. The mortality results of the
experiment are
provided in Table M below.
TABLE M
Status of Survivors/ Percent
Guinea Pigs Challenged Survival
Combination 6/6 100%
Immunized
EP-Immunized 5/6 83%
Sham-Immunized 3/6 50%
Following the conclusion of the weight monitoring study, the surviving
animals were sacrificed by hypercarbia and the right lung and spleen of each
animal
was assayed for viable M. tuberculosis using the protocol of Example 5. The
results of
the counts, including the 3 animals that died the last week of the experiment,
are
presented in Table N below in terms of mean colony forming units (CFU)
standard
error (SE).
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TABLE N
Mean CFU SE
Guinea Pig n
Status Right Lung Spleen =
Sham-immunized 6 8.9 5.4 x 107 1.3 0.7 x 107
Immunized 6 3.4 1.7 x 106 1.8 0.6 x 106
EP-immunized 6 1.7 0.7 x 107 5.0 2.8 x 106
The log difference between the concentration of bacilli in the lung of the
animals immunized with the combination of purified proteins and that of the
sham-
immunized animals was 1.4 while the log difference of bacilli in the spleen
was 0.9.
Paralleling this, on gross inspection at autopsy immunized animals had
markedly
decreased lung involvement with tuberculosis compared with sham-immunized
controls. Positive control animals immunized with the bulk extracellular
preparation
(EP) of Example I showed 0.7 log more bacilli in the lung and .5 log more
bacilli in the
spleen than animals immunized with the Combination I mixture of purified
extracellular
proteins.
EXAMPLE 14
IMMUNOPROTECTION ANALYSIS OF COMBINATION I VACCINE AT Low DOSES THROUGH
INTRADERMAL AND SUBCUTANEOUS DELIVERY
While Example 13 confirmed that Combination I proteins demonstrated
immunoprotection in animals immunized intradermally with 100 g of each
protein (30
+ 32A + 16 + 23 + 71) 3 times, 4 weeks apart, an alternative study was
conducted to
demonstrate the immunoprotective capacity of lower doses of Combination I
proteins,
specifically 20 g or 2 g of each protein. As in Example 13, guinea pigs (6
animals
per group) were immunized with Combination I proteins (30 + 32A + 16 + 23 +
71)
intradermally in SAF 4 times, 3 weeks apart. Animals received either 20 g or
each
protein per immunization or 2 g of each protein per immunization. Control
animals
were sham-immunized utilizing the previous protocol. Three weeks later, the
animals
were challenged with aerosolized M. tuberculosis and weights were measured
weekly
for 9 weeks. All immunized animals survived to the end of the experiment while
one
sham-immunized animal died before the end of the experiment. As the following
results illustrate, doses 5 fold and even 50 fold lower than those of Example
13
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protected immunized animals from aerosolized M. tuberculosis and that delivery
by
both the intradermal and subcutaneous route was effective.
Compared with guinea pigs immunized with 20 g of each protein of
Combination I, sham-immunized animals lost 12 % of their total body weight
during
the 9 weeks of the experiment (weights were normalized to just before
challenge).
Compared with guinea pigs immunized with 2 g of each protein of Combination
I,
sham-immunized animals lost 11% of their normalized total body weight. Thus,
guinea
pigs immunized intradermally with low doses of Combination I proteins were
protected
against weight loss after aerosol challenge with M tuberculosis.
Similarly, guinea pigs immunized intradermally with low doses of
Combination I proteins also were protected against splenomegaly associated
with
dissemination of M tuberculosis to the spleen. As shown in Table 0, whereas
animals
immunized with 20 g or 2 g of each protein of Combination I had spleens
weighing
an average of 4.6 1.2g and 4.0 0.8g (Mean SE), respectively, sham-
immunized
animals had spleens weighing an average of 9.6 1.8g (Table 1), or more than
twice as
much.
TABLE 0
Spleen Weight (g)
Status of Guinea Pies n Mean SE
Sham-Immunized 5 9.6 1.8
Immunized (20 g) 6 4.6 1.2
Immunized (2 g) 6 4.0 0.8
Guinea pigs immunized intradermally with low doses of Combination I
proteins also had fewer CFU of M tuberculosis in their spleens As shown ' : 'L
b1 D
y . < A. J11V N'n.:~ aae L ,
when compared with sham-immunized animals, guinea pigs immunized with 20 g or
2
g of each protein of Combination I had an average of 0.6 and 0.4 log fewer
CFU,
respectively, in their spleens.
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TABLE P
CFU in Spleen Log
Guinea Pig Status Mean SE Difference
Sham-Immunized 5 3.1 2.3 x 106
Immunized (20 g) 6 8.1 2.4 x 105 -0.6
Immunized (2 g) 6 1.2 0.6 x 106 -0.4
Moreover, guinea pigs immunized subcutaneously with Combination I
proteins were also protected against weight loss, splenomegaly, and growth of
M tuberculosis in the spleen. In the same experiment described in Example 14,
guinea
pigs were also immunized subcutaneously rather than intradermally with 20 g
of
Combination I proteins, 4 times, 3 weeks apart. These animals were protected
from
challenge almost as much as the animals immunized intradermally with 20 g of
Combination I proteins.
EXAMPLE 15
RESPONSE OF COMBINATION I AND COMBINATION II IMMUNIZED GUINEA PIGS TO
CHALLENGE WITH COMBINATION I AND COMBINATION II
Additional studies were performed to ascertain whether other
combinations of majorly abundant extracellular products of M tuberculosis
would
provide protective immunity as well. One study utilized guinea pigs which were
immunized with a vaccine comprising two combinations - Combination I (71, 32A,
30,
23, and 16) and Combination II (32A, 30, 24, 23, and 16). Combination II is
believed
to comprise up to 62% of the total extracellular protein normally present in
M tuberculosis supernatants. Animals (6 per group) were immunized four times
with
100 g of each protein in Combination I or II in SAF, 3 weeks apart. Negative
control
animals were sham-immunized with equivalent volumes of SAF and buffer on the
same
schedule.
As in Example 12, the animals were tested for cutaneous delayed-type
hypersensitivity to determine if the animals developed a measurable immune
response
following vaccination. Animals immunized with Combination II had 16.8 1.3 mm
(Mean SE) erythema and 12.8 1.2 mm induration in response to skin-testing
with
Combination II whereas sham-immunized animals had only 1.3 0.8 mm erythema
and
0.3 3 mm induration in response to Combination II. Thus, animals immunized
with
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Combination II had greater than 12 fold more erythema and greater than 40 fold
more
induration than controls. By way of comparison, animals immunized with
Combination
I had 21.3 2.0 mm erythema and 15.8 0.1 mm induration in response to skin-
testing
with Combination I, whereas sham-immunized animals had only 6.4 0.8 mm
5 erythema and 2.6 0.7 mm induration in response to Combination I. Thus,
animals
immunized with Combination I had greater than 3 fold more erythema and greater
than
6 fold more induration than controls. The difference from controls for
Combination II
proteins was even greater than that for Combination I proteins.
In the same experiment, animals immunized with a lower dose of
10 Combination II proteins (20 g of each protein vs. 100 g) also developed
strong
cutaneous hypersensitivity to Combination II. They had 21.0 2.0 mm erythema
and
15.3 0.9 mm induration in response to Combination II, whereas the sham-
immunized
animals had only 1.3 0.8 mm erythema and 0.3 0.3 mm induration, as noted
above.
Thus, animals immunized with a lower dose of Combination II proteins had
greater than
15 16 fold erythema and greater than 50 fold more induration than controls, a
difference
that was even greater than for animals immunized with the higher dose of
Combination
II proteins.
EXAMPLE 16
20 IMMUNOPROTECTIVE ANALYSIS OF COMBINATION I AND II
VACCINE AGAINST AEROSOLIZED M. TUBERCULOSIS
Three weeks after the last immunization, the guinea pigs used for the
preceding hypersensitivity assay were challenged with aerosolized M.
tuberculosis,
25 Erdman strain as in Example 13 and weighed weekly for 7 weeks. As in
Example 13, 6
animals were in each group. During the first 7 weeks after challenge, sham-
immunized
animals lost an average of 19.5g. In contrast, animals immunized with
Combination II
(100 g of each protein) gained 52.4 g and animals immunized with Combination
II at a
lower dose (20 g of each protein) gained an average of 67.2g. By way of
contrast,
30 animals immunized with Combination I gained 68g. Thus, compared with guinea
pigs
immunized with Combination II (100 g), sham-immunized animals lost 11 % of
their
total body weight. Compared with guinea pigs immunized with Combination II at
a
lower dose (20 4'g), sham-immunized animals lost 14% of their total body
weight.
Compared with animals immunized with Combination I, sham-immunized animals
also
35 lost 14% of their total body weight.
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EXAMPLE 17
RESPONSE OF GUINEA PIGS IMMUNIZED WITH COMBINATIONS III THROUGH XII
TO A CHALLENGE WITH THE SAME VACCINE OR ITS COMPONENTS
Additional experiments were performed to demonstrate the effectiveness
of various combinations of M. tuberculosis majorly abundant extracellular
products. In
these studies, Hartley type guinea pigs were immunized intradermally with
vaccines
comprising combinations of 2 or more majorly abundant extracellular products
purified
as in Example 2. The purified extracellular products are identified using
their apparent
molecular weight as determined by SDS-PAGE. The guinea pigs were immunized
with
the following combinations of majorly abundant extracellular products.
Combination Protein Constituents
III 30 + 32A + 32B + 16 + 23
IV 30 + 32A
V 30 + 32B
VI 30 + 16
VII 30 + 23
VIII 30 + 71
IX 30+23.5
X 30+12
XI 30+24
XII 30 + 58
Each combination vaccine included 100 g of each listed protein. The
combination vaccines were volumetrically adjusted and injected intradermally
in the
adjuvant SAF. As before the guinea pigs were immunized four times, three weeks
apart.
A cutaneous hypersensitivity assay was performed to determine if the
animals had developed a measurable immune response following vaccination with
the
Combinations III to XII. Groups of six guinea pigs were shaved over the back
and
injected intradermally with the same combination of purified extracellular
products to
which they were immunized. For this challenge 10 g of each of the proteins in
the
combination were injected. All injections were performed using a total volume
of 0.1
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ml. Sham-immunized controls, which had been immunized with SAF only were also
skin-tested with Combinations III to XII, again using 10 g of each protein in
the
respective combination. The diameters of erythema and induration at skin tests
sites
were measured 24 hours after injection as described in Example 3.
The results of these measurements are presented in Table Q below. Data
are again reported in terms of mean measurement values for the group
standard error
(SE) as determined using traditional methods.
TABLE 0
Diameter of Skin Reaction (mm)
Vaccine Skin Test
Combination Combination Erythema Induration
III III 12.2 2.0 6.8 0.8
IV IV 9.9 0.5 6.3 0.2
V V 13.0 1.1 8.1 0.7
VI VI 19.2 1.2 12.4 0.5
VII VII 14.3 1.0 8.7 0.4
VIII VIII 18.9 1.1 12.6 0.8
IX IX 17.0 0.9 12.1 0.9
X X 19.3 1.4 13.6 1.2
XI XI 18.3 1.2 12.4 0.8
XII XII 17.7 0.9 14.0 1.2
Sham III 4.8 0.9 2.0 0.0
Sham IV 4.3 1.1 2.0 0.0
Sham V 5.0 0.5 2.0 0.0
Sham VI 4.5 0.3 2.0 0.0
Sham VII 4.5 0.3 2.0 0.0
Sham VIII 3.3 0.3 2.3 0.3
Sham IX 3.7 0.3 2.0 0.0
Sham X 3.7 0.4 2.0 0.0
Sham XI 3.7 0.2 2.0 0.0
Sham XII 3.8 0.2 2.0 0.0
The results clearly demonstrate that a strong cell-mediated immune
response was generated to each of the combinations of purified extracellular
proteins.
The immunized guinea pigs showed erythema at least twice and usually 3 fold or
more
that of controls for all combinations. Further, the immunized guinea pigs
showed
induration at least 3 fold that of controls for all combinations.
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EXAMPLE 18
IMMUNOPROTECTIVE ANALYSIS OF COMBINATIONS III-XII
AGAINST AEROSOLIZED M TUBERCULOSIS
To demonstrate the prophylactic efficacy of these exemplary
combinations of purified extracellular products, guinea pigs immunized with
Combinations III through XII were challenged with M tuberculosis three weeks
after
the last immunization using the protocol of Example 4.
Consistent with earlier results guinea pigs immunized with
Combinations III through XII were all protected against death after challenge.
At 4
weeks after challenge, 2 of 6 sham-immunized animals (33%) died compared with
0
animals in groups immunized with Combinations IV-XII and 1 of 6 animals (17%)
in
the group immunized with Combination III. At 10 weeks after challenge, 50% of
the
sham-immunized animals had died compared with 0 deaths in the animals in
groups
immunized with Combinations IX and XII (0%), 1 of 6 deaths (17%) in the
animals in
the groups immunized with Combination III, IV, V, VI, X, and XI, 1 of 5 deaths
(20%)
in the animals immunized with Combination VIII, and 2 of 6 deaths (33%) in the
animals immunized with Combination VII.
Guinea pigs that died before the end of the observation period were
autopsied and examined for evidence of gross tuberculosis lesions. Lesions
were found
in all animals which expired during the study.
Following the conclusion of the mortality study, the surviving animals
were sacrificed by hypercarbia and the spleen of each animal was assayed for
viable
M. tuberculosis using the protocol of Example 5. The results are presented in
Table R
below in terms of mean colony forming units (CFU) along with the log decrease
from
the sham immunized animals. An asterisk next to the CFU value indicates that
spleen
counts were zero on one animal in each group. For purposes of calculation,
zero counts
were treated as 103 CFU per spleen or 3 logs.
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TABLE R
Vaccine CFU in Spleen Log Decrease
Group (Mean Log) from Sham
III 5.99 .5
IV 5.41 1.1
V 6.27 .3
VI <5.80* >.7
VII <5.61 * >.9
VIII 6.47 .1
IX <5.85* >.7
X <5.74* >.8
XI 5.93 .6
XII 6.03 .5
Sham 6.53 --
Animals immunized with Combinations III, IV, VI, VII, IX, X, XI, and
XII had at least 0.5 log fewer colony forming units of M tuberculosis in their
spleens
on the average than the sham-immunized controls. In particular, combinations
IV and
VII proved to be especially effective, reducing the average number of colony
forming
units by roughly a factor of ten. Animals immunized with Combinations V and
VIII
had 0.3 and 0.1 log fewer colony forming units (CFU), respectively, in their
spleens on
average, than sham-immunized controls. This dramatic reduction in colony
forming
units in the animals immunized in accordance with the teachings of the present
invention once again illustrates the immunoprotective operability of the
present
invention.
EXAMPLE 19
RESPONSE OF GUINEA PIGS IMMUNIZED WITH 3 DIFFERENT DOSAGES OF COMBINATION
XIII TO A CHALLENGE WITH COMBINATION XIII
To further define the operability and scope of the present invention as
well as to demonstrate the efficacy of additional combinations of purified
extracellular
products, guinea pigs were immunized as before using alternative vaccination
dosages.
Specifically, 50 g, 100 gg and 200 g of an alternative combination of 3
majorly
abundant extracellular products identified as Combination XIII and comprising
the 30
KD, 32(A) KD, and 16 KD proteins. As with the preceding examples, groups of
animals were immunized intradermally 4 times, 3 weeks apart with the
alternative
dosages of Combination XIII in SAF.
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A cutaneous hypersensitivity assay was performed to determine if the
animals had developed a measurable immune response following vaccination. The
animals were shaved over the back and injected intradermally with Combination
XIII
containing 10.0 g of each of the purified extracellular products. All
injections were
5 performed using a total volume of 0.1 ml. Sham-immunized controls were also
skin-
tested with the same dosage of Combination XIII. The diameters of erythema and
induration at skin- test sites were measured 24 hours after injection.
The results are presented in Table S below in terms of mean
measurement values for the group standard error (SE) as determined using
traditional
10 methods
TABLE S
Diameter of Skin Reaction (mm)
Vaccine Vaccine
Combination Dose E ema Induration
XIII 50 17.8 1.3 13.2 1.0
XIII 100 11.2 0.9 7.3 0.4
XIII 200 10.0 0.7 7.0 0.4
Sham 0 5.7 0.5 0.2 0.2
Once again, these results clearly demonstrate that a strong cell-mediated
15 immune response to Combination XIII was generated in animals immunized with
each
of the three dosages of Combination XIII. The immunized animals exhibited
erythema
about two to three times that of controls. Even more strikingly, the immunized
animals
exhibited induration at least 35 fold that of control animals which exhibited
a minimal
response in all cases.
EXAMPLE 20
IMMUNOPROTECTIVE ANALYSIS OF COMBINATION XIII IN THREE DIFFERENT DOSAGES
AGAINST AEROSOLIZED M TUBERCULOSIS
To further demonstrate the protective immunity aspects of the vaccines
of the present invention at various dosages, the immunized guinea pigs (6 per
group)
used for the preceding cutaneous hypersensitivity assay were challenged with
aerosolized M tuberculosis three weeks after the last immunization. The
aerosol
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challenge was performed using the protocol detailed in Example 4. A control
group of
12 sham-immunized animals was challenged simultaneously.
Results of the weekly weight determinations following challenge are
graphically represented in Figure 10 and distinctly show guinea pigs immunized
with
each of the three dosages of Combination XIII were protected from weight loss.
Animals immunized with the higher dosages of Combination XIII (100 and 200 g)
actually showed a net gain in weight and animals immunized with the lower
dosage (50
g) showed a relatively small loss in weight. In contrast, the sham immunized
animals
lost approximately 22% of their total body weight in the weeks immediately
after
challenge and averaged a loss of 182 g over the 10 week observation period.
Table U below illustrates the percent weight change for immunized and
control animals as determined by taking the mean weight at the end of the
challenge,
subtracting the mean weight at the start of the challenge and dividing the
result by the
mean weight at the start of the challenge. Similarly, the percent protection
was
determined by subtracting the mean percent weight loss of the controls from
the mean
percent weight gain or loss of the immunized animals.
TABLE U
Immunogen Dosage % Weight % Protection from
Change Weight Loss
Combination XIII 50 -4% 18%
Combination XIII 100 +7% 29%
Combination XIII 200 +5% 27%
Sham Sham -22% -
Table U shows that the sham-immunized animals lost a considerable
amount of weight (18% - 29%) over the monitoring period compared with the
immunized animals. Figure 10 provides a more graphic illustration of the net
weight
loss for each group of immunized animals versus sham-control animals plotted
at
weekly intervals over the ten week monitoring period. As loss of body mass or
"consumption" is one of the classical side effects of tuberculosis, these
results indicate
that the growth and proliferation of tubercle bacilli in the immunized animals
was
inhibited by the three different dosages of the exemplary combination vaccine
of the
present invention.
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EXAMPLE 21
IMMUNOPROTECTIVE ANALYSIS OF COMBINATIONS XIV-XVIII
AGAINST CHALLENGE WITH COMBINATIONS XIV-XVIII
To further demonstrate the scope of the present invention and the broad
range of effective vaccines which may be formulated in accordance with the
teachings
thereof, five additional combination vaccines, Combinations XIV through XVIII,
were
tested in guinea pigs. Identified by the apparent molecular weight of the
purified
extracellular products determined using SDS-PAGE, the composition of each of
the
combination vaccines is given below.
Combination Protein Constituents
XIV 30, 32A, 16, 32B, 24, 23, 45
XV 30, 32A, 16, 32B, 24, 23, 45,
23.5, 12
XVI 30, 32A, 16, 32B, 24, 23
XVII 30, 32A, 16, 32B, 24, 71
XVIII
I 30, 32A, 16, 23, 71
In addition to the new combination vaccines and appropriate controls,
Combination I was also used in this series of experiments. Guinea pigs were
immunized intradermally with 50 gg of each protein of Combination XIV or XV
and
with 100 p.g of each protein of Combinations I, XVI, XVII, and XVIII all in
SAF
adjuvant. The animals were immunized a total of four times, with each
injection three
weeks apart.
A cutaneous hypersensitivity assay was performed to determine if the
animals had developed a measurable immune response following vaccination using
the
previously discussed protocol. Guinea pigs were shaved over the back and
injected
intradermally with the same combination of purified extracellular proteins to
which they
were immunized. For each challenge the appropriate combination vaccine
containing
10 g of each protein was injected. All injections were performed using a
total volume
of 0.1 ml. Sham-immunized controls were also skin-tested with the same dosage
of
each combination. The diameters of erythema and induration at skin test sites
were
measured at 24 hours after injection as described in Example 3.
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The results of these measurements are presented in Table V below,
reported in terms of mean measurement values for the group standard error
(SE) as
determined using traditional methods.
TABLE V
Diameter of Skin Reaction (mm)
Vaccine Skin Test
Combination Combination Erythema Induration
XIV XIV 13.3 0.7 9.1 0.4
XV XV 10.4 0.4 6.5 0.4
XVI XVI 8.0 1.8 5.1 1.0
XVII XVII 9.4 0.9 6.1 1.1
XVIII XVIII 13.6 1.2 8.7 0.7
I I 10.0 0.3 6.7 0.2
Sham XIV 5.5 1.6 0.4 0.2
Sham XV 6.1 0.5 0.4 0.2
Sham XVI 4.6 1.4 0.4 0.2
Sham XVII 5.7 1.2 0.2 0.2
Sham XVIII 2.1 1.1 0 0
Sham I 6.0 1.2 0.6 0.2
These results clearly demonstrate that a strong cell-mediated immune
response was generated to Combinations XIV through XVIII, and, as before, to
Combination I. Immunized animals exhibited erythema about twice that of
controls.
Even more strikingly, the immunized animals exhibited induration at least 10
fold
greater than the sham-immunized controls which exhibited a minimal response in
all
cases.
EXAMPLE 22
IMMUNOPROTECTIVE ANALYSIS OF COMBINATIONS XIV-XVIII
AND COMBINATION I AGAINST AEROSOLIZED M TUBERCULOSIS
To confirm the immunoreactivity of the combination vaccines of
Example 21 and to demonstrate their applicability to infectious tuberculosis,
the
immunized guinea pigs used for the preceding cutaneous hypersensitivity assay
were
challenged with aerosolized M tuberculosis three weeks after the last
immunization and
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monitored using the protocol of Example 4. A control group of 12 sham-
immunized
animals, the same as used in Example 20, was similarly challenged. The results
of these
challenge are graphically represented in Figure 11 and shown in Table W
directly
below.
Percent weight change was determined by taking the mean weight at the
end of the challenge, subtracting the mean weight at the start of the
challenge and
dividing the result by the mean weight at the start of the challenge.
Similarly, the
percent protection was determined by subtracting the mean percent weight loss
of the
controls from the mean percent weight gain or loss of the immunized animals.
TABLE W
Immunogen % Weight % Protection from
Change Weight Loss
Combination XIV 3% 25%
Combination XV -4% 18%
Combination XVI -15% 7%
Combination XVII -11% 11%
Combination XVIII -12% 10%
Combination I -11% 11%
Sham -22%
As shown in Table W, guinea pigs immunized with each of the
combination vaccines were protected from weight loss. Sham-immunized animals
lost
approximately 22% of their total combined body weight. In contrast the
prophylactic
effect of the combination vaccines resulted in actual weight gain for one of
the test
groups and a reduced amount of weight loss in the others. Specifically,
animals
immunized with Combination XIV evidenced a 3% weight gain while those animals
immunized with the other combinations lost only 4% to 15% of their total
combined
weight.
These results are shown graphically in Figure 11 which plots weekly
weight determinations in terms of net weight gain or loss for each group of
animals
following aerosolized challenge. This statistically significant difference
between the
net weight loss for the immunized animals and the sham-immunized controls
shown in
Figure 11 provides further evidence for the immunoprophylactic response
generated by
the combination vaccines of the present invention.
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EXAMPLE 23
CELL-MEDIATED IMMUNITY IN GUINEA PIGS IMMUNIZED WITH
THREE DIFFERENT ADJUVANTS
5 In order to further demonstrate the broad applicability and versatility of
the vaccine formulations of the present invention, immunogenic studies were
conducted
using different adjuvants. Specifically three different immunogens, purified
30 KD
protein, Combination I (30, 32A, 16, 23, 71) and Combination XIII (30, 32A,
16) were
each formulated using three different adjuvants, Syntex Adjuvant Formulation I
(SAF),
10 incomplete Freund's adjuvant (IFA) and Monophosphoryl Lipid A containing
adjuvant
(MPL). Such adjuvants are generally known to enhance the immune response of an
organism when administered with an immunogen.
Guinea pigs were immunized intradermally with 100 gg of each protein
comprising Combinations I and XIII and approximately 100 g of purified 30 KD
15 protein in each of the three different adjuvant formulations. The guinea
pigs were
immunized with each formulation a total of three times with injections three
weeks
apart.
Following immunization, a cutaneous hypersensitivity assay was
performed to determine if the guinea pigs had developed a measurable immune
20 response. Guinea pigs were shaved over the back and injected intradermally
with the
same immunogen to which they had been immunized. For the challenge, 10 jig of
each
protein in Combinations I and XIII or 10 g of purified 30 KD protein was
injected in a
total volume of 100 l. Sham-immunized guinea pigs, vaccinated with one of the
three
adjuvants, were skin-tested with each of the immunogen formulations containing
the
25 same adjuvant. The diameters of erythema and induration at skin test sites
were
measured 24 hours after challenge as described in Example 3.
The results of these measurements are presented in Table X below. As
previously discussed data are reported in terms of mean measurement values for
the
group standard error as determined using accepted statistical techniques.
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TABLE X
Diameter of Skin Reaction (mm)
Skin Test
Vaccine Adjuvant Reagent Erythema Induration
30 SAF 30 10.7 1.6 5.8 1.5
30 IFA 30 8.8 0.7 4.6 0.7
30 MPL 30 10.2 1.7 5.3 1.5
XIII SAF XIII 7.3 0.5 4.1 0.5
XIII IFA XIII 6.8 0.9 3.5 0.5
XIII MPL XIII 6.3 0.4 3.4 0.3
I SAF I 6.9 0.6 4.0 0.3
I IFA I 6.8 0.2 3.6 0.3
I MPL I 7.4 0.4 3.9 0.5
Sham SAF 30 0.7 0.7 1.0 0
Sham IFA 30 0 0 0 0
Sham MPL 30 0 0 0 0
Sham SAF XIII 1.0 1.0 1.0 0
Sham IFA XIII 0 0 0.3 0.3
Sham MPL XIII 0 0 0 0
Sham SAF I 4.7 0.3 1.0 0
Sham IFA I 2.0 1.0 0.7 0.3
Sham MPL I 1.0 1.0 0.7 0.3
As shown in the data presented in Table X, the combination vaccines and
purified extracellular products of the present invention provide a strong cell-
mediated
immunogenic response when formulated with different adjuvants. Moreover, each
one
of the three adjuvants provided about the same immunogenic response for each
respective immunogen. In general, the immunized guinea pigs exhibited erythema
diameters approximately seven to ten times that of the sham-immunized guinea
pigs
while indurations were approximately four to six times greater than measured
in the
control animals.
The ability of the present invention to provoke a strong immunogenic
response in combination with different adjuvants facilitates vaccine
optimization. That
is, adjuvants used to produce effective vaccine formulations in accordance
with the
teachings herein may be selected based largely on consideration of secondary
criteria
such as stability, lack of side effects, cost and ease of storage. These and
other criteria,
not directly related to the stimulation of an immune response, are
particularly important
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when developing vaccine formulations for widespread use under relatively
primitive
conditions.
EXAMPLE 24
IMMUNOPROTECTIVE ANALYSIS OF COMBINATIONS XIX-XXVIII
AGAINST CHALLENGE WITH COMBINATIONS XIX-XXVIII
The broad scope of the present invention was further demonstrated
through the generation of an immune response using ten additional combination
vaccines, Combinations XIX through XXVIII. In addition to the new combination
vaccines and appropriate controls, Combinations IV and XIII were also used as
positive
controls to provoke an immune response in guinea pigs. Identified by the
apparent
molecular weight of the purified extracellular products determined using SDS-
PAGE,
the composition of each of the combination vaccines is given below.
Combination Protein Constituents
XIX 30, 32A, 23
XX 30, 32A, 23.5
XXI 30, 32A, 24
XXII 30,32A, 71
XXIII 30,32A, 30,32A,16,23
XXIV 30, 32A, 16, 23.5
XXV 30,32A, 30,32A,16,24
XXVI 30,32A, 16,71
XXVII 30, 32A, 16, 32B
XXVIII 30, 32A, 16, 45
IV 30,32A
XIII 30, 32A, 16
The guinea pigs were immunized a total of four times, with each
injection three weeks apart. Each combination vaccine used to immunize the
animals
consisted of 100 g of each protein in SAF adjuvant to provide a total volume
of
0.1 ml.
Using the protocol discussed in Example 3, a cutaneous hypersensitive
assay was performed to determine if the animals had developed a measurable
immune
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78
response following vaccination with the selected combination vaccine. The
guinea pigs
were shaved over the back and injected intradermally with the same combination
of
purified extracellular proteins with which they were immunized. The protein
combinations used to challenge the animals consisted of 10 g of each protein.
Sham
immunized controls were also skin-tested with the same dosage of each
combination.
As in Example 3, the diameters of erythema and induration at the skin test
sites were
measured at 24 hours after injection.
The results of these measurements are presented in Table Y below,
reported in terms of mean measurement values for the group of animals +
standard
error.
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TABLE Y
Diameter of Skin Reaction (mm)
Vaccine Skin Test
Combination Combination Erythema Induration
XIX XIX 8.5 0.6 3.9 0.3
XX XX 8.2 0.3 3.7 0.3
XXI XXI 11.1 1.1 4.5 0.4
XXII XXII 9.4 0.8 4.3 0.4
XXIII XXIII 8.3 1.1 3.0 0.3
XXIV XXIV 8.5 0.9 3.4 0.5
XXV XXV 7.9 0.5 3.2 0.4
XXVI XXVI 8.9 0.7 3.3 0.5
XXVII XXVII 7.2 1.0 2.8 0.5
XXVIII XXVIII 8.5 0.5 2.8 0.3
IV IV 9.0 0.9 4.1 0.3
XIII XIII 9.4 0.9 4.3 0.3
Sham XIX 4.0 2.6 1.0 0
Sham XX 1.3 1.3 1.0 0
Sham XXI 3.5 1.0 1.3 1.3
Sham XXII 1.3 1.3 1.0 1.0
Sham XXIII 0 0 1.0 0
Sham XXIV 0 0 1.0 0
Sham XXV 0 0 1.0 0
Sham XXVI 2.3 2.3 2.0 1.0
Sham XXVII 0 0 1.0 0
Sham XXVIII 2.0 1.2 1.0 0
Sham IV 2.8 1.6 1.0 0
Sham XIII 1.5 1.5 1.0 0
The results presented in Table Y explicitly show that a strong cell-
mediated immune response was generated to Combinations XIX through XXVIII when
challenged with the same immunogens. As before, a strong cell-mediated immune
response was also provoked by Combinations IV and XIII. The erythema exhibited
by
the immunized guinea pigs was at least twice, and generally proved to be and
more then
four fold greater than, the reaction provoked in the corresponding sham
immunized
control animals. Similarly, the induration exhibited by the immunized animals
was at
least twice, and generally three to four times greater than, that of the non-
immunized
controls. The substantially stronger immune response generated among the
animals
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immunized in accordance with the teachings of the present invention once again
illustrates the immunoprotective operability of the combination vaccines of
the present
invention.
Those skilled in the art will also appreciate additional benefits of the
5 vaccines and methods of the present invention. For example, because
individual
compounds or selected combinations of highly purified molecular species are
used for
the subject vaccines rather than whole bacteria or components thereof, the
vaccines of
the present invention are considerably less likely to provoke a toxic response
when
compared with prior art attenuated or killed bacterial vaccines. Moreover, the
10 molecular vaccines of the present invention are not life threatening to
immunocompromised individuals. In fact, the compositions of the present
invention
may be used therapeutically to stimulate a directed immune response to a
pathogenic
agent in an infected individual.
Selective use of majorly abundant extracellular products or their
15 immunogenic analogs also prevents the development of an opsonizing humoral
response which can increase the pathogenesis of intracellular bacteria. As the
protective
immunity generated by this invention is directed against unbound proteins, any
opsonic
response will simply result in the phagocytosis and destruction of the majorly
abundant
extracellular product rather than the expedited inclusion of the parasitic
bacteria.
20 Moreover, the selective use of purified extracellular products reduces the
potential for
generating a response which precludes the use of widely used screening and
control
techniques based on host recognition of immunogenic agents. Unlike prior art
vaccines,
the screening tests could still be performed using an immunoreactive molecule
that is
expressed by the pathogen but not included in the vaccines made according to
the
25 present invention. The use of such an immunogenic determinant would only
provoke a
response in those individuals which had been exposed to the target pathogen
allowing
appropriate measures to be taken.
Another advantage of the present invention is that purified extracellular
products are easily obtained in large quantities and readily isolated using
techniques
30 well known in the art as opposed to the attenuated bacteria and bacterial
components of
prior art vaccines. Since the immunoreactive products of the present invention
are
naturally released extracellularly into the surrounding media for most
organisms of
interest, removal of intracellular contaminants and cellular debris is
simplified. Further,
as the most prominent or majorly abundant extracellular products or
immunogenic
35 analogs thereof are used to stimulate the desired immune response,
expression levels
and culture concentrations of harvestable product is generally elevated in
most
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81
production systems. Accordingly, whatever form of production is employed,
large scale
isolation of the desired products is easily accomplished through routine
biochemical
procedures such as chromatography or ultrafiltration. These inherent
attributes and
molecular characteristics of the immunogenic determinants used in the present
invention greatly facilitate the production of a consistent, standardized,
high quality
composition for use on a large scale.
Alternatively, the use of purified molecular compounds based on the
immunogenic properties of the most prominent or majorly abundant extracellular
products of target pathogens also makes the large scale synthetic generation
of the
immunoactive vaccine components of the present invention relatively easy. For
instance, the extracellular products of interest or their immunogenic analogs
may be
cloned into a non-pathogenic host bacteria using recombinant DNA technology
and
harvested in safety. Molecular cloning techniques well known in the art may be
used
for isolating and expressing DNA corresponding to the extracellular products
of
interest, their homologs or any segments thereof in selected high expression
vectors for
insertion in host bacteria such as Escherichia coli. Exemplary techniques may
be found
in II R. Anon, Synthetic Vaccines 31-77, 1987, Tam et al., "Incorporation of T
and B
Epitopes of the Circumsporozoite Protein in a Chemically Defined Synthetic
Vaccine
Against Malaria,: J. Exp. Med. 171:299-306, 1990, and Stover et al.,
"Protective
Immunity Elicited by Recombinant Bacille Calmette-Guerin (BCG) Expressing.
Outer
Surface Protein A (OspA) Lipoprotein: A Candidate Lyme Disease Vaccine," J.
Exp.
Med. 178:197-209 (1993).
Similarly, the extracellular proteins, their analogs, homologs or
immunoreactive protein subunits may be chemically synthesized on a large scale
in a
relatively pure form using common laboratory techniques and automated
sequencer
technology. This mode of production is particularly attractive for
constructing peptide
subunits or lower molecular weight analogs corresponding to antigenic
determinants of
the extracellular products. Exemplary techniques for the production of smaller
protein
subunits are well known in the art and may be found in II R. Anon, Synthetic
Vaccines
15-30, 1987, and in A. Streitwieser, Jr., Introduction to Organic Chemistry
953-55,
1985 (3d ed.). Alternative techniques may be found in Gross et al.,
"Nonenzymatic
Cleavage of Peptide Bonds: The Methionine Residues in Bovine Pancreatic
Ribonuclease," The Journal of Biological Chemistry 237(6), 1962, Mahoney,
"High-
Yield Cleavage of Tryptophanyl Peptide Bonds by o-Iodosobenzoic Acid,"
Biochemistry 18(17), 1979, and Shoolnik et al., "Gonococcal Pili," Journal of
Experimental Medicine 159, 1984. Other immunogenic techniques such as anti-
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82
idiotyping or directed molecular evolution using peptides, nucleotides or
other
molecules such as mimetics can also be employed to generate effective,
immunoreactive compounds capable of producing the desired prophylactic
response.
Nucleic acid molecules useful for the practice of the present invention
may be expressed from a variety of vectors, including, for example, viral
vectors such
as herpes viral vectors (e.g., U.S. Patent No. 5,288,641), retroviruses (e.g.,
EP 0,415,731; WO 90/07936, WO 91/0285, WO 94/03622; WO 93/25698;
WO 93/25234; U.S. Patent No. 5,219,740; WO 89/09271; WO 86/00922;
WO 90/02797; WO 90/02806; U.S. Patent No. 4,650,764; U.S. Patent No.
5,124,263;
U.S. Patent No. 4,861,719; WO 93/11230; WO 93/10218; Vile and Hart, Cancer
Res.
53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al.,
Cancer
Res. 53:83-88, 1993; Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba
et al.,
J. Neurosurg. 79:729-735, 1993), pseudotyped viruses, adenoviral vectors
(e.g.,
WO 94/26914, WO 93/9191; Kolls et al., PNAS 91(l):215-219, 1994; Kass-Eisler
et
al., PNAS 90(24):11498-502, 1993; Guzman et al., Circulation 88(6):2838-48,
1993;
Guzman et al., Cir. Res. 73(6):1202-1207, 1993; Zabner et al., Cell 75(2):207-
216,
1993; Li et al., Hum. Gene Ther. 4(4):403-409, 1993; Caillaud et al., Eur. J.
Neurosci.
5(10):1287-1291, 1993; Vincent et al., Nat. Genet. 5(2):130-134, 1993; Jaffe
et al.,
Nat. Genet. 1(5):372-378, 1992; and Levrero et al., Gene 101(2):195-202,
1991),
adenovirus-associated viral vectors (Flotte et al., PNAS 90(22):10613-10617,
1993),
parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457-463, 1994), and
pox virus
vectors (Panicali and Paoletti, PNAS 79:4927-4931, 1982).
The nucleic acid molecules (or vectors, i.e., an assembly capable of
directing the expression of a sequence of interest) may be introduced into
host cells by a
wide variety of mechanisms, including, for example, transfection, including,
for
example, DNA linked to killed adenovirus (Michael et al., J.Biol. Chem.
268(10):6866-6869, 1993; and Curiel et al., Hum. Gene Ther. 3(2):147-154,
1992),
cytofectin-mediated introduction (DMRIE-DOPE, Vical, Calif.), direct DNA
injection
(Acsadi et al., Nature 352:815-818, 1991); DNA ligand (Wu et al., J. Biol.
Chem.
264:16985-16987, 1989); lipofection (Feigner et al., Proc. Natl. Acad. Sci.
USA
84:7413-7417, 1989); liposomes (Pickering et al., Circ. 89(l):13-21, 1994; and
Wang et
al., PNAS 84:7851-7855, 1987); microprojectile bombardment (Williams et al.,
PNAS
88:2726-2730, 1991); and direct delivery of nucleic acids which encode the
enzyme
itself, either alone (Vile and hart, Cancer Res. 53:3860-3864, 1993), or
utilizing PEG-
nucleic acid complexes (see also WO 93/18759; WO 93/04701; WO 93/07283 and
WO 93/07282).
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83
As an additional alternative, DNA or other genetic material encoding one
or more genes capable of inducing the expression of one or more of the
extracellular
products, homologs, analogs, or subunits of the present invention can be
directly
injected into a mammalian host utilizing so called "naked DNA" techniques.
Following
the in vivo introduction and the resultant uptake of the genetic construct by
the host's
cells the host will begin the endogenous production of the one or more encoded
immunoreactive products engendering an effective immune response to subsequent
challenge. As those skilled in the art will appreciate, coupling the genetic
construct to
eucaryotic promoter sequences and/or secretion signals may facilitate the
endogenous
expression and subsequent secretion of the encoded immunoreactive product or
products. Exemplary techniques for the utilization of naked DNA as a vaccine
can be
found in International Patent No. WO 9421797 A (Merck & Co. Inc. and ViCal
Inc.),
International Patent Application No. WO 9011092 (ViCal Inc.), and Robinson,
"Protection Against a Lethal Influenza Virus Challenge by Immunization with a
Hemagglutinin-Expressing Plasmid DNA," Vaccine 11:9, 1993, and in Ulmer et
al.,
"Heterologous Protection Against Influenza by Injection of DNA Encoding a
Viral
Protein," Science 259, 1993, incorporated by reference herein.
Alternatively, techniques for the fusion of a strongly immunogenic
protein tail have been disclosed in Tao et al., "Idiotype/Granulocyte-
Macrophage
Colony-Stimulating Factor Fusion Protein as a Vaccine for B-Ceo Lymphoma,"
Nature
362, 1993, and for T-cell epitope mapping in Good et al., "Human T-Cell
Recognition
of the Circumsporozoite Protein of Plasmodium falciparum: Immunodominant T-
Cell
Domains Map to the Polymorphic Regions of the Molecule," Proc. Nat]. Acad.
Sci.
USA 85, 1988, and Gao et al., "Identification and Characterization of T Helper
Epitopes
in the Nucleoprotein of Influenza A Virus," J. Imm. 143(9), 1989.
As many bacterial genera exhibit homology, the foregoing examples are
provided for the purposes of illustration and are not intended to limit the
scope and
content of the present invention or to restrict the invention to the genus
Mycobacterium
or to particular species or serogroups therein or to vaccines against
tuberculosis alone.
It should also be reemphasized that the prevalence of interspecies homology in
the
DNA and corresponding proteins of microorganisms enables the vaccines of the
present
invention to induce cross-reactive immunity. Because the immunodominant
epitopes of
the majorly abundant extracellular products may provide cross-protective
immunity
against challenge with other serogroups and species of the selected genera,
those skilled
in the art will appreciate that vaccines directed against one species may be
developed
using the extracellular products or immunogenic analogs of another species.
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For example, M bovis is between 90% and 100% homologous with
M. tuberculosis and is highly cross-reactive in terms of provoking an immune
response.
Accordingly, vaccines based on abundant extracellular products of M bovis or
other
Mycobacterium can offer various degrees of protection against infection by
M tuberculosis and vice versa. Thus, it is contemplated as being within the
scope of
the present invention to provide an immunoprophylactic response against
several
bacterial species of the same genera using an highly homologous immunogenic
determinant of an appropriate majorly abundant extracellular product.
It should also be emphasized that the immunogenic determinant selected
to practice the present invention may be used in many different forms to
elicit an
effective protective or immunodiagnostic immune response. Thus the mode of
presentation of the one or more immunogenic determinants of selected majorly
abundant extracellular products to the host immune system is not critical and
may be
altered to facilitate production or administration. For example, the vaccines
of the
present invention may be formulated using whole extracellular products or any
immunostimulating fraction thereof including peptides, protein subunits,
immunogenic
analogs and homologs as noted above. In accordance with the teachings of the
present
invention, effective protein subunits of the majorly abundant extracellular
products of
M. tuberculosis can be identified in a genetically diverse population of a
species of
mammal. The resultant immunodominant T-cell epitopes identified should be
recognized by other mammals including humans and cattle. These immunodominant
T-cell epitopes are therefore useful for vaccines as well as for
immunodiagnostic
reagents. An exemplary study identifying the immunodominant T-cell epitopes of
the
KD major secretory protein of M tuberculosis was conducted as follows.
EXAMPLE 25
IMMUNODOMINANT EPITOPE MAPPING OF THE 30 KD PROTEIN
Fifty-five synthetic peptides (15-mers) covering the entire native 30 KD
protein and overlapping by 10 amino acids were used for splenic lymphocyte
proliferation assays to identify the immunodominant T-cell epitopes of the 30
KD major
secretory protein of M tuberculosis 55. The sequence of each 15-mer synthetic
peptide
utilized is given below in conjunction with an identification number (1-55)
corresponding to the antigen peptide sequence numbers in Figures 12a and b as
well as
an identification of the amino acid residues and relative position of each
sequence.
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No. Residues Peptide Sequence Seq ID No.
1 1 - 15 FSRPGLPVEYLQVPS 37
2 6- 20 LPVEYLQVPSPSMGR 38
3 11 - 25 LQV PS PSMGRD I KVQ 39
4 16- 30 P S M G R D I KVQFQSGG 40
5 21 - 35 D I KVQFQSGGNNSPA 41
6 26- 40 FQSGGNNSPAVYLLD 42
7 31 - 45 N N S P A V Y L L D G L R AQ 43
8 36 - 50 VYLLDGLRAQDDYNG 44
9 41 - 55 G L R A Q D D Y N G W D I NT 45
10 46 - 60 D D Y N G W D I NTPAFEW 46
11 51 - 65 WD I NTPAFEWYYQSG 47
12 56 - 70 P A F E W Y Y Q S G L S I VM 48
13 61 - 75 Y Y Q S G L S I VMPVGGQ 49
14 66 - 80 LS I VMPVGGQSSFYS 50
15 71 - 85 PVGGQSSFYSDWYSP 51
16 76 - 90 SSFYSDWYSPACGKA 52
17 81 - 95 D W Y S PACGKAGCQTY 53
18 86 - 100 ACGKAGCQTYKWETF 54
19 91 - 105 G C Q T Y K W E T F L T S E L 55
20 96 - 110 K W E T F L T S E L P Q W L S 56
21 101 - 115 LTS EL PQWLSANRAV 57
22 106 - 120 PQWLSANRAVKPTGS 58
23 111 - 125 ANRAVKPTGSAAIGL 59
24 116- 130 KPTGSAAIGLSMAGS 60
25 121 - 135 AAI G L S M A G S S A M I L 61
26 126 - 140 S M A G S S A M I L A A Y H P 62
27 131 - 145 SAM I LAAYHPQQF I Y 63
28 136 - 150 A A Y H P Q Q F I YAGS LS 64
29 141 - 155 QQFIYAGSLSALLDP 65
30 146 - 160 A G S L S A L L D P S Q G M G 66
31 151 - 165 A L L D P S Q G M G P S L IG 67
32 156 - 170 S Q G M G P S L IGLAMGD 68
33 161 - 175 PSL I GLAMGDAGGYK 69
34 166 - 180 LAMGDAGGYKAADMW 70
35 171 - 185 AGGYKAADMWGPSSD 71
36 176 - 190 AADMWGPSSDPAWER 72
37 181 - 195 GPSSDPAWERNDPTQ 73
38 186-200 P A W E R N D P T Q Q I P K L 74
39 191 -205 N D P T Q Q I PKLVANNT 75
40 196-210 QI PKLVANNTRLWVY 76
41 201 - 215 VANNTRLWVYCGNGT 77
42 206 - 220 RLWVYCGNGTPNELG 78
43 211 - 225 CGNGTPNELGGAN I P 79
44 216-230 PNELGGAN I P A E F L E 80
45 221 -235 GAN I PAEFLENFVRS 81
46 226-240 AEFLENFVRSSNLKF 82
47 231 -245 NFVRSSNLKFQDAYN 83
48 236 -250 SNLKFQDAYNAAGGH 84
49 241 - 255 QDAYNAAGGHNAVFN 85
50 246 -260 AAGGHNAVFNFPPNG 86
51 251 -265 NAVFNFPPNGTHSWE 87
52 256 -270 FPPNGTHSWEYWGAQ 88
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53 261 - 275 THSWEYWGAQLNAMK 89
54 266- 280 Y W G A Q L N A M K G D L Q S 90
55 271 - 285 LNAMKGDLQSSLGAG 91
Splenic lymphocytes were obtained from outbred male Hartley strain
guinea pigs (Charles River Breeding Laboratories) that had been immunized
intradermally 3-4 times with 100 g of purified 30 KD protein emulsified in
SAF
(Allison and Byars, 1986). Control animals received phosphate buffered saline
in SAF.
Cell mediated immune responses were evaluated by skin testing as described
above.
Lymphocytes were seeded in 96-well tissue culture plates (Falcon Labware) and
incubated in triplicate with the synthetic 15-mer peptides at 20 g ml-',
purified 30 KD
protein at 20 g ml-', purified protein derivative [(PPD); Connaught
Laboratories LTD]
at 20 g ml-', or concanavalin A at 10 g ml-' for 2 days in the presence of
10 U
polymyxin B. Subsequently, cells were labeled for 16 h with 1 p.Ci [3H]
thymidine
(New England Nuclear) and then harvested (Breiman and Horwitz, 1987). A
positive
proliferative response was defined as follows: (dpm of antigen) - (dpm of
medium) >_ 1
500 and (dpm of antigen)/(dpm of medium) >_ 1.2. Immunodominant epitopes
recognized by greater than 25% of the guinea pigs immunized with purified
M tuberculosis 30 KD protein are presented in Table Z below and graphically
illustrated in Figures 12a and 12b.
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TABLE Z
Inclusive Amino
Acids for
Peptide No. Mature Protein Seq ID No.
1 1- 15 37
2 6- 20 38
3 11- 25 39
21- 35 41
6 26- 40 42
13 61- 75 49
21 101-115 57
26 126 - 140 62
27 131-145 63
31 151-165 67
33 161-175 69
36 176 - 190 72
37 181-195 73
41 201 - 215 77
45 221 - 235 81
49 241 - 255 85
53 261 - 275 89
The results presented in Table Z identify the immunodominant T-cell
epitopes of the 30 KD major secretory protein of M tuberculosis. Those skilled
in the
5 art will appreciate that earlier investigators have studied the 30 KD
protein of M. bovis
which is highly related to M tuberculosis protein. However, these earlier
studies of the
M bovis protein differ markedly from the foregoing study in that the prior art
studied
actual patients, BCG vaccines, patients with tuberculosis, or PPD-positive
individuals.
Because the response to this protein in such individuals is often weak, the
prior art
epitope mapping studies were difficult and of questionable accuracy. In
contrast, the
study of Example 25 utilized outbred guinea pigs immunized with purified
protein,
thereby focusing the immune system on this single protein and producing a very
strong
cell-mediated immune response. Moreover, these guinea pigs were studied within
a few
weeks of immunization, at the peak of T-cell responsiveness.
In accordance with the teachings of the present invention one or more of
the immunodominant epitopes identified above can be incorporated into a
vaccine
against tuberculosis. For example, individual immunodominant epitopes can be
synthesized and used individually or in combination in a multiple antigen
peptide
system. Alternatively, two or more immunodominant epitopes can be linked
together
chemically. The peptides, either linked together or separately, can be
combined with an
appropriate adjuvant and used in subunit vaccines for humans or other mammals.
In
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88
addition, the immunodominant epitopes can be used in new immunodiagnostic
reagents
such as new skin tests.
Those skilled in the art will also appreciate that DNA encoding the
peptides can be synthesized and used to express the peptides, individually or
collectively, or can be combined in a DNA vaccine injected directly into
humans or
other mammals. A construct consisting of only the immunogenic epitopes (or the
DNA
coding therefor) would focus the immune response on the protective portions of
the
molecule. By avoiding irrelevant or even immunosuppressive epitopes such a
construct
may induce a stronger and more protective immune response.
Smaller protein subunits of the majorly abundant extracellular products,
molecular analogs thereof, genes encoding therefore, and respective
combinations
thereof are within the scope of the present invention as long as they provoke
effective
immunoprophylaxis or function as an immunodiagnostic reagent. Moreover,
recombinant protein products such as fusion proteins or extracellular products
modified
through known molecular recombinant techniques are entirely compatible with
the
teachings of the present invention. In addition, immunogenically generated
analogs of
the selected immunoactive determinants or peptides and nucleotides derived
using
directed evolution are also within the scope of the invention. Moreover, the
selected
immunoactive determinants can be modified so as to bind more tightly to
specific MHC
molecules of humans or other species or be presented more efficiently by
antigen
presenting cells. Further, the selected immunoactive determinants can be
modified so
as to resist degradation in the vaccinated host.
Similarly, the formulation and presentation of the immunogenic agent to
the host immune system is not limited to solutions of proteins or their
analogs in
adjuvant. For example, the immunogenic determinant derived from the
appropriate
extracellular proteins may be expressed by M tuberculosis, different species
of
Mycobacteria, different species of bacteria, phage, mycoplasma or virus that
is non-
pathogenic and modified using recombinant technology. In such cases the whole
live
organism may be formulated and used to stimulate the desired response.
Conversely,
large scale vaccination programs in hostile environments may require very
stable
formulations without complicating adjuvants or additives. Further, the vaccine
formulation could be directed to facilitate the stability or immunoreactivity
of the active
component when subjected to harsh conditions such as lyophilization or oral
administration or encapsulation. Accordingly, the present invention
encompasses vastly
different formulations of the immunogenic determinants comprising the subject
vaccines depending upon the intended use of the product.
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89
Those skilled in the art will appreciate that vaccine dosages should be
determined for each pathogen and host utilizing routine experimentation. At
present, it
is believed that the lowest practical dosage will be on the order of 0.1 g
though
dosages of 2.0 g, 20.0 g, 100 g and even 1 mg may be optimum for the
appropriate
system. The proper dosage can be administered using any conventional
immunization
technique and sequence known in the art.
EXAMPLE 26
EXPRESSION OF RECOMBINANT 30 KDA PROTEIN
For the expression of the mature 30 kDa protein, the gene encoding the
30 kDa protein was engineered such that the initiator phenylalanine of the
mature
protein was fused to a glycine residue artificially inserted at the Ncol site
or carboxyl
terminus of the pelB leader sequence in pET22b (Novagen, Madison, WI) (see
Figure
13). This strategy provided a fusion molecule from which the mature 30 kDa
protein
could be easily released and led to the expression of relatively large
quantities of
recombinant 30 kDa protein over a period of 4 hours. Thereafter, expression of
recombinant protein reached a plateau. Expression of the recombinant molecules
continued for up to 8 hours without exerting serious detrimental effects on
the bacterial
culture. A typical yield from 1 liter of E. coli culture was approximately 50
mg,
amounting to nearly 25% of the total cell protein.
To achieve expression of recombinant 30 kDa protein in its full-length or
truncated version, constructs in pET22b were expressed in E. coli
BL21(DE3)pLysS
upon induction with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG). Samples
of
induced cultures were taken at hourly intervals for up to 8 hours and aliquots
of the
culture supernatants and cell pellets were run on 12.5% denaturing
polyacrylamide gels
and stained with Coomassie brilliant blue R. Recombinant protein was purified
as
described by Horwitz et al. ("Protective immunity against tuberculosis induced
by
vaccination with major extracellular proteins of Mycobacterium tuberculosis,"
Proc.
Natl. Acad. Sci. USA 92:1530-1534, 1995), with the exception that all
chromatography
steps included the addition of 8 M urea to the buffers. The purified
recombinant protein
was dialyzed against phosphate buffered saline and remained soluble.
The mature 30 kDa protein was expressed in the pET22b vector either
with its own or the plasmid encoded pelB leader peptide. The results of the
electrophoresis of the cell pellets are shown in Figure 14. Lanes A and B show
CA 02222000 1997-11-24
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Coomassie stained protein extracts upon IPTG induction of bacteria carrying
the
pET22b vector with the mature 30 kDa protein gene fused to the pe1B leader DNA
sequence (A) and the pET22b vector with the full-length 30 kDa protein gene
(B). Lane
C shows mature 30 kDa protein isolated from M. tuberculosis culture filtrates
as a
5 reference. Lanes D, E, and F show a Western blot analysis of the same
proteins as in A,
B, and C probed with anti-30/32A-B kDa complex specific antibodies. Lane G,
protein
extract from E. coli cultures carrying the pET22b vector alone, probed with
the same
antibodies. Positions of full-length and mature 30 kDa proteins are marked 30W
and
30M, respectively, and these recombinant proteins are further identified by
their first 5
10 or 7 N-terminal amino acids. Numbers on the left refer to molecular mass
standards in
kDa.
EXAMPLE 27
EXPRESSION OF SOLUBLE, PROCESSED, EXTRACELLULAR,
15 M TUBERCULOSIS 30 KDA MAJOR SECRETORY PROTEIN USING THE
PLASMID PSMT3 IN MYCOBACTERIUMSMEGMATIS AND MYCOBACTERIUM VACCAE
This example is directed to demonstrating the expression and secretion
of the M tuberculosis 30 kDa major secretory protein in a mycobacterium. We
used
20 the pSMT3 plasmid (Dr. Douglas B. Young, Dept. Medical Microbiology, St.
Mary's
Hospital Medical School, Norfolk Place, London, W2 1PG, United Kingdom, a 5.7
kb
(kilo base pairs) plasmid with both E. coli (column El ori) and mycobacterium
(Mycobacterium fortuitum plasmid pAL5000 ori) origins of replication, a
hygromycin
resistance marker, a hsp60 promoter (Mycobacterium bovis BCG heat shock
protein
25 promoter sequence), and a multicloning site downstream of the hsp60
promoter. The
expression system is shown diagrammatically in Figure 15.
The insert consisted of a 4.7 kb HinDIII - BamHI genomic DNA
fragment from M tuberculosis Erdman strain containing the sequence for the 30
kDa
protein. The insert was cloned into pSMT3 in E. coli DH5a and recombinant
plasmid
30 DNA was transformed into M. smegmatis 1-2c and M vaccae R877R (National
Collection of Type Cultures (NCTC) 11659) by electroporation at a setting of
6250
V/cm and 25 mFarad. M smegmatis 1-2c is a cured isolate of strain M smegmatis
mc26, which is a single cell isolate of ATCC 607 (American Type Culture
Collection)
which was prepared from M smegmatis mc26 by the procedure described in Zhang
35 et al., Molecular Microbiology 5(2):381-391, 1991. M smegmatis mc26 was
isolated
from ATCC 607 by the procedure described in Jacobs et al., Nature 327:532-535,
1987.
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91
Using 1 mg of recombinant plasmid DNA and approximately 4 x 109 CFU of
Mycobacteria, this method yielded 100-200 hygromycin-resistant transformants.
The
transformants were stable in broth culture and constitutively expressed the
M. tuberculosis 30 kDa protein, yielding approximately 10 mg processed
protein/L of
culture. Most importantly, the protein was soluble and approximately 90% of
the
expressed protein was secreted in the culture supernatant (see Figure 16).
The electrophoresis results shown in Figure 16 were obtained as follows.
Supernatant fluid from each of 5 recombinant M. smegmatis clones containing
the
pSMT3 construct with the M tuberculosis 30 kDa gene was subjected to SDS-PAGE
(sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis (5
rightmost
lanes). The major protein (arrow in Figure 16) is the recombinant mature
M. tuberculosis 30 kDa major secretory protein. The left most lane depicts
molecular
mass standards (66, 45, 36, 29, 24, 20, 14 kDa). The recombinant protein
migrates just
above the 29 kDa marker.
Western blot analysis was used to confirm that the major extracellular
protein in the culture supernatant was the recombinant mature M. tuberculosis
30 kDa
major secretory protein. The results are shown in Figure 17. In Figure 17, the
proteins
depicted in the four rightmost lanes of Figure 16 were subjected to SDS-PAGE
and
blotted onto nitrocellulose (4 rightmost lanes). The blot was probed with
rabbit
polyclonal antibody specific to the M tuberculosis 30/32 kDa protein complex.
Only
the recombinant M tuberculosis 30 kDa protein is stained (arrow). The lane to
the left
contains prestained molecular mass markers (106, 80, 49.5, 32.5, 27.5, and
18.5 kDa).
The recombinant protein migrates between the 32.5 and 27.5 kDa mass standards.
In addition, N-terminal sequence analysis of the first 6 N-terminal amino
acids yielded FSRPGL, confirming that the N-terminal sequence was identical to
that of
the mature M tuberculosis 30 kDa protein.
Two constructs in pET20 (Novagen, Madison, WI), one for the mature
kDa protein and the other for the 32A kDa protein, failed to yield expression
of
either protein in E. coli. The isolation of pKK233 is described by Amann and
Brosius,
30 Gene 40:183-190, 1985. pTrc99A (Pharmacia Biotech, Sweden) may be used in
place
of the pKK233 vector. Three different constructs in pKK233 - one for the full-
length
30 kDa protein, one for the full-length 32 kDa protein, and one for the mature
30 kDa
protein - failed to yield expression of any of the proteins in E. coli.
One construct in pRSET-A for the mature 30 kDa protein yielded a
fusion protein in E. coli, but the 30 kDa protein could not be cleaved free of
this fusion
protein with enterokinase. Similarly, two constructs in pTrx-Fus, one for the
mature 30
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92
kDa protein and one for the mature 32 kDa protein, yielded fusion proteins in
E. coli
from which the M. tuberculosis proteins could not be efficiently cleaved with
enterokinase. A summary of the suitability of various expression systems is
set forth in
Table AA. All of the inserts are for the 32A kDa protein.
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93
A+R'I ~Y3y td
N U
gn.
p W W
a ~ ~ z
='w y=~w =y'w -'w = o o o
a a 2 E A E U ~3 ~3
M Ca.
z o o ,, ~~ - - o o 0 0
p w c o = 0 o w c E w E w w a.
g8 S z z z z go Ho ~o~
H U) H ti y y V H
r- al
M ~ . C ~ U . U M O~G :0 a~ C7 C7 C7 C7
COD a e= e e e e e G e N C C
Fpy.. 09C+D N 'VMh U ,~MM N
C^ p M lh
irl i~l u `.S o is o
M M V V U V
f U U U U
w w > j > ~ U w
I i H w `$ W w x x z z
z z z z z z ` F a
a c7 C N ¾ Q G A.
F F m m FF F E F aD pp o
~ G.. W F[]' ~[ ~ ~ ~ Y ,may i [[N~+
M tell rTi rTi M t ~l = t Cn y F"
m '~ z z z z' o W '~ '~
z z Pi v v g
t r,
CD cq
m
t to m m m m m ~ M m m M m m C> ell > > . 3 7 `L
W FL W W
FNS, .D .D [~.`~:( .D O O m m a 9 9
G d G 6 0. 6 n' G d n 6 6
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94
z z z z z 0
00
00
mo mo
m
~y y
~` m m
y
ao
V M ~+
s s
c
z z z z
z z a
H H ae o N ~
o N ~ ~ ~ Q
Sz 1'
Q Q ,v m m
d N U
LL LL W
M fMf M M M *0
d ~ Q.
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As can be seen from Table AA, not all constructs resulted in protein
expression. A leader sequence in front of the structural gene was required for
expression. Thus, one pET22b construct containing the mature 30 kDa protein
gene
and one construct containing the mature 32A kDa protein gene failed to express
either
5 protein. Successful expression in pET22b of the 30 and 32A kDa M.
tuberculosis
proteins was obtained by adding the respective leader sequence of the protein
in front of
the structural gene. For both proteins, this resulted in expression of both
the full-length
and processed protein. These constructs were relatively stable in E. coli,
i.e., they
expressed the recombinant proteins after 2 or 3 subcultures but not after
additional
10 subculturing.
Successful expression in pET22b of the 30 and 32A kDa M tuberculosis
proteins was also obtained by adding the E. coli-derived pe1B leader sequence
in front
of the structural gene for each protein. Expression levels in these constructs
were even
higher than in those utilizing the respective leader sequences of the 30 or
32A kDa
15 proteins. However, a drawback of the pe1B constructs was their instability.
These
constructs lost their ability to express any recombinant protein after one
subculture.
A brief discussion of general approaches to the expression of desired
proteins is set forth above in Example 24.
Smaller protein subunits of the majorly abundant extracellular products
20 and molecular analogs thereof are within the scope of the present invention
as long as
they provoke effective immunoprophylaxis. Moreover, recombinant protein
products
such as fusion proteins or extracellular products modified through known
molecular
recombinant techniques are entirely compatible with the teachings of the
present
invention. In addition, immunogenically generated analogs of the selected
25 immunoactive determinants such as anti-idiotype antibodies, or peptides and
nucleotides derived using directed evolution are also within the scope of the
invention.
Similarly, the formulation and presentation of the immunogenic agent to
the host immune system is not limited to solutions of proteins or their
analogs in
adjuvant. For example, the immunogenic determinant derived from the
appropriate
30 extracellular proteins may be expressed on a different species of bacteria,
phage,
mycoplasma or virus that is non-pathogenic and modified using recombinant
technology. In such cases the whole live organism may be formulated and used
to
stimulate the desired response. Conversely, large scale vaccination programs
in hostile
environments may require very stable formulations without complicating
adjuvants or
35 additives. Further, the vaccine formulation could be directed to facilitate
the stability or
immunoreactivity of the active component when subjected to harsh conditions
such as
CA 02222000 1997-11-24
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96
lyophilization or oral administration or encapsulation. Accordingly, the
present
invention encompasses vastly different formulations of the immunogenic
determinants
comprising the subject vaccines depending upon the intended use of the
product.
Those skilled in the art will appreciate that vaccine dosages should be
determined for each pathogen and host utilizing routine experimentation. At
present, it
is believed that the lowest practical dosage will be on the order of 0.1 mg
though
dosages of 2.0 mg, 20.0 mg, 100 mg and even 1 mg may be optimum for the
appropriate system. The proper dosage can be administered using any
conventional
immunization technique and sequence known in the art.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Horwitz, Marcus A. and Harth, Gunter
(ii) TITLE OF INVENTION: ABUNDANT EXTRACELLULAR PRODUCTS
AND METHODS FOR THEIR PRODUCTION AND USE
(iii) NUMBER OF SEQUENCES: 95
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Kurt A. MacLean
(B) STREET: 2029 Century Park East. Suite 3800
(C) CITY: Los Angeles
(D) STATE: California
(E) COUNTRY: U.S.A.
(F) ZIP: 90067-3024
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1Ø
Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/568.357
(B) FILING DATE: 06-DEC-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/551,149
(B) FILING DATE: 31-OCT-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/447,398
(B) FILING DATE: 23-MAY-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/289.667
(B) FILING DATE: 12-AUG-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/156,358
(B) FILING DATE: 23-NOV-1993
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98
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MacLean, Kurt A.
(B) REGISTRATION NUMBER: 31,118
(C) REFERENCE/DOCKET NUMBER: 118-119
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (714) 263-8250
(B) TELEFAX: (714) 263-8260
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Asn Ser Lys Ser Val
1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr Asp Arg Val Ser
1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Ala Arg Ala Val Gly
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Thr Glu Lys Thr Pro
1 5
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Asp Pro Glu Pro Ala
1 5
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Phe Ser Arg Pro Gly
1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Phe Ser Arg Pro Gly
1 5
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
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Phe Ser Arg Pro Gly
1 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ala Pro Tyr Glu Asn
1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Pro Lys Thr Tyr
1 5
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(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ala Glu Thr Tyr Leu
1 5
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Ala Tyr Pro Ile Thr
1 5
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(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ala Asp Pro Arg Leu
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Phe Asp Thr Arg Leu
1 5
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
Pro Ser Met Gly Arg Asp Ile Lys Val Gin Phe Gin Ser Gly Gly
16 20 25 30
Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp
31 35 40
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Phe Ser Arg Pro Giy Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
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Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gin Ser Gly Gly
16 20 25 30
Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp
31 35 40
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
Ala Ser Met Gly Arg Asp Ile
16 20
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Phe Asp Thr Arg Leu Met Arg Leu Glu Asp Glu Met Lys Glu Gly
1 5 10 15
Arg Tyr Glu Val Arg Ala Glu Leu Pro Gly Val Asp Pro Asp Lys
16 20 25 30
Asp Val Asp Ile Met Val Arg Asp Gly Gln Leu Thr Ile Lys Ala
31 35 40 45
Glu Arg Thr
46
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Ala Asp Pro Arg Leu Gln Phe Thr Ala Thr Thr Leu Ser Gly Ala
1 5 10 15
Pro Phe Asp Lys Ala Ser Leu Gln Gly Lys Pro Ala Val Leu Trp
21 20 25 30
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Ala Asp Pro Arg Leu Gln Phe Thr Ala Thr Thr Leu Ser Gly Ala
1 5 10 15
Pro Phe Asp Lys Ala Ser Leu Gln Gly Lys Pro Ala Val Leu Trp
16 20 25 30
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ala Tyr Pro Ile Thr Gly Cys Leu Gly Ser Glu Leu Thr Met Thr
1 5 10 15
Asp Thr Val Gly Gln Val Val Leu Gly Trp Lys Val Ser Asp Leu
16 20 25 30
Phe Lys Ser Thr Ala Val Ile Pro Gly Tyr Thr Val Xaa Glu Gln
31 35 40 45
Gln Ile
46
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(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ala Tyr Pro Ile Thr Asx Lys Leu Gly Ser Glu Leu Thr Met Thr
1 5 10 15
Asp Thr Val Gly Gin Val Val Leu Gly Trp Lys Val Ser Asp Leu
16 20 25 30
Tyr Lys Ser Thr Ala Val Ile Pro Gly Tyr Thr Val Xaa Glu Gin
31 35 40 45
Gln Ile
46
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Ala Glu Thr Tyr Leu Pro Asp Leu Asp Trp Asp Tyr Gly Ala Leu
1 5 - 10 15
Glu Pro His Ile Ser Gly Gln
16 20
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Ala Pro Lys Thr Tyr Xaa Glu Glu Leu Lys Gly Thr Asp
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Ala Pro Tyr Glu Asn Leu Met Asx Pro Ser Pro Ser Met Gly Arg
1 5 10 15
Asp Ile Pro Val Ala Phe Leu Ala Gly Gly Pro His Ala Val Tyr
16 20 25 30
Leu Leu Asp Ala Phe Asn Ala Gly Pro Asp Val Ser Asn Trp Val
31 35 40 45
Thr Ala Gly Asn Ala Met Met Thr Leu Ala Xaa Lys Gly Ile Cys
46 50 55 60~
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Ala Pro Tyr Glu Asn Leu Met Val Pro Ser Pro Ser Met Gly Arg
1 5 10 15
Asp Ile Pro Val Ala Phe Leu Ala Gly Gly Pro His Ala Val Tyr
16 20 25 30
Leu Leu Asp Ala Phe Asn Ala Gly Pro Asp Val Ser Asn Trp Val
31 35 40 45
Thr Ala Gly Asn Ala Met Met Thr Leu Ala Xaa Lys Gly Ile Ser
46 50 55 60
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
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(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
Pro Ser Met Gly Arg Asp Ile Lys Val Gin Phe Gln Ser Gly Gly
16 20 25 30
Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp
31 35 40
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gin Val Pro Ser
1 5 10 15
Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly
16 20 25 30
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Asn Asn Ser Pro Xaa Leu Tyr Leu Leu Asp
31 35 40
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
Ala Xaa Met Gly Arg Asp Ile
16 20
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
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Asp Pro Glu Pro Ala Pro Pro Val Pro Asp Asp Ala Ala Ser Pro
1 5 10 15
Pro Asp Asp Ala Ala Ala Pro Pro Ala Pro Ala Asp Pro Pro Xaa
16 20 25 30
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Thr Glu Lys Thr Pro Asp Asp Val Phe Lys Leu Ala Lys Asp Glu
1 5 10 15
Lys Val Leu Tyr Leu
16 20
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Ala Arg Ala Val Gly Ile
1 5
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Thr Asp Arg Val Ser Val Gly Asn
1 5
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
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Asn Ser Lys Ser Val Asn Ser Phe Gly Ala His Asp Thr Leu Lys
1 5 10 15
Val Xaa Glu Arg Lys Arg Gln
16 20
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 978 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
ATGACAGACG TGAGCCGAAA GATTCGAGCT TGGGGACGCC GATTGATGAT 50
CGGCACGGCA GCGGCTGTAG TCCTTCCGGG CCTGGTGGGG CTTGCCGGCG 100
GAGCGGCAAC CGCGGGCGCG TTCTCCCGGC CGGGGCTGCC GGTCGAGTAC 150
CTGCAGGTGC CGTCGCCGTC GATGGGCCGC GACATCAAGG TTCAGTTCCA 200
GAGCGGTGGG AACAACTCAC CTGCGGTTTA TCTGCTCGAC GGCCTGCGCG 250
CCCAAGACGA CTACAACGGC TGGGATATCA ACACCCCGGC GTTCGAGTGG 300
TACTACCAGT CGGGACTGTC GATAGTCATG CCGGTCGGCG GGCAGTCCAG 350
CTTCTACAGC GACTGGTACA GCCCGGCCTG CGGTAAGGCT GGCTGCCAGA 400
CTTACAAGTG GGAAACCTTC CTGACCAGCG AGCTGCCGCA ATGGTTGTCC 450
GCCAACAGGG CCGTGAAGCC CACCGGCAGC GCTGCAATCG GCTTGTCGAT 500
GGCCGGCTCG TCGGCAATGA TCTTGGCCGC CTACCACCCC CAGCAGTTCA 550
TCTACGCCGG CTCGCTGTCG GCCCTGCTGG ACCCCTCTCA GGGGATGGGG 600
CCTAGCCTGA TCGGCCTCGC GATGGGTGAC GCCGGCGGTT ACAAGGCCGC 650
AGACATGTGG GGTCCCTCGA GTGACCCGGC ATGGGAGCGC AACGACCCTA 700
CGCAGCAGAT CCCCAAGCTG GTCGCAAACA ACACCCGGCT ATGGGTTTAT 750
TGCGGGAACG GCACCCCGAA CGAGTTGGGC GGTGCCAACA TACCCGCCGA 800
GTTCTTGGAG AACTTCGTTC GTAGCAGCAA CCTGAAGTTC CAGGATGCGT 850
ACAACCCCGC GGGCGGGCAC AACGCCGTGT TCAACTTCCC GCCCAACGGC 900
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ACGCACAGCT GGGAGTACTG GGGCGCTCAG CTCAACGCCA TGAAGGGTGA 950
= CCTGCAGAGT TCGTTAGGCG CCGGCTGA 978
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1017 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
ATGCAGCTTG TTGACAGGGT TCGTGGCGCC GTCACGGGTA TGTCGCGTCG 50
ACTCGTGGTC GGGGCCGTCG GCGCGGCCCT AGTGTCGGGT CTGGTCGGCG 100
CCGTCGGTGG CACGGCGACC GCGGGGGCAT TTTCCCGGCC GGGCTTGCCG 150
GTGGAGTACC TGCAGGTGCC GTCGCCGTCG ATGGGCCGTG ACATCAAGGT 200
CCAATTCCAA AGTGGTGGTG CCAACTCGCC CGCCCTGTAC CTGCTCGACG 250
GCCTGCGCGC GCAGGACGAC TTCAGCGGCT GGGACATCAA CACCCCGGCG 300
TTCGAGTGGT ACGACCAGTC GGGCCTGTCG GTGGTCATGC CGGTGGGTGG 350
CCAGTCAAGC TTCTACTCCG ACTGGTACCA GCCCGCCTGC GGCAAGGCCG 400
GTTGCCAGAC TTACAAGTGG GAGACCTTCC TGACCAGCGA GCTGCCGGGG 450
TGGCTGCAGG CCAACAGGCA CGTCAAGCCC ACCGGAAGCG CCGTCGTCGG 500
TCTTTCGATG GCTGCTTCTT CGGCGCTGAC GCTGGCGATC TATCACCCCC 550
AGCAGTTCGT CTACGCGGGA GCGATGTCGG GCCTGTTGGA CCCCTCCCAG 600
GCGATGGGTC CCACCCTGAT CGGCCTGGCG ATGGGTGACG CTGGCGGCTA 650
CAAGGCCTCC GACATGTGGG GCCCGAAGGA GGACCCGGCG TGGCAGCGCA 700
ACGACCCGCT GTTGAACGTC GGGAAGCTGA TCGCCAACAA CACCCGCGTC 750
TGGGTGTACT GCGGCAACGG CAAGCCGTCG GATCTGGGTG GCAACAACCT 800
GCCGGCCAAG TTCCTCGAGG GCTTCGTGCG GACCAGCAAC ATCAAGTTCC 850
AAGACGCCTA CAACGCCGGT GGCGGCCACA ACGGCGTGTT CGACTTCCCG 900
GACAGCGGTA CGCACAGCTG GGAGTACTGG GGCGCGCAGC TCAACGCTAT 950
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GAAGCCCGAC CTGCAACGGG CACTGGGTGC CACGCCCAAC ACCGGGCCCG 1000
CGCCCCAGGG CGCCTAG 1017
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Phe Ser Arg Pro Gly Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val Gln
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Pro Ser Met Gly Arg Asp Ile Lys Val Gln Phe Gln Ser Gly Gly
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Asp Ile Lys Val Gln Phe Gln Ser Giy Gly Asn Asn Ser Pro Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn 15r
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Trp Asp Ile Asn Thr Pro Ala Phe Glu Trp Tyr Tyr Gin Ser Gly
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Pro Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val Gly Gly Gln
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Leu Ser Ile Val Met Pro Val Gly Gly Gln Ser Ser Phe Tyr Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Pro Val Gly Gly Gln Ser Ser Phe Tyr leer Asp Trp Tyr Ser Pro
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
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(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys Gln Thr Tyr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
Ala Cys Gly Lys Ala Gly Cys Gin Thr Tyr Lys Trp Glu Thr Phe
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Lys Trp Glu Thr Phe Leu Thr Ser Glu Leu Pro Gln Trp Leu Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
Leu Thr Ser Glu Leu Pro Gin Trp Leu Ser Ala Asn Arg Ala Val
10 15
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE: =
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Ala Asn Arg Ala Val Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu
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1 5 10 15
(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
Lys Pro Thr Gly Ser Ala Ala Ile Gly Leu Ser Met Ala Gly Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala Ala Tyr His Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
Ser Ala Met Ile Leu Ala Ala Tyr His Pro Gln Gln Phe Ile Tyr
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
Ala Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
Ala Gly Ser Leu Ser Ala Leu Leu Asp Pro Ser Gln Gly Met Gly
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
Ala Leu Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
Pro Ser Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp 5 10 15
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser Asp
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
Ala Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tubercu7osis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu Trp Val Tyr
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tubercu7osis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly 5 10 15
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly Ala Asn Ile Pro
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
Pro Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
Ala Glu Phe Leu Glu Asn Phe Val Arg Ser Ser Asn Leu Lys Phe
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
Asn Phe Val Arg Ser Ser Asn Leu Lys Phe Gin Asp Ala Tyr Asn
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly His
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
Gln Asp Ala Tyr Asn Ala Ala Gly Gly His Asn Ala Val Phe Asn
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
Ala Ala Gly Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp Glu
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
Phe Pro Pro Asn Gly Thr His Ser Trp Glu Tyr Trp Gly Ala Gln
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:89:.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
Thr His Ser Trp Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys
1 5 10 15
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(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:91:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: C-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Mycobacterium tuberculosis
(B) STRAIN: Erdman
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
Leu Asn Ala Met Lys Gly Asp Leu Gln Ser Ser Leu Gly Ala Gly
1 5 10 15
CA 02222000 1997-11-24
WO 96/37219 PCT/US96/07781
145
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 432 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
ATGGCGGCCA TCGCGACCTT TGCGGCACCG GTCGCGTTGG CTGCCTATCC 50
CATCACCGGA AAACTTGGCA GTGAGCTAAC GATGACCGAC ACCGTTGGCC 100
AAGTCGTGCT CGGCTGGAAG GTCAGTGATC TCAAATCCAG CACGGCAGTC 150
ATCCCCGGCT ATCCGGTGGC CGGCCAGGTC TGGGAGGCCA CTGCCACGGT 200
CAATGCGATT CGCGGCAGCG TCACGCCCGC GGTCTCGCAG TTCAATGCCC 250
GCACCGCCGA CGGCATCAAC TACCGGGTGC TGTGGCAAGC CGCGGGCCCC 300
GACACCATTA GCGGAGCACT ATCCCCCAAG GCGAACAATC GACCGGAAAA 350
TCTACTTCGA TGTCACCGGC CCATCGCCAA CCATCGTCGC GATGAACGAC 400
GGATGGAGGA TCTGCTGATT TGGGAGCCGT AG 432
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1437 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
GTGACGGAAA AGACGCCCGA CGACGTCTTC AAACTTGCCA AGGACGAGAA GGTCGAATAT 60
GTCGACGTCC GGTTCTGTGA CCTGCCTGGC ATCATGCAGC ACTTCACGAT TCCGGCTTCG 120
GCCTTTGACA AGAGCGTGTT TGACGACGGC TTGGCCTTTG ACGGCTCGTC GATTCGCGGG 180
TTCCAGTCGA TCCACGAATC CGACATGTTG CTTCTTCCCG ATCCCGAGAC GGCGCGCATC 240
GACCCGTTCC GCGCGGCCAA GACGCTGAAT ATCAACTTCT TTGTGCACGA CCCGTTCACC 300
CTGGAGCCGT ACTCCCGCGA CCCGCGCACC ATCGCCCGCA AGGCCGAGAA CTACCTGATC 360
CA 02222000 1997-11-24
WO 96/37219 PCT/US96/07781
146
AGCACTGGCA TCGCCGACAC CGCATACTTC GGCGCCGAGG CCGAGTTCTA CATTTTCGAT 420
TCGGTGAGCT TCGACTCGCG CGCCAACGGC TCCTTCTACG AGGTGGACGC CATCTCGGGG 480
TGGTGGAACA CCGGCGCGGC GACCGAGGCC GACGGCAGTC CCAACCGGGG CTACAAGGTC 540
CGCCACAAGG GCGGGTATTT CCCAGTGGCC CCCAACGACC AATACGTCGA CCTGCGCGAC 600
AAGATGCTGA CCAACCTGAT CAACTCCGGC TTCATCCTGG AGAAGGGCCA CCACGAGGTG 660
GGCAGCGGCG GACAGGCCGA GATCAACTAC CAGTTCAATT CGCTGCTGCA CGCCGCCGAC 720
GACATGCAGT TGTACAAGTA CATCATCAAG AACACCGCCT GACAAAACGG CAAAACGGTC 780
ACGTTCATGC CCAAGCCGCT GTTCGGCGAC AACGGGTCCG GCATGCACTG TCATCAGTCG 840
CTGTGGAAGG ACGGGGCCCC GCTGATGTAC GACGAGACGG GTTATGCCGG TCTGTCGGAC 900
ACGGCCCGTC ATTACATCGG CGGCCTGTTA CACCACGCGC CGTCGCTGCT GGCCTTCACC 960
AACCCGACGG TGAACTCCTA CAAGCGGCTG GTTCCCGGTT ACGAGGCCCC GATCAACCTG 1020
GTCTATAGCC AGCGCAACCG GTCGGCATGC GTGCGCATCC CGATCACCGG CAGCAACCCG 1080
AAGGCCAAGC GGCTGGAGTT CCGAAGCCCC GACTCGTCGG GCAACCCTTA TCTGGCGTTC 1140
TCGGCCATGC TGATGGCAGG CCTGGACGGT ATCAAGAACA AGATCGAGCC GCAGGCGCCC 1200
GTCGACAAGG ATCTCTACGA GCTGCCGCCG GAAGAGGCCG CGAGTATCCC GCAGACTCCG 1260
ACCCAGCTGT CAGATGTGAT CGACCGTCTC GAGGCCGACC ACGAATACCT CACCGAAGGA 1320
GGGGTGTTCA CAAACGACCT GATCGAGACG TGGATCAGTT TCAAGCGCGA AAACGAGATC 1380
GAGCCGGTCA ACATCCGGCC GCATCCCTAC GAATTCGCGC TGTACTACGA CGTTTAA 1437
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
GTGCGCATCA AGATCTTCAT GCTGGTCACG GCTGTCGTTT TGCTCTGTTG TTCGGGTGTG 60
GCCACGGCCG CGCCCAAGAC CTACTGCGAG GAGTTGAAAG GCACCGATAC CGGCCAGGCG 120
TGCCAGATTC AAATGTCCGA CCCGGCCTAC AACATCAACA TCAGCCTGCC CAGTTACTAC 180
CA 02222000 1997-11-24
WO 96/37219 PCT/US96/07781
147
CCCGACCAGA AGTCGCTGGA AAATTACATC GCCCAGACGC GCGACAAGTT CCTCAGCGCG 240
GCCACATCGT CCACTCCACG CGAAGCCCCC TACGAATTGA ATATCACCTC GGCCACATAC 300
CAGTCCGCGA TACCGCCGCG TGGTACGCAG GCCGTGGTGC TCAAGGTCTA CCAGAACGCC 360
GGCGGCACGC ACCCAACGAC CACGTACAAG GCCTTCGATT GGGACCAGGC CTATCGCAAG 420
CCAATCACCT ATGACACGCT GTGGCAGGCT GACACCGATC CGCTGCCAGT CGTCTTCCCC 480
ATTGTGCAAG GTGAACTGAG CAAGCAGACC GGACAACAGG TATCGATAGC GCCGAATGCC 540
GGCTTGGACC CGGTGAATTA TCAGAACTTC GCAGTCACGA ACGACGGGGT GATTTTCTTC 600
TTCAACCCGG GGGAGTTGCT GCCCGAAGCA GCCGGCCCAA CCCAGGTATT GGTCCCACGT 660
TCCGCGATCG ACTCGATGCT GGCCTAG 687
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 900 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
ATGAAGGGTC GGTCGGCGCT GCTGCGGGCG CTCTGGATTG CCGCACTGTC ATTCGGGTTG 60
GGCGGTGTCG CGGTAGCCGC GGAACCCACC GCCAAGGCCG CCCCATACGA GAACCTGATG 120
GTGCCGTCGC CCTCGATGGG CCGGGACATC CCGGTGGCCT TCCTAGCCGG TGGGCCGCAC 180
GCGGTGTATC TGCTGGACGC CTTCAACGCC GGCCCGGATG TCAGTAACTG GGTCACCGCG 240
GGTAACGCGA TGAACACGTT GGCGGGCAAG GGGATTTCGG TGGTGGCACC GGCCGGTGGT 300
GCGTACAGCA TGTACACCAA CTGGGAGCAG GATGGCAGCA AGCAGTGGGA CACCTTCTTG 360
TCCGCTGAGC TGCCCGACTG GCTGGCCGCT AACCGGGGCT TGGCCCCCGG TGGCCATGCG 420
GCCGTTGGCG CCGCTCAGGG CGGTTACGGG GCGATGGCGC TGGCGGCCTT CCACCCCGAC 480
CGCTTCGGCT TCGCTGGCTC GATGTCGGGC TTTTTGTACC CGTCGAACAC CACCACCAAC 540
GGTGCGATCG CGGCGGGCAT GCAGCAATTC GGCGGTGTGG ACACCAACGG AATGTGGGGA 600
GCACCACAGC TGGGTCGGTG GAAGTGGCAC GACCCGTGGG TGCATGCCAG CCTGCTGGCG 660
CAAAACAACA CCCGGGTGTG GGTGTGGAGC CCGACCAACC CGGGAGCCAG CGATCCCGCC 720
CA 02222000 1997-11-24
WO 96/37219 PCTIUS96/07781
148
GCCATGATCG GCCAAGCCGC CGAGGCGATG GGTAACAGCC GCATGTTCTA CAACCAGTAT 780
CGCAGCGTCG GCGGGCACAA CGGACACTTC GACTTCCCAG CCAGCGGTGA CAACGGCTGG 840
GGCTCGTGGG CGCCCCAGCT GGGCGCTATG TCGGGCGATA TCGTCGGTGC GATCCGCTAA 900
