U.S. patent application number 11/643714 was filed with the patent office on 2007-04-26 for method of preventing t cell-mediated responses by the use of the major histocompatibility complex class ii analog protein (map protein) from staphylococcus aureus.
Invention is credited to Eric Brown, Magnus Hook, Lawrence Lee.
Application Number | 20070092533 11/643714 |
Document ID | / |
Family ID | 22989503 |
Filed Date | 2007-04-26 |
United States Patent
Application |
20070092533 |
Kind Code |
A1 |
Brown; Eric ; et
al. |
April 26, 2007 |
Method of preventing T cell-mediated responses by the use of the
major histocompatibility complex class II analog protein (map
protein) from Staphylococcus aureus
Abstract
A method of immunomodulating the T cell response in
Staphylococcal bacteria is provided wherein an effective amount of
the Map protein from Staphylococcus aureus is administered to a
host to prevent or suppress the T cell response. The present method
may be utilized with either the Map protein or an effective
subdomain or fragment thereof such as the Map19 protein. The
present invention is advantageous in that suppression or prevention
of the T cell response in a host can prevent or ameliorate a wide
variety of the pathogenic conditions such as toxic shock syndrome
and other diseases associated with the T cell-mediated response
caused by staphylococcal infection, and is particularly useful in
cases where patients have had chronic or recurrent infections from
S. aureus or other staphylococcal bacteria.
Inventors: |
Brown; Eric; (Houston,
TX) ; Lee; Lawrence; (Houston, TX) ; Hook;
Magnus; (Houston, TX) |
Correspondence
Address: |
STITES & HARBISON PLLC
1199 NORTH FAIRFAX STREET
SUITE 900
ALEXANDRIA
VA
22314
US
|
Family ID: |
22989503 |
Appl. No.: |
11/643714 |
Filed: |
December 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10041775 |
Jan 10, 2002 |
7153515 |
|
|
11643714 |
Dec 22, 2006 |
|
|
|
60260523 |
Jan 10, 2001 |
|
|
|
Current U.S.
Class: |
424/190.1 |
Current CPC
Class: |
C07K 14/31 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
424/190.1 |
International
Class: |
A61K 39/02 20060101
A61K039/02 |
Claims
1. A method of preventing or modulating a T cell-mediated response
in a host comprising administering to the host an isolated S.
aureus Map protein having an amino acid sequence as set forth in
SEQ ID NO:6 in an amount effective to prevent or modulate said T
cell-mediated response in the host.
2. The method according to claim 1 wherein the T cell-mediated
response is delayed-type hypersensitivity (DTH).
3. The method according to claim 1 wherein the Map protein is
encoded by a nucleotide sequence according to SEQ ID NO:5.
4. A method of treating or preventing a pathological condition
associated with overstimulation of T cells in a human or animal
patient comprising administering to the patient an isolated S.
aureus Map protein having an amino acid sequence according to SEQ
ID NO:6 in an amount effective to treat or prevent said condition
associated with overstimulation of T cells.
5. The method according to claim 4 wherein the condition associated
with overstimulation of T cells is toxic shock syndrome.
6. The method according to claim 4 wherein the Map protein is
encoded by a nucleotide sequence according to SEQ ID NO:5 or
degenerates thereof.
7. A method of treating or preventing a T cell lymphoproliferative
disease in a human or animal patient comprising administering to
the patient an isolated S. aureus Map protein having the amino acid
sequence according to SEQ ID NO:6, in an amount effective to treat
or prevent a T cell lymphoproliferative disease.
8. The method according to claim 7 wherein the Map protein is
encoded by a nucleotide sequence according to SEQ ID NO:5 or
degenerates thereof.
9. A pharmaceutical composition for preventing or modulating a T
cell-mediated response to a staphylococcal infection comprising an
isolated S. aureus Map protein having the amino acid sequence
according to SEQ ID NO:6 in an amount effective to prevent or
modulate a T cell-mediated response and a pharmaceutically
acceptable vehicle, carrier or excipient.
10. The pharmaceutical composition of claim 6 wherein the Map
protein is encoded by a nucleotide sequence according to SEQ ID NO:
5 or degenerates thereof.
11. A method of reducing or eliminating T cell-mediated
proliferation in a patient in need of such treatment comprising
administering to the patent an isolated S. aureus Map protein
according to SEQ ID NO: 6 in an amount effective to reduce or
eliminate T cell proliferation in the patient following a
staphylococcal infection.
12. The method according to claim 11 wherein the Map protein is
encoded by a nucleotide sequence according to SEQ ID NO:5 or
degenerates thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 10/041,775, filed Jan. 10, 2002, and claims
the benefit of U.S. Provisional application Ser. No. 60/260,523,
filed Jan. 10, 2001, said applications incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates in general to the utilization
of major histocompatibility complex class II analog protein, or
"Map" protein, and its biologically effective fragments and domains
thereof, in therapeutic methods to combat bacterial infection and
associated immunological disruption, and in particular to the use
of the Map protein and effective or active fragments thereof,
including the Map19 protein, in methods of suppressing or
modulating T cell-mediated responses to infection from
staphylococcal bacteria such as Staphylococcus aureus, so as to be
useful in the treatment of and protection against S. aureus
infections.
BACKGROUND OF THE INVENTION
[0003] Staphylococcus aureus (SA) is an opportunistic pathogen that
can cause a wide spectrum of infections from superficial local skin
infections to life-threatening systemic infections that can affect
internal organs and tissues. In addition, bacterial arthritis, as
well as acute and chronic osteomyelitis caused by haematogenous
spread or by direct inoculation in open trauma or surgical
intervention such as internal fixation or joint replacement, affect
hundreds of thousands of patients each year (1-6). SA is also a
major cause of infections associated with indwelling medical
devices, such as catheters and prosthesis (6). The cost to society
in patient care, which often involves extended hospital stays and
repeated surgery, can be estimated at several billion dollars per
year. With the documented emergence of multidrug resistance SA
strains, the threat of this widely distributed pathogen is now
appreciated and novel therapies for treatment and prevention are
needed.
[0004] The successful colonization of the host is a process
required for most microorganisms, including S. aureus, to cause
infections in animals and humans. Microbial adhesion is the first
crucial step in a series of events that can eventually lead to
disease. Pathogenic microorganisms colonize the host by attaching
to host tissues or serum conditioned implanted biomaterials, such
as catheters, artificial joints, and vascular grafts, through
specific adhesins present on the surface of the bacteria.
MSCRAMM.TM.s (Microbial Surface Components Recognizing Adhesive
Matrix Molecules) are a family of cell surface adhesins that
recognize and specifically bind to distinct components in the
host's extracellular matrix. Once the bacteria have successfully
adhered and colonized host tissues, their physiology is
dramatically altered and damaging components such as toxins and
proteolytic enzymes are secreted. Moreover, adherent bacteria often
produce a biofilm and quickly become more resistant to the killing
effect of most antibiotics.
[0005] S. aureus is thus known to express a repertoire of different
MSCRAMM.TM.s that can act individually or in concert to facilitate
microbial adhesion to specific host tissue components. A search for
such MCSRAMM's which recognized host components uncovered a 72-kDa
protein identified as the major histocompatibility complex class II
analog protein, or "Map" protein, a surface localized protein
expressed by virtually every S. aureus strain (7). Cloning and
sequencing of the gene encoding the Map protein revealed a protein
consisting of roughly 110-amino acid-long domains repeated six
times with each domain containing a 31 amino acid-long subdomain
with homology to MHC Class II. If conservative amino acid
substitutions were included, the respective subdomains were 61, 65,
52, 59, 52 and 45% similar to the amino-terminal end of the b chain
of many MHC class II proteins from different mammalian species
(8).
[0006] However, previous studies varied with regard to how the Map
protein affected immune function, and thus it would be highly
desirable to utilize the Map protein so as to affect host immune
responses, such as on T cells, so as to reduce or prevent infection
from bacteria such as S. aureus.
SUMMARY OF THE INVENTION
[0007] Accordingly, it is an object of the present invention to
provide a method of utilizing the S. aureus Map protein, or
effective fragments and domains thereof, to immunomodulate S.
aureus so as to protect against and treat staphylococcal
infections.
[0008] It is also an object of the present invention to provide and
utilize binding subdomains of the S. aureus MAP protein, including
the Map19 protein, in methods of protecting against staphylococcal
infections or reducing their persistence and virulence.
[0009] It is also an object of the present invention to provide
isolated Map proteins and active fragments and regions therefrom to
prevent T cell-mediated responses in Staphylococcal bacteria and
thus reduce or prevent occurrence and spread of bacterial
diseases.
[0010] These and other objects are provided by virtue of the
present invention which provides methods of utilizing the Map
protein and/or its binding subdomains or other effective fragments
thereof, in immunomodulating Staphylococcal bacteria so as to be
able to treat or prevent Staphylococcus infection. Use and
administration of an effective amount of the Map protein or its
effective subdomains or fragments thereof can thus be utilized in a
host to reduce T cell proliferation and achieve a significant
reduction in T cell-mediated processes such as delayed-type
hypersensitivity (DTH). Suitable compositions and vaccines based on
the isolated MAP protein and its effective regions and subdomains,
such as the Map10 and Map19 proteins, as well as methods for their
use, are also contemplated by the present invention.
[0011] These embodiments and other alternatives and modifications
within the spirit and scope of the disclosed invention will become
readily apparent to those skilled in the art from reading the
present specification and/or the references cited herein, all of
which are incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0012] FIGS. 1a-1c are graphic representations showing the
Map-induced inhibition of DTH in accordance with the present
invention. DpbA-immunized mice were treated with either native Map
(A) or recombinant Map19 (B-C) on the day of immunization (day 0)
and on days 2, 4, and 6 post immunization. On day 7, BALB/c (A-B)
and C3H/Hen (C) mice were challenged with DbpA and footpads were
measured 0 and 24 h after challenge. Mice treated with supernatant
from Map.sup.+SA (A) or recombinant Map19 (B-C) had a significantly
reduced DTH response compared to immunized and challenged mice
(p<0.0001*; Student's t test). Data are expressed as the
mean.+-.SE of 5 mice.
[0013] FIG. 2 shows the Map19 dose-response for inhibition of DTH
in accordance with the present invention. DbpA-immunized mice were
treated with various doses of Map19 (25-200 .mu.g) or ACE19 (200
.mu.g) as described previously. On day 7, mice were challenged with
DbpA and footpads were measured 0 and 24 h after challenge.
Significant values are indicated by an * (Students t test). Data
are expressed as the mean.+-.SE of 5 mice.
[0014] FIG. 3 is a graphic representation of tests showing that
adoptively transferred T cells from Map-treated mice do not elicit
a DTH response in naive mice. DbpA-immunized mice were treated with
either Map19 or SdrF as described above. On day 7, mice were
sacrificed and spleens were harvested and enriched for T cells by
nylon wool purification. 5.times.10.sup.7 cells were injected i.p.
into syngeneic recipients. 24 h later, recipient mice were
challenged with DbpA and the DTH response was assessed as described
above. DbpA-immunized and DbpA-immunized, SdrF-treated mice
developed a significant DTH response compared to unimmunized but
challenged mice (p<0.04*; Student's t test). DbpA-immunized,
Map19-treated mice had a significantly reduced DTH response
compared to the other treatment groups (p<0.001**; Student's t
test). Data are expressed as the mean.+-.SE of 5 mice.
[0015] FIG. 4 is a graphic representation of the Map-induced
inhibition of T cell proliferation using the method of the present
invention. In this test, BAT2.2 T cell proliferation was measured
after 40 h in culture in the presence of APCs and antigen in the
presence of various proteins. 100 .mu.g of each protein was added
per well. Data are expressed as the mean absorbance.+-.SE of
triplicate wells.
[0016] FIG. 5 shows Map-induced apoptosis of BAT2.2 T cells in
accordance with the present invention. BAT2.2 cells (5 U I-2/ml)
were incubated in media alone (lane 1), or in the presence of
either 100 .mu.g Map19 (lane 2) or ACE40 (lane 3). DNA from U937
cells were used as a positive control (lane 5).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0017] In accordance with the present invention, there are provided
methods and immunogenic compositions for suppressing, preventing or
immunomodulating T cell-mediated responses caused by staphylococcal
bacteria by the use of an immunogenic, immunologically effective
amount of an isolated natural or recombinant Map protein or an
immunologically active fragment or domain therefrom such as the
Map19 protein. The MAP protein is a surface localized protein
expressed by virtually every S. aureus strain. McGavin et al (7)
originally identified the 72 kDa surface protein, from S. aureus
strain FDA 574, that binds a variety of host proteins including
BSP, fibrinogen, fibronectin, vitronectin, and thrombospondin. The
gene, designated map, was cloned and sequenced (U.S. Pat. No.
5,648,240, incorporated herein by reference). The gene encoding the
MAP protein is shown in SEQ ID NO:5 along with the region coding
for the MAP protein (SEQ ID NO: 6).
[0018] Reinfection of humans with SA is one of the hallmarks of
diseases caused by this pathogen and the roles of acquired and
innate immunity in protection against infection vary with the many
manifestations of disease resulting from SA infections (25-28).
While SA infections affecting the skin appear to be exacerbated by
strong cellular responses, it is clear that cellular immunity is
critical in orchestrating the clearance of systemic SA infections
and in preventing reinfection with the same or similar pathogens
(29-33). One possible reason for recurring SA infections is the
reduction in chemotactic, phagocytic and bactericidal functions of
polymorphonuclear leukocytes from patients with chronic or
recurrent SA infections (27, 30, 33). Whether this is a function of
the bacterial infection or a preexisting condition in these
individuals is not known (27, 30, 33).
[0019] Regardless, the presence of SA-immunoregulatory molecules
suggests that these bacteria have the potential to counteract or
evade host defense mechanisms. Both superantigens and protein A
produced by SA during an infection serve immune-evasion functions.
Superantigens can activate between about 5-20% of T cells by
directly binding to both the major histocompatibility complex (MHC)
class II molecules on antigen-presenting cells and to the T cell
receptor (TCR) on T cells. This interaction can initiate apoptosis
in T cells and thymocytes in vivo and in vitro. The in vivo effects
of such massive T cell stimulation often results in disease (e.g.,
toxic shock syndrome and food poisoning in humans) (34). Protein A,
while less harmful to the host compared to superantigens, may also
serve as a means of immune evasion by binding to the Fc fragment of
immunoglobulins (i.e. IgG) resulting in loss of antibody
function.
[0020] As shown herein, the Map protein and its effective regions
or subdomains such as Map19 appear to function as an immune
modulator with the capacity to affect host immune responses during
SA infections. In addition to its role as a bacterial adhesin, Map
has the capacity to interfere with T cell activation and/or
proliferation facilitating SA survival in mammals (8, 11, 24, 35,
36) and can serve to potentiate survival in mammals of varied
genetic backgrounds.
[0021] Studies in accordance with the present invention have shown
that Map serves as an immunomodulatory protein as evidenced in
double infection studies in which a primary infection with
Map.sup.-SA conferred significant protection against reinfection
with Map.sup.+SA. This contrasts significantly with SA-induced
pathology from mice receiving primary and secondary infections with
Map.sup.+SA. Accordingly, T cell-mediated responses in
Map.sup.+SA-infected mice appear to be abrogated by the presence of
Map compared to Map.sup.-SA-infected mice which develop
cell-mediated immunity over the course of infection. Map.sup.-SA
infection, which is cleared over time, results in a memory response
capable of controlling a secondary Map.sup.+SA infection. That a
primary Map.sup.-SA infection conferred significant but not
complete protection against Map.sup.+SA challenge suggested that
the delicate balance between an anamnestic response and
Map-mediated immunomodulation could be affected by the challenge
dose. Inhibition of DTH responses directly or as a result of
adoptively transferred T cells from Map-treated mice combined with
the in vitro effects of Map on T cell proliferation, have evidenced
a direct involvement of Map with T cells resulting in apoptosis.
Flow cytometric analysis of fluorescein isothiocyanate
(FITC)-labeled Map19 revealed binding to 100% of BAT2.2 T
cells.
[0022] Additional tests of nylon wool-purified naive T cells
cultured in the presence of Map19 were not induced to either
proliferate or undergo apoptosis. Furthermore, proliferation of
naive T cells as a result of incubation with concanavalin A or by
antibody-cross-linking of the TCR was not inhibited by Map. This
result evidenced that activated T cells but not naive T cells are
susceptible to Map and that T cell proliferation via `non
classical` pathways bypasses the Map-mediated inhibition of T cell
proliferation. The present data provides evidence that Map
functions as an immunoregulatory protein during SA infections and
it appears that this protein is yet another weapon used by SA to
escape immune recognition and clearance.
[0023] Accordingly, in accordance with the present invention,
methods of utilizing an immunologically effective amount of the Map
protein or its effective regions or subdomains such as Map19 are
provided which can be used to treat or prevent T cell-mediated
responses to staphylococcal bacteria, such as DTH.
[0024] In addition, the administration of immunologically effective
amounts of an isolated and/or purified S. aureus Map protein or one
of its effective regions such as Map19 can be utilized in methods
of treating or preventing pathological conditions associated with
overstimulation of T cells such as toxic shock syndrome and food
poisoning. In accordance with the present invention, a method is
provided which comprises administering to a human or animal patient
in need of such treatment an immunologically effective amount of an
isolated natural or recombinant Map protein. By Map protein is
meant the whole natural or recombinant Map protein, or any
effective or otherwise immunologically active fragment, fraction,
domain, subdomain or region thereof which also has effective
immunogenic properties so as to prevent or suppress a T
cell-mediated response in the patient. In accordance with the
invention, one such region is the Map19 protein, the nucleic acid
sequence of which is provided herein as SEQ ID NO:1, and the amino
acid sequence is provided as SEQ ID NO:2. Accordingly, the present
invention also relates to methods of administering immunologically
effective amounts of an isolated and/or purified S. aureus Map19
protein so as to be utilized in methods of treating or preventing
pathological conditions associated with T cell proliferation or
other T cell-mediated responses such as those caused by infection
from S. aureus.
[0025] As would be recognized by one of ordinary skill in the art,
immunogenic compositions containing an immunogenically effective
amount of the Map protein or the Map19 protein can be prepared and
administered to a human or animal patient in need of such
treatment, particularly those patients requiring treatment or
prevention of pathological conditions and other diseases associated
with the T cell-mediated response, such as patients with chronic or
recurrent SA infections. By effective amount, it would be
recognized that the preferred dose for administration of a
composition containing the Map or Map19 protein in accordance with
the present invention is that amount will be effective in
preventing or modulating the T cell response caused by a
staphylococcal infection, and one would readily recognize that this
amount will vary greatly depending on the nature of the infection
and the condition of a patient. Accordingly, an "effective amount"
of a pharmaceutical or immunological agent to be used in accordance
with the invention is intended to mean a nontoxic but sufficient
amount of the agent, such that the desired prophylactic,
immunological or therapeutic effect is produced.
[0026] As one of skill in the art would recognize, the exact amount
of an effective composition that is required will thus vary from
subject to subject, depending on the species, age, and general
condition of the subject, the severity of the condition being
treated, the particular carrier or adjuvant being used and its mode
of administration, and the like. Accordingly, the "effective
amount" of any particular composition will vary based on the
particular circumstances, and an appropriate effective amount may
be determined in each case of application by one of ordinary skill
in the art using only routine experimentation. The dose should be
adjusted to suit the individual to whom the composition is
administered and will vary with age, weight and metabolism of the
individual. The compositions may additionally contain stabilizers
or pharmaceutically acceptable preservatives, such as thimerosal
(ethyl(2-mercaptobenzoate-S) mercury sodium salt) (Sigma Chemical
Company, St. Louis, Mo.).
[0027] The compositions of the invention may also be used as
vaccines which will be useful in generating antibodies in a host
patient to treat or prevent a staphylococcal infection. As would be
recognized by one skilled in this art, a vaccine may be prepared
for administration in a number of suitable ways, such as by
parenteral (i.e., intramuscular, intradermal or subcutaneous)
administration or nasopharyngeal (i.e., intranasal) administration,
and the general composition comprises the effective isolated Map or
Map19 protein along with a pharmaceutically acceptable vehicle,
carrier or excipient. In one such mode, the vaccine is injected
intramuscularly, e.g., into the deltoid muscle, however, the
particular mode of administration will depend on the nature of the
bacterial infection and the condition of the patient. The vaccine
is thus preferably combined any of a variety of pharmaceutically
acceptable vehicles, carriers or excipients, such as water or a
buffered saline, that would be well known to those of ordinary
skill in the art. In addition, the vaccine may be lyophilized for
resuspension at the time of administration or in solution.
[0028] In carrying out the method of the present invention, the
isolation and/or purification of the Map protein or of the Map19
protein, or other active fragments or domains of the Map protein,
can be accomplished in a number of suitable ways as would be
recognized by one skilled in the art. For example, both the Map
protein (SEQ ID NO: 6) and the Map19 protein (SEQ ID NO: 2) may be
produced recombinantly using conventional techniques well known in
the industry. With regard to the Map protein, the production of
this protein is disclosed for example in U.S. Pat. No. 5,648,240,
incorporated herein by reference. With regard to the Map19 protein,
one such suitable method would be through expression in E. coli
(e.g., JM101 from Qiagen, Chatsworth, Calif.) harboring the
appropriate plasmid (11-16). In this method, E. coli was grown at
37.degree. C. in LB containing the appropriate antibiotics until
they reached an A.sub.600 of 0.6 (17).
Isopropyl-.beta.-D-thiogalactopyranoside (IPTG) (Life Technologies)
was added to a final concentration of 0.2 mM, and the cells were
incubated at 37.degree. C. for an additional 4 hours. Cells from a
1 L culture were harvested by centrifugation and resuspended in 10
ml "binding buffer" (BB) (20 mM Tris HCl, 0.5 M NaCl, 15 mM
imidazole, pH 8.0) and lysed in a French pressure cell at 11,000
pounds/inch.sup.2 (13). The lysate was centrifuged at
40,000.times.g for 15 min and the supernatant filtered through a
0.45 .mu.m filter. A 1 ml iminodiacetic acid Sepharose column
(Sigma, St. Louis, Mo.) was charged with 75 mM NiCl.sub.2.6H.sub.2O
and equilibrated with BB. The filtered supernatant was applied to
the column and washed with 10 volumes of BB, then 10 volumes of BB
containing 60 mM imidazole. The bound proteins were eluted with BB
containing 200 mM imidazole, dialyzed against PBS containing 10 mM
EDTA, then dialyzed against PBS (13). Protein concentrations were
determined by the Bicinchoninic Acid (BCA) Protein Assay (Pierce)
and proteins were stored at -20.degree. C. until use.
[0029] In addition to obtaining isolates of the Map protein through
recombinant means, natural isolates of the Map protein may be
obtained for use in the present invention by a number of suitable
means as well. For example, the natural Map protein can be
extracted using standard methods. In one such suitable method,
Map.sup.+SA and Map.sup.-SA were grown overnight as described
above. Bacteria were pelleted by centrifugation and resuspended in
1 M LiCl (one tenth of the original media volume). The suspension
was incubated at 42.degree. C. with shaking for two hours. The
bacteria were pelleted and the supernatant was removed and
quantified for protein by UV spectrophotometry using 1 M LiCl as a
blank. Extracted proteins were diluted to 0.2-mg/ml in PBS and
passed through a 0.45-micron filter for sterilization prior to i.p.
injection (7).
[0030] As indicated above, and in the examples below, the method of
the present invention is carried by administering effective amounts
to human or animal patients so as to achieve the desired
prophylactic, immunological or therapeutic effect immunization
against staphylococcal infections, e.g. the reduction or modulation
of the T cell response thereto, and such effective amounts would be
determined through routine means as indicated above for a
particular patient based on factors such as type and size of
patient, type of infection, level of virulence, etc.
[0031] In short, the present invention can thus be utilized
advantageously as a means of treating or preventing otherwise
persistent, chronic or recurrent infections from staphylococcal
bacteria such as S. aureus and will be useful in suppressing or
modulating the T cell-mediated responses in a human or animal
patient so as to treat, prevent or ameliorate a wide variety of
conditions caused by the T cell-mediated responses to
staphylococcal infection.
EXAMPLES
[0032] The following examples are provided which exemplify aspects
of the preferred embodiments of the present invention. It should be
appreciated by those of skill in the art that the techniques
disclosed in the examples which follow represent techniques
discovered by the inventors to function well in the practice of the
invention, and thus can be considered to constitute preferred modes
for its practice. However, those of skill in the art should, in
light of the present disclosure, appreciate that many changes can
be made in the specific embodiments which are disclosed and still
obtain a like or similar result without departing from the spirit
and scope of the invention.
Example 1
[0033] Overview
[0034] Staphylococcus aureus (SA) expresses a 72-kDa protein with
the capacity to bind to a variety of extracellular matrix
components (ECM), suggesting that at least one role for this
protein involves adherence and colonization of host tissues.
Analysis of Map, however, also revealed homologies to a segment of
the peptide-binding groove of the b chain of the major
histocompatability class (MHC) II mammalian proteins. Map-deficient
SA (Map.sup.- SA) were generated to examine Map's role in the
infection process. Map.sup.-SA-infected mice presented with
significantly reduced levels of arthritis, osteomylitis, and
abscess formation compared to Map.sup.+SA-infected control animals.
Furthermore, Map.sup.-SA-infected mice challenged with Map.sup.+SA
were significantly protected against SA-induced pathology compared
to mice infected and challenged with Map.sup.+SA. Native and
recombinant forms of Map were tested for their ability to interfere
with T cell response in vivo and in vitro. T cells or mice treated
with recombinant Map had reduced levels of T cell proliferation and
significant reduction of the delayed-type hypersensitivity (DTH)
response to challenge antigen, respectively. The data presented
here evidence a role for Map as an immunomodulatory protein which
may play a role in persistent SA infections and thus may function
to potentiate SA survival in mammals by affecting the host's
cellular immune responses.
[0035] Background
[0036] Staphylococcus aureus (SA) is an opportunistic pathogen that
can cause a wide spectrum of infections from superficial local skin
infections to life-threatening systemic infections that can affect
internal organs and tissues. In addition, bacterial arthritis, as
well as acute and chronic osteomyelitis caused by haematogenous
spread or by direct inoculation in open trauma or surgical
intervention such as internal fixation or joint replacement, affect
hundreds of thousands of patients each year (1-6). SA is also a
major cause of infections associated with indwelling medical
devices, such as catheters and prosthesis (6). The cost to society
in patient care, which often involves extended hospital stays and
repeated surgery, can be estimated at several billion dollars per
year. With the documented emergence of multidrug resistance SA
strains, the threat of this widely distributed pathogen is now
appreciated and novel therapies for treatment and prevention are
needed.
[0037] A search for SA adhesins recognizing host components
uncovered a 72-kDa protein capable of binding a variety of host
proteins (7). Cloning and sequencing of this gene revealed a
protein consisting of 110-amino acid-long domains repeated six
times with each domain containing a 31 amino acid-long subdomain
with homology to MHC class II. If conservative amino acid
substitutions were included, the respective subdomains were 61, 65,
52, 59, 52, and 45% similar to the amino-terminal end of the
.quadrature. chain of many MHC class II proteins from different
mammalian species (8).
[0038] The present work supports a role for Map as an
immunomodulatory protein. Mice infected with SA genetically
manipulated to be deficient in Map (Map.sup.-SA) have significantly
reduced levels of arthritis and abscess formation (heart and
kidneys) following reinfection with wild-type SA (Map.sup.+SA)
compared to mice infected and reinfected with Map.sup.+SA or mice
receiving a single inoculum of Map.sup.+SA. Evidence linking
interactions between Map and T cells came from experiments in which
nude mice were infected with Map.sup.-SA. The severity of
osteomyelitis and arthritis was greater in nude mice compared to
genotype controls infected with SA.sup.-Map, suggesting not only a
role for T cells in protection against SA infections but also a
role for Map in circumventing T cell-mediated immunity. Testing the
hypothesis that Map acts to interfere with cellular immunity,
various T cell-mediated responses were measured in vivo and in
vitro in the presence of Map. DTH, which is a CD4.sup.+-mediated
response, was significantly reduced in Map-treated mice and T cell
proliferation in vitro was significantly reduced in the presence of
Map, likely as a function of Map-induced apoptosis. These data
evidence that Map is a virulence factor whose abilities to
potentially alter T cell function in vivo may affect SA persistence
and survival and may function in facilitating recurring SA
infections.
Materials and Methods
[0039] Mice
[0040] Specific pathogen-free (MTV.sup.-) BALB/c and C3H/Hen mice
were purchased from Harlan Sprague Dawley, Indianapolis, Ind. The
animals were maintained in facilities approved by the American
Association for Accreditation of Laboratory Animal Care in
accordance with current regulations and standards of the United
States Department of Agriculture, Department of Health and Human
Services, and National Institutes of Health. All animal procedures
were approved by the Institutional Animal Care and Use Committee.
Female mice were 8-10 weeks old at the start of each
experiment.
[0041] Expression and Purification of Recombinant Proteins
[0042] Recombinant Map19, DbpA SdrF, M55, CNA, ACE19 and ACE40 were
expressed in E. coli (JM101) (Qiagen, Chatsworth, Calif.) harboring
the appropriate plasmid (11-16). E. coli was grown at 37.degree. C.
in LB containing the appropriate antibiotics until they reached an
A.sub.600 of 0.6 (17). Isopropyl-.beta.-D-thiogalactopyranoside
(IPTG) (Life Technologies) was added to a final concentration of
0.2 mM, and the cells were incubated at 37.degree. C. for an
additional 4 hours. Cells from a 1 L culture were harvested by
centrifugation and resuspended in 10 ml "binding buffer" (BB) (20
mM Tris HCl, 0.5 M NaCl, 15 mM imidazole, pH 8.0) and lysed in a
French pressure cell at 11,000 pounds/inch.sup.2 (13). The lysate
was centrifuged at 40,000.times.g for 15 min and the supernatant
filtered through a 0.45 .mu.m filter. A 1 ml iminodiacetic acid
Sepharose column (Sigma, St. Louis, Mo.) was charged with 75 mM
NiCl.sub.2.6H.sub.2O and equilibrated with BB. The filtered
supernatant was applied to the column and washed with 10 volumes of
BB, then 10 volumes of BB containing 60 mM imidazole. The bound
proteins were eluted with BB containing 200 mM imidazole, dialyzed
against PBS containing 10 mM EDTA, then dialyzed against PBS (13).
Protein concentrations were determined by the Bicinchoninic Acid
(BCA) Protein Assay (Pierce) and proteins were stored at
-20.degree. C. until use.
[0043] Quantitation of S. aureus and Intravenous Injections
[0044] Map.sup.+SA and Map.sup.-SA (strain Newman 8325) were grown
overnight in Lennox broth (LB) (Difco, Detroit, Mich.) media at
37.degree. C. with shaking and used in all infection experiments.
50 .mu.l of this culture was used to inoculate 10 ml of fresh LB in
a 250 ml Erlenmeyer flask. The new cultures were grown as above
until the optical density reached 0.5 at 600 nm with a 1-cm quartz
cuvette. Aliquots of each culture were quantified for colony
forming units (CFU). The remainder of each culture was washed three
times in sterile PBS. The cultures, based on prior growth-curve
determinations, were diluted to approximate 2.times.10.sup.7
CFU/ml. Mice were in injected i.v. with 1.times.10.sup.7 S. aureus
in 0.5 ml PBS and monitored for up to eight weeks. At the
conclusion of the experiment, mice were sacrificed and the joints
were examined histologically for arthritis development as described
previously (18, 19).
[0045] Extraction of Map from Staphylococcus aureus
[0046] Map.sup.+SA and Map.sup.-SA were grown overnight as
described above. Bacteria were pelleted by centrifugation and
resuspended in 1 M LiCl (one tenth of the original media volume).
The suspension was incubated at 42.degree. C. with shaking for two
hours. The bacteria were pelleted and the supernatant was removed
and quantified for protein by UV spectrophotometry using 1 M LiCl
as a blank. Extracted proteins were diluted to 0.2-mg/ml in PBS and
passed through a 0.45-micron filter for sterilization prior to i.p.
injection (7).
[0047] In Vitro Proliferation of BAT2.2 T Cells
[0048] The Borrelia burgdorferi-specific T cell line BAT2.2 was
stimulated with whole, inactive Borrelia and antigen presenting
cells (APC) as described previously (18, 20). Briefly,
1.times.10.sup.5 BAT2.2 T cells were cultured in 96-well
flat-bottom plates (Costar, Cambridge Mass.) along with
3.times.10.sup.5 mitomycin-treated APC in complete medium (CTL)
(RPMI 1640 containing 2 mM L-glutamine, 100 units/ml penicillin,
100 .mu.g/ml streptomycin, 50 .mu.g/ml gentamicin, 0.2 mM
nonessential amino acids, 11 .mu.g/ml sodium pyruvate, 0.02 M
N-2-hydroxyethylpiperaxine-N'-2ethanesulfonic acid, and
5.times.10.sup.-5 N 2-mercaptoethanol+10% heat-inactivated fetal
bovine serum), and Borrelia (2 .mu.g) in the presence of various
proteins. Each treatment group was done in triplicate in a final
volume of 200 .mu.l complete medium. 10, 50, and 100 .mu.g of each
protein was added to each well and the T cells were allowed to
proliferate for 24-48 hours at 37.degree. C. 4 h before the end of
the proliferation period, 20 .mu.l/well of
3-{4,5-Dimethylthizol-2-y}-2,5diphenyl-tetraxolium bromide (MTT) (5
mg/ml) was added to each well. After 4 h incubation at 37.degree.
C., 100 .mu.l of solubilization buffer (0.04 N HCl in isopropanol)
was added to each well and absorbance measured at 590 nm. Data are
expressed as mean.+-.SE of the mean of triplicate wells.
[0049] Delayed Type Hypersensitivity (DTH) Assay
[0050] Mice were immunized with 20 .mu.g of decorin binding protein
A (DbpA) in complete Freund's adjuvant (day 0) (19). 7 days post
immunization, mice were challenged with 2.5 .mu.g DbpA (13). DbpA
was administered in 50 .mu.l of PBS. At the time of immunization,
days 2, 4, and 6 post immunization, mice were injected i.p. with
100 .mu.g of native Map (N-Map) extracted from Map.sup.+SA,
supernatant from Map.sup.-SA, or with 100 .mu.g of the recombinant
proteins Map19, SdrF, M55 or ACE40 in 500 .mu.l of PBS (11-15, 21).
The footpads were measured before challenge and 24 h later, using a
spring-loaded micrometer (Mitutoyo, Tokyo, Japan). Mice were
anesthetized with Metofane.TM. during footpad measurements
(22).
[0051] Adoptive T Cell Transfer
[0052] BALB/c mice (5 mice/group) were immunized with DbpA and were
treated with recombinant Map19 or recombinant ACE19 as described
above. The day after the last Map19 or ACE40 treatment mice were
sacrificed and the spleens from each treatment group were enriched
for T cells by passage over nylon wool columns as described
previously (20). 24 h after i.p. injection of T cells
(5.times.10.sup.7 nylon wool-enriched T cells/mouse in 500 .mu.l
complete media), mice were challenged in the hind footpads with
DbpA and the DTH response was assessed as described above.
[0053] Map-Induced Apoptosis of BAT2.2 Cells
[0054] 2.times.10.sup.6 BAT2.2 T cells/well (5 U I-2/ml) were
incubated in the presence of Map19 or ACE19 in a final volume of
200 .mu.l complete media and examined for apoptosis using an
Apoptotic DNA Ladder Kit (Roche Molecular Biochemicals,
Indianapolis, Ind.) according to manufacturers instructions. 100
.mu.g of each protein was used and apoptosis measured after a 24 h
incubation at 37.degree. C. DNA was treated with 2 .mu.g/ml RNase
(DNase free) for 20 min. at room temperature before examination by
agarose gel electrophoresis.
[0055] Flow Cytometry
[0056] Nylon wool enriched T cells (1.times.10.sup.6/tube) were
washed in PBS containing 3% FBS and stained with the following
monoclonal antibodies: fluorescein isothiocyanate (FITC)-conjugated
anti-mouse CD8a (Ly2) and phycoerythrin (PE)-conjugated anti-mouse
CD4 (L3T4) (PharMingen, San Diego, Calif.). The cells were
incubated with the directly conjugated antibodies for 1 h at
4.degree. C. and then washed and analyzed on a Coulter EpicProfile
(Coulter Corp., Miami, Fla.).
Results
[0057] Experimental S. aureus Infection
[0058] Infection parameters that resulted in high degrees of
arthritis incidence were used to examine what role Map played in SA
infection (23). BALB/c mice were injected in the tail i.v. with
1.times.10.sup.7 SA and sacrificed 4 weeks later for histological
examination of hind tibiotarsal joints. These preliminary
experiments revealed that Map.sup.-SA-infected mice had both a
reduced frequency and severity of arthritis compared to
Map.sup.+SA-infected controls. The hypothesis that Map acted as an
immunomodulator resulting in impaired immunity to SA with a
concomitant inability to respond to a challenge infection was
tested by infecting mice with Map.sup.-SA and Map.sup.+SA
respectively, and challenging both groups with Map.sup.+SA 4 weeks
later. Significant differences were observed in abscess formation
in hearts and kidneys between the Map.sup.-/Map.sup.+-infected
group and the Map.sup.+/Map.sup.+- and -/Map.sup.+-infected groups
(Table I). Less than 50% of hearts and 25% of kidneys from
Map.sup.-/Map.sup.+ infected mice presented with abscesses compared
to >75% abscess formation in both hearts and kidneys from
Map.sup.+/Map.sup.+ and -/Map.sup.+ infected mice (Table I).
Significant differences were also observed in arthritis and
osteomyelitis scores and frequencies (Table II). Arthritis was
prevalent in 54% of mice infected with Map.sup.-/Map.sup.+ compared
to >80% incidence in Map.sup.+/Map.sup.+ and -/Map.sup.+
infected mice (Table II). The mean arthritis and osteomyelitis
scores recorded were also more than 2 times less in
Map.sup.-/Map.sup.+ infected mice compared to scores from
Map.sup.+/Map.sup.+- and -/Map.sup.+-infected mice (Table II).
[0059] Map-Mediated Inhibition of Delayed-Type-Hypersensitivity
(DTH)
[0060] The similarity between Map and the peptide-binding region of
class II MHC combined with the high levels of Map recoverable from
the surface of SA prompted experiments designed to address the
question regarding the potential role of Map on cellular immunity
(7, 8). DTH responses are initiated and mediated by CD4.sup.+ T
cells in response to recall antigens and result in specific,
measurable inflammation at the site of challenge. Mice immunized
with recombinant decorin-binding protein A (DbpA) emulsified in
complete Freund's adjuvant (CFA) developed a significant DTH
response to DbpA as measured by footpad swelling 7 days post
immunization (FIG. 1) (19). However, mice treated with native Map
(.sup.+Map Supernatant) or recombinant Map19 on the day of
immunization (day 0) and days 2, 4 and 6 post immunization had a
significantly reduced DTH response to DbpA compared to control mice
(FIG. 1). Neither supernatants from Map.sup.-SA (FIG. 1a) or
recombinant control protein ACE19 had any measurable effects on the
DTH response to DbpA (FIG. 1b-c). Map19's inhibitory effects were
not affected by genetic differences since the DTH response was
diminished in both BALB/c and C3H/Hen mice following immunization
and challenge (FIGS. 1b and c, respectively).
[0061] Map Time Course and Dose Response for DTH Inhibition
[0062] Both the induction and elicitation of the DTH response were
affected by Map treatment since Map19 injected either before or
after immunization resulted in a significant reduction in the DTH
response (Table III). Although all Map19-treated mice had a
significantly reduced response to DbpA challenge following
immunization, mice receiving Map19 on both the day of immunization
and challenge (in addition to d2 and d4, Experiment I Table III)
had the greatest reduction in footpad swelling compared to control
mice (13.7.+-.1.46 vs. 34.75.+-.3.47 mm.times.10.sup.-2,
respectively) (Experiment I, Table III). The hypothesis that Map19
could act to prevent DTH by interfering with either the induction
or elicitation of DTH was tested by comparing challenge responses
in untreated mice to groups either treated with Map19 every other
day (starting on the day of immunization) or to mice treated with
Map19 only on the day of immunization and challenge (Experiment 2,
Table III). Map19-treated mice had a significantly reduced DTH
response compared to untreated or ACE40-treated controls
(Experiment II, Table III). Since mice treated only on the days of
immunization and challenge had a significantly reduced DTH response
indistinguishable from the response observed in mice treated with
Map19 every other day, it evidenced that Map19's inhibitory
activity correlated with T cell activation and that it's capacity
to interfere with T cell function was maximal during the T cell
activation stages of DTH. Doses of Map in the excess of 100 .mu.g
did not further reduce the DTH response, however, 25 .mu.g, the
lowest dosed tested in this experiment, still significantly reduced
the DTH response (FIG. 2).
[0063] Adoptively Transferred T Cells from Map-Treated Mice
[0064] Mice immunized with DbpA were either left untreated or
injected i.p. with either Map19 or the recombinant control protein
SdrF on the day of immunization (day 0) and on days 2, 4, and 6
post immunization. On day 7, mice were sacrificed and single cell
suspensions from whole spleens were prepared and enriched for T
cells by passage over nylon wool columns (20). Adoptive transfer of
nylon wool-purified T cells from Map19-treated mice did not elicit
a DTH response to DbpA in naive recipients compared to mice
adoptively transferred with enriched T cells from control groups
(FIG. 3). Flow cytometric analysis of cells nylon wool-collected
cells revealed a profile that was 46.83.+-.0.92% CD4.sup.+,
31.63.+-.0.96% CD8.sup.+, 1.2.+-.0.26% CD4.sup.+ CD8.sup.+, and
20.4.+-.1.33% CD4.sup.- CD8.sup.-. These data are expressed as the
mean percentage of positive cells.+-.SE for the 3 groups
examined.
[0065] INHIBITION of T Cell Proliferation and Apoptosis Induction
In Vitro.
[0066] Recombinant Map10 (SEQ ID NOS. 3 and 4) and Map19 (SEQ ID
NOS. 1 and 2) were tested for their ability to inhibit the
proliferation of the Borrelia-specific T cell line BAT 2.2 (8, 20).
T cell proliferation was measured at 40 h after plating in the
presence of mitomycin C-treated syngeneic antigen presenting cells
(APC) and inactive Borrelia (iBb) (20). Proliferation was measured
as a function of tetrazolium blue production following a 4 h
incubation in the presence of MTT. BAT 2.2 cells in the presence of
either Map10 or 19 but not in the presence of recombinant control
proteins CNA or M55 were inhibited from proliferating (FIG. 4)
(24). BAT 2.2 incubated in the presence of Borrelia only were
plotted as baseline as the control group with the highest
background proliferation (FIG. 4). In a similar experiment, BAT2.2
cells in the presence of IL-2 were cultured in the presence of
Map19 for 24 h. DNA extracted from BAT2.2 T cells incubated in the
presence of Map19 was examined for fragmentation by gel
electrophoresis (FIG. 5). DNA fragmentation comparable to
apoptotic-positive control DNA (lane 5) was only observed in DNA
extracted from Map19-treated T cells (lane 3) but not untreated
(lane 2) or ACE40-treated (lane 4) T cells (FIG. 5).
Summary
[0067] Reinfection of humans with SA is one of the hallmarks of
diseases caused by this pathogen and the roles of acquired and
innate immunity in protection against infection vary with the many
manifestation of disease resulting from SA infections (25-28).
While SA infections affecting the skin appear to be exacerbated by
strong, cellular responses, it is clear that cellular immunity is
critical in orchestrating the clearance of systemic SA infections
and in preventing reinfection with the same or similar pathogens
(29-33). One possible reason for recurring SA infections is the
reduction in chemotactic, phagocytic and bactericidal functions of
polymorphonuclear leukocytes from patients with chronic or
recurrent SA infections (27, 30, 33). Whether this is a function of
the bacterial infection or a preexisting condition in these
individuals is not known (27, 30, 33). Regardless, the presence of
SA-immunoregulatory molecules suggests that these bacteria have the
potential to counteract or evade host defense mechanisms. Both
superantigens and protein A produced by SA during an infection
serve immune-evasion functions. Superantigens can activate between
5-20% of T cells by directly binding to both the major
histocompatibility complex (MHC) class II molecules on
antigen-presenting cells and to the T cell receptor (TCR) on T
cells. This interaction can initiate apoptosis in T cells and
thymocytes in vivo and in vitro. The in vivo effects of such
massive T cell stimulation often results in disease (i.e. toxic
shock syndrome and food poising in humans) (34). Protein A, while
less harmful to the host compared to superantigens, may also serve
as a means of immune evasion by binding to the Fc fragment of
immunoglobulins (i.e. IgG) resulting in loss of antibody
function.
[0068] The present series of tests supported the idea that Map may
function as an immune modulator with the capacity to affect host
immune responses during SA infections. In addition to its potential
role as a bacterial adhesin; our tests showed that Map apparently
has the capacity to interfere with T cell activation and/or
proliferation facilitating SA survival in mammals (8, 11, 24, 35,
36). Sequence analysis of the SA genome revealed 5 open-reading
frames encoding Map-like proteins (14). While only one of these Map
proteins (SA1751) had a >80% homology to Newman stain Map (8,
14), the presence of other Map-like proteins suggested a critical
role for Map in SA survival; perhaps the potential to encode a
variety of MHC II-like proteins can serve to potentiate survival in
mammals of varied genetic backgrounds.
[0069] Additional evidence suggesting Map serves as an
immunomodulatory protein stemmed from double infection studies in
which a primary infection with Map.sup.-SA conferred significant
protection against reinfection with Map.sup.+SA. This contrasts
significantly with SA-induced pathology from mice receiving primary
and secondary infections with Map.sup.+SA. Accordingly, it appears
that T cell-mediated responses in Map.sup.+SA-infected mice are
abrogated by the presence of Map compared to Map.sup.-SA-infected
mice which develop cell-mediated immunity over the course of
infection. Map.sup.-SA infection, which is cleared over time,
results in a memory response capable of controlling a secondary
Map.sup.+SA infection. That a primary Map.sup.-SA infection
conferred significant but not complete protection against
Map.sup.+SA challenge suggested that the delicate balance between
an anamnestic response and Map-mediated immunomodulation could be
affected by the challenge dose. Our tests showed inhibition of DTH
responses directly or as a result of adoptively transferred T cells
from Map-treated mice, and this combined with the in vitro effects
of Map on T cell proliferation evidenced a direct involvement of
Map with T cells resulting in apoptosis. Flow cytometric analysis
of fluorescein isothiocyanate (FITC)-labeled Map19 revealed binding
to 100% of BAT2.2 T cells (data not shown).
[0070] In additional tests evidencing the effect on the Map protein
on T cell-mediated responses, nylon wool-purified naive T cells
cultured in the presence of Map19 were not induced to either
proliferate or undergo apoptosis. Furthermore, proliferation of
naive T cells as a result of incubation with concanavalin A or by
antibody-cross-linking of the TCR was not inhibited by Map. This
evidence that activated T cells but not naive T cells are
susceptible to Map and that T cell proliferation via `non
classical` pathways bypasses the Map-mediated inhibition of T cell
proliferation. Based on Map's effects on cellular immune responses
in vivo and in vitro, it appears that this protein is yet another
weapon used by SA to escape immune recognition and clearance.
Accordingly, in accordance with the present invention, the
administration of effective amounts of the Map protein or its
active regions or fragments such as Map19 appears to be useful in
achieving the suppression or modulation of T cell-mediated
responses to a host cell against S. aureus and thus may be useful
in methods to prevent or reduce the persistence or virulence of
infection by staphylococcal bacteria. TABLE-US-00001 TABLE I
Abscess formation in heart and kidneys harvested from Map.sup.- and
Map.sup.+SA-infected mice.sup.A Tissue Examined.sup.B Infecting
Strains Heart Kidneys Map-/Map.sup.+ 12/26 (46%).sup.C,D 13/52
(25%).sup.E Map.sup.+/Map.sup.+ 17/19 (89%) 33/38 (86%)
--/Map.sup.+ 29/31 (94%) 48/62 (77%) .sup.ABALB/c mice were
infected i.v. with either 1 .times. 10.sup.7 Map.sup.+ or
Map.sup.-SA strain Newman or left untreated. 4 weeks post primary
infection, mice from all groups received 1 .times. 10.sup.7
Map.sup.+SA i.v. 4 weeks latter hearts and kidneys were examined
grossly and histologically for abscess formation. .sup.BThe data
are pooled observations from three separate experiments. .sup.Cp
< .005 versus Map.sup.+/Map.sup.+ group; Fisher's exact test.
.sup.Dp < .0001 versus --/Map.sup.+; Fisher's exact test.
.sup.Ep < .0001 versus Map.sup.+/Map.sup.+ and -Map.sup.+
groups; Fisher's exact test.
[0071] TABLE-US-00002 TABLE II Histological examination of joints
harvested from Map.sup.- and Map.sup.+SA-infected mice.sup.A
Infecting Strains Mean Arthritis Rating Arthritis Frequency (%)
Mean Ostomylitis Score Osteomyelitis Frequency (%) Map-/Map+
0.84.sup.B 14/26 (54%).sup.C 0.57.sup.B 6/26.sup.D (23%) Map+/Map+
1.65 18/21 (86%) 1.95 14/21 (66%) -/Map+ 2.06 28/32 (88%) 1.48
14/32 (44%) .sup.ABALB/c mice were infected i.v. with either 1
.times. 10.sup.7 Map.sup.- or Map.sup.+SA strain Newman or left
untreated. 4 weeks post primary infection, mice from all groups
received 1 .times. 10 Map.sup.+SA i.v. 4 weeks latter, the right
hind limb joint was harvested and examined histologically for
arthritis and osteomylitis. .sup.Bp < 0.05 versus control
groups; Student's t test. .sup.Cp < 0.05 versus control groups;
Fisher's exact test. .sup.Dp < 0.005 versus +Map/+Map group;
Fisher's exact test.
[0072] TABLE-US-00003 TABLE III Histological examination of joints
harvested from SA.sup.-Map or SA.sup.+Map-infected nude mice.sup.A
Infecting Strains Mean Arthritis Rating Arthritis Frequency (%)
Mean Ostomylitis Score Osteomyelitis Frequency (%) nu/+/Map.sup.+SA
2.86 7/7 (100%) 2.29 5/7 (71%) nu/+/Map.sup.-SA 1.33 8/9
(89%).sup.C 0.44.sup.B 3/9 (33%) nu/nu/Map.sup.+SA 2.43 8/8 (100%)
2.62 8/8 (100%) nu/nu/Map.sup.-SA 2.10 7/10 (70%) 1.20 6/10 (60%)
.sup.AHsd nu/nu and nu/+ mice were infected i.v. with either 1
.times. 10.sup.7 Map.sup.-or Map.sup.+SA strain Newman or left
untreated. 4 weeks post primary infection, mice from all groups
received 1 .times. 10.sup.7 Map.sup.+SA i.v. 4 weeks latter, the
right hind limb joint was harvested and examined histologically for
arthritis and osteomylitis. .sup.Bp < 0.05 versus
nu/+/Map.sup.+SA; Student's t test.
[0073] TABLE-US-00004 TABLE IV The Effect of Map19 Treatment at
Various Times Before and After Immunization on the Elicitation of
DTH.sup.A Time Course Treatment.sup.B d -6 d -4 d -2 d 0.sup.E d 2
d 4 d 7.sup.F Mean Footpad Swelling.sup.C SF.sup.D Exp. I Map19 + +
+ +IM CH 18.75.sup.G .+-.3.26 Map19 + + +IM CH 22.75.sup.G .+-.2.66
Map19 + +IM CH 20.25.sup.G .+-.1.93 Map19 +IM CH 23.00.sup.G
.+-.1.36 Map19 +IM + CH 17.75.sup.H .+-.2.06 Map19 +IM + + CH
23.62.sup.G .+-.3.48 Map19 +IM + + +CH 13.75.sup.I .+-.1.46 -- CH
5.50.sup.I .+-.1.24 -- IM CH 34.75.sup. .+-.3.47 Exp. II Map19 +IM
+ + +CH 13.30.sup.I .+-.1.50 Map19 +IM +CH 10.10.sup.I .+-.0.82
ACE40 +IM + + +CH 26.60.sup.J .+-.2.83 ACE40 +IM +CH 26.75.sup.J
.+-.1.73 -- CH 3.10.sup.I .+-.0.67 -- IM CH 33.56 .+-.3.04
.sup.ABALB/c mice were immunized with DbpA on day zero. .sup.B+
Indicates treatment with 100 .mu.g of recombinant Map19 at various
time points prior to and after immunization. Control mice in Exp I
were treated with ACE40 in a parallel experiment and had DTH
responses similar to control mice (data not shown). .sup.CFootpads
were measured at 0 and 24 h after challenge. The data are expressed
as the mean footpad swelling of five mice/group. .sup.DStandard
Error .sup.EMice were immunized with 5 .mu.g of DbpA emulsified in
CFA i.p. .sup.F7 days after immunization the mice were challenged
in both hind footpads with 2 .mu.g of DpbA in 50 .mu.l of PBS.
.sup.Gp < 0.05; Students t test compared to IM and CH control.
.sup.Hp < 0.005; Students t test compared to IM and CH control.
.sup.Ip < p < 0.0001; Students t test compared to IM and CH
control. .sup.JNot significant compared to IM and CH control.
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259:1695-1702.
Sequence CWU 1
1
6 1 603 DNA Staphylococcus aureus CDS (1)..(603) 1 atg aga gga tcg
cat cac cat cac cat cac gga tcc cag att cca tat 48 Met Arg Gly Ser
His His His His His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 aca atc
act gtg aat ggt aca agc caa aac att tta tca agc tta aca 96 Thr Ile
Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30
ttt aat aag aat caa caa att agt tat aaa gat ata gag aat aaa gtt 144
Phe Asn Lys Asn Gln Gln Ile Ser Tyr Lys Asp Ile Glu Asn Lys Val 35
40 45 aaa tca gtt tta tac ttt aat aga ggt att agt gat atc gat tta
aga 192 Lys Ser Val Leu Tyr Phe Asn Arg Gly Ile Ser Asp Ile Asp Leu
Arg 50 55 60 ctt tct aag caa gca aaa tac acg gtt cat ttt aag aat
gga aca aaa 240 Leu Ser Lys Gln Ala Lys Tyr Thr Val His Phe Lys Asn
Gly Thr Lys 65 70 75 80 aga gtt gtc gat ttg aaa gca ggc att cac aca
gcc gac tta atc aat 288 Arg Val Val Asp Leu Lys Ala Gly Ile His Thr
Ala Asp Leu Ile Asn 85 90 95 aca agt gac att aaa gca att agt gtt
aac gta gat act aaa aag caa 336 Thr Ser Asp Ile Lys Ala Ile Ser Val
Asn Val Asp Thr Lys Lys Gln 100 105 110 gtg aaa gat aaa gag gca aaa
gca aat gtt caa gtg ccg tat aca atc 384 Val Lys Asp Lys Glu Ala Lys
Ala Asn Val Gln Val Pro Tyr Thr Ile 115 120 125 act gtg aat ggt aca
agc caa aac att tta tca aac tta aca ttt aaa 432 Thr Val Asn Gly Thr
Ser Gln Asn Ile Leu Ser Asn Leu Thr Phe Lys 130 135 140 aag aat cag
caa att agt tat aaa gat tta gag aat aat gta aaa tca 480 Lys Asn Gln
Gln Ile Ser Tyr Lys Asp Leu Glu Asn Asn Val Lys Ser 145 150 155 160
gtt tta aaa tca aac aga ggt ata act gat gta gat tta aga ctt tca 528
Val Leu Lys Ser Asn Arg Gly Ile Thr Asp Val Asp Leu Arg Leu Ser 165
170 175 aaa caa gcg aaa ttt aca gtt aat ttt aaa aat ggc acg aaa aaa
gtt 576 Lys Gln Ala Lys Phe Thr Val Asn Phe Lys Asn Gly Thr Lys Lys
Val 180 185 190 atc gat ttg aaa gca ggc att tat tga 603 Ile Asp Leu
Lys Ala Gly Ile Tyr 195 200 2 200 PRT Staphylococcus aureus 2 Met
Arg Gly Ser His His His His His His Gly Ser Gln Ile Pro Tyr 1 5 10
15 Thr Ile Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser Ser Leu Thr
20 25 30 Phe Asn Lys Asn Gln Gln Ile Ser Tyr Lys Asp Ile Glu Asn
Lys Val 35 40 45 Lys Ser Val Leu Tyr Phe Asn Arg Gly Ile Ser Asp
Ile Asp Leu Arg 50 55 60 Leu Ser Lys Gln Ala Lys Tyr Thr Val His
Phe Lys Asn Gly Thr Lys 65 70 75 80 Arg Val Val Asp Leu Lys Ala Gly
Ile His Thr Ala Asp Leu Ile Asn 85 90 95 Thr Ser Asp Ile Lys Ala
Ile Ser Val Asn Val Asp Thr Lys Lys Gln 100 105 110 Val Lys Asp Lys
Glu Ala Lys Ala Asn Val Gln Val Pro Tyr Thr Ile 115 120 125 Thr Val
Asn Gly Thr Ser Gln Asn Ile Leu Ser Asn Leu Thr Phe Lys 130 135 140
Lys Asn Gln Gln Ile Ser Tyr Lys Asp Leu Glu Asn Asn Val Lys Ser 145
150 155 160 Val Leu Lys Ser Asn Arg Gly Ile Thr Asp Val Asp Leu Arg
Leu Ser 165 170 175 Lys Gln Ala Lys Phe Thr Val Asn Phe Lys Asn Gly
Thr Lys Lys Val 180 185 190 Ile Asp Leu Lys Ala Gly Ile Tyr 195 200
3 396 DNA Staphylococcus aureus CDS (1)..(396) 3 atg aga gga tcg
cat cac cat cac cat cac gga tcc cag att cca tat 48 Met Arg Gly Ser
His His His His His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 aca atc
act gtg aat ggt aca agc caa aac att tta tca agc tta aca 96 Thr Ile
Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30
ttt aat aag aat caa caa att agt tat aaa gat ata gag aat aaa gtt 144
Phe Asn Lys Asn Gln Gln Ile Ser Tyr Lys Asp Ile Glu Asn Lys Val 35
40 45 aaa tca gtt tta tac ttt aat aga ggt att agt gat atc gat tta
aga 192 Lys Ser Val Leu Tyr Phe Asn Arg Gly Ile Ser Asp Ile Asp Leu
Arg 50 55 60 ctt tct aag caa gca aaa tac acg gtt cat ttt aag aat
gga aca aaa 240 Leu Ser Lys Gln Ala Lys Tyr Thr Val His Phe Lys Asn
Gly Thr Lys 65 70 75 80 aga gtt gtc gat ttg aaa gca ggc att cac aca
gcc gac tta atc aat 288 Arg Val Val Asp Leu Lys Ala Gly Ile His Thr
Ala Asp Leu Ile Asn 85 90 95 aca agt gac att aaa gca att agt gtt
aac gta gat act aaa aag caa 336 Thr Ser Asp Ile Lys Ala Ile Ser Val
Asn Val Asp Thr Lys Lys Gln 100 105 110 gtg aaa gat aaa gag gca aaa
gca aat gtt gtc gac ctg cag cca agc 384 Val Lys Asp Lys Glu Ala Lys
Ala Asn Val Val Asp Leu Gln Pro Ser 115 120 125 tta att agc tga 396
Leu Ile Ser 130 4 131 PRT Staphylococcus aureus 4 Met Arg Gly Ser
His His His His His His Gly Ser Gln Ile Pro Tyr 1 5 10 15 Thr Ile
Thr Val Asn Gly Thr Ser Gln Asn Ile Leu Ser Ser Leu Thr 20 25 30
Phe Asn Lys Asn Gln Gln Ile Ser Tyr Lys Asp Ile Glu Asn Lys Val 35
40 45 Lys Ser Val Leu Tyr Phe Asn Arg Gly Ile Ser Asp Ile Asp Leu
Arg 50 55 60 Leu Ser Lys Gln Ala Lys Tyr Thr Val His Phe Lys Asn
Gly Thr Lys 65 70 75 80 Arg Val Val Asp Leu Lys Ala Gly Ile His Thr
Ala Asp Leu Ile Asn 85 90 95 Thr Ser Asp Ile Lys Ala Ile Ser Val
Asn Val Asp Thr Lys Lys Gln 100 105 110 Val Lys Asp Lys Glu Ala Lys
Ala Asn Val Val Asp Leu Gln Pro Ser 115 120 125 Leu Ile Ser 130 5
2433 DNA Staphylococcus aureus CDS (71)..(2137) 5 aaaaaataga
gaaagtctgg ctataattaa gttgcaatca cgaattatca taaaaaagga 60
gtgataattt atg aaa ttt aag tca ttg att aca aca aca tta gca tta 109
Met Lys Phe Lys Ser Leu Ile Thr Thr Thr Leu Ala Leu 1 5 10 ggc gtt
ata gca tca aca gga gca aac tta gat act aac gaa gca tct 157 Gly Val
Ile Ala Ser Thr Gly Ala Asn Leu Asp Thr Asn Glu Ala Ser 15 20 25
gcc gca gct aag caa ata gat aaa tca tca agt tca tta cac cat gga 205
Ala Ala Ala Lys Gln Ile Asp Lys Ser Ser Ser Ser Leu His His Gly 30
35 40 45 tat tct aaa ata cag att cca tat aca atc act gtg aat ggt
aca agc 253 Tyr Ser Lys Ile Gln Ile Pro Tyr Thr Ile Thr Val Asn Gly
Thr Ser 50 55 60 caa aac att tta tca agc tta aca ttt aat aag aat
caa caa att agt 301 Gln Asn Ile Leu Ser Ser Leu Thr Phe Asn Lys Asn
Gln Gln Ile Ser 65 70 75 tat aaa gat ata gag aat aaa gtt aaa tca
gtt tta tac ttt aat aga 349 Tyr Lys Asp Ile Glu Asn Lys Val Lys Ser
Val Leu Tyr Phe Asn Arg 80 85 90 ggt att agt gat atc gat tta aga
ctt tct aag caa gca aaa tac acg 397 Gly Ile Ser Asp Ile Asp Leu Arg
Leu Ser Lys Gln Ala Lys Tyr Thr 95 100 105 gtt cat ttt aag aat gga
aca aaa aga gtt gtc gat ttg aaa gca ggc 445 Val His Phe Lys Asn Gly
Thr Lys Arg Val Val Asp Leu Lys Ala Gly 110 115 120 125 att cac aca
gct gac tta atc aat aca agt gac att aaa gca att agt 493 Ile His Thr
Ala Asp Leu Ile Asn Thr Ser Asp Ile Lys Ala Ile Ser 130 135 140 gtt
aac gta gat act aaa aag caa gtg aaa gat aaa gag gca aaa gca 541 Val
Asn Val Asp Thr Lys Lys Gln Val Lys Asp Lys Glu Ala Lys Ala 145 150
155 aat gtt caa gtg ccg tat aca atc act gtg aat ggt aca agc caa aac
589 Asn Val Gln Val Pro Tyr Thr Ile Thr Val Asn Gly Thr Ser Gln Asn
160 165 170 att tta tca aac tta aca ttt aaa aag aat cag caa att agt
tat aaa 637 Ile Leu Ser Asn Leu Thr Phe Lys Lys Asn Gln Gln Ile Ser
Tyr Lys 175 180 185 gat tta gag aat aat gta aaa tca gtt tta aaa tca
aac aga ggt ata 685 Asp Leu Glu Asn Asn Val Lys Ser Val Leu Lys Ser
Asn Arg Gly Ile 190 195 200 205 act gat gta gat tta aga ctt tca aaa
caa gcg aaa ttt aca gtt aat 733 Thr Asp Val Asp Leu Arg Leu Ser Lys
Gln Ala Lys Phe Thr Val Asn 210 215 220 ttt aaa aat ggc acg aaa aaa
gtt atc gat ttg aaa gca ggc att tat 781 Phe Lys Asn Gly Thr Lys Lys
Val Ile Asp Leu Lys Ala Gly Ile Tyr 225 230 235 aca gcg aac tta atc
aat aca ggc ggt att aaa aat atc aat ata aat 829 Thr Ala Asn Leu Ile
Asn Thr Gly Gly Ile Lys Asn Ile Asn Ile Asn 240 245 250 gta gaa act
aaa aag caa gcg aaa gat aaa gaa gca aaa gta aat aat 877 Val Glu Thr
Lys Lys Gln Ala Lys Asp Lys Glu Ala Lys Val Asn Asn 255 260 265 caa
gtg cca tat tca att aat tta aat ggt aca aca act aat att caa 925 Gln
Val Pro Tyr Ser Ile Asn Leu Asn Gly Thr Thr Thr Asn Ile Gln 270 275
280 285 tct aat tta gca ttt tca aat aaa cct tgg aca aat tac aaa aat
tta 973 Ser Asn Leu Ala Phe Ser Asn Lys Pro Trp Thr Asn Tyr Lys Asn
Leu 290 295 300 aca aca aag gta aaa tca gta ttg aaa tct gac aga ggc
gtt agt gaa 1021 Thr Thr Lys Val Lys Ser Val Leu Lys Ser Asp Arg
Gly Val Ser Glu 305 310 315 cgt gat ttg aaa cac gca aag aaa gcg tat
tac act gtt tac ttt aaa 1069 Arg Asp Leu Lys His Ala Lys Lys Ala
Tyr Tyr Thr Val Tyr Phe Lys 320 325 330 aat ggt ggt aaa aga gtg ata
cat tta aac tcg aat att tat aca gct 1117 Asn Gly Gly Lys Arg Val
Ile His Leu Asn Ser Asn Ile Tyr Thr Ala 335 340 345 aac tta gtt cat
gcg aaa gat gtt aag aga att gaa gtt act gta aaa 1165 Asn Leu Val
His Ala Lys Asp Val Lys Arg Ile Glu Val Thr Val Lys 350 355 360 365
aca gtt tcg aaa gta aaa gcg gag cgt tat gta cca tat aca att gca
1213 Thr Val Ser Lys Val Lys Ala Glu Arg Tyr Val Pro Tyr Thr Ile
Ala 370 375 380 gta aat gga gca tca aat cca act tta tca gat tta aaa
ttt aca ggt 1261 Val Asn Gly Ala Ser Asn Pro Thr Leu Ser Asp Leu
Lys Phe Thr Gly 385 390 395 gac tca cgt gta agc tac agt gat atc aag
aaa aaa gtt aaa tca gta 1309 Asp Ser Arg Val Ser Tyr Ser Asp Ile
Lys Lys Lys Val Lys Ser Val 400 405 410 ttg aaa cat gat aga ggt atc
ggt gaa cgt gaa tta aaa tat gcc gaa 1357 Leu Lys His Asp Arg Gly
Ile Gly Glu Arg Glu Leu Lys Tyr Ala Glu 415 420 425 aaa gca act tat
aca gta cat ttt aaa aat gga aca aaa aaa gtg att 1405 Lys Ala Thr
Tyr Thr Val His Phe Lys Asn Gly Thr Lys Lys Val Ile 430 435 440 445
aat tta aac tct aat att agt caa ctg aat ctg ctt tat gtc aaa gat
1453 Asn Leu Asn Ser Asn Ile Ser Gln Leu Asn Leu Leu Tyr Val Lys
Asp 450 455 460 att aaa aat ata gat atc gat gtt aaa act ggg gca aaa
gcg aaa gtc 1501 Ile Lys Asn Ile Asp Ile Asp Val Lys Thr Gly Ala
Lys Ala Lys Val 465 470 475 tat agc tat gta cca tac aca atc gca gta
aat ggg aca aca aca cct 1549 Tyr Ser Tyr Val Pro Tyr Thr Ile Ala
Val Asn Gly Thr Thr Thr Pro 480 485 490 att gca tca aaa cta aaa ctt
tcg aat aaa caa tta att ggt tat caa 1597 Ile Ala Ser Lys Leu Lys
Leu Ser Asn Lys Gln Leu Ile Gly Tyr Gln 495 500 505 gat tta aat aaa
aaa gtt aaa tca gtt tta aaa cat gat aga ggt atc 1645 Asp Leu Asn
Lys Lys Val Lys Ser Val Leu Lys His Asp Arg Gly Ile 510 515 520 525
aat gat att gaa ttg aaa ttt gcg aaa caa gca aag tat act ata cac
1693 Asn Asp Ile Glu Leu Lys Phe Ala Lys Gln Ala Lys Tyr Thr Ile
His 530 535 540 ttt aaa aat gga aag aca caa gtc gtt gac ctt aaa tca
gat atc ttt 1741 Phe Lys Asn Gly Lys Thr Gln Val Val Asp Leu Lys
Ser Asp Ile Phe 545 550 555 aca aga aat tta ttc agt gtc aaa gat att
aaa aag att gat att aat 1789 Thr Arg Asn Leu Phe Ser Val Lys Asp
Ile Lys Lys Ile Asp Ile Asn 560 565 570 gtg aaa caa caa tct aaa tct
aat aaa gcg ctt aat aaa gtg act aac 1837 Val Lys Gln Gln Ser Lys
Ser Asn Lys Ala Leu Asn Lys Val Thr Asn 575 580 585 aaa gcg act aaa
gtg aag ttt cca gta acg ata aat gga ttt tca aat 1885 Lys Ala Thr
Lys Val Lys Phe Pro Val Thr Ile Asn Gly Phe Ser Asn 590 595 600 605
tta gtt tca aat gaa ttt gcg ttt tta cat cca cat aaa ata aca aca
1933 Leu Val Ser Asn Glu Phe Ala Phe Leu His Pro His Lys Ile Thr
Thr 610 615 620 aac gac ttg aat gct aaa ctt aga cta gcg tta cga agc
gat caa ggt 1981 Asn Asp Leu Asn Ala Lys Leu Arg Leu Ala Leu Arg
Ser Asp Gln Gly 625 630 635 att act aaa cat gat att gga ctt tct gaa
cgc act gtg tat aaa gtg 2029 Ile Thr Lys His Asp Ile Gly Leu Ser
Glu Arg Thr Val Tyr Lys Val 640 645 650 tat ttt aaa gac gga tca tca
aaa tta gaa gac tta aaa gct gcg aaa 2077 Tyr Phe Lys Asp Gly Ser
Ser Lys Leu Glu Asp Leu Lys Ala Ala Lys 655 660 665 caa gat tca aaa
gta ttt aaa gca act gac att aaa aaa gta gac att 2125 Gln Asp Ser
Lys Val Phe Lys Ala Thr Asp Ile Lys Lys Val Asp Ile 670 675 680 685
gaa att aaa ttt taatctttaa ttttatatta aggcatctca caatagtggg 2177
Glu Ile Lys Phe gtgcctttta catttgtaga gatgtgatac ttgaagtgat
ttgccgcacg tttgataaat 2237 ttatctaagg catatcaagt tatgttagga
gagatgtata aaactaatta ggtatagcga 2297 ttgacaagtt gctgaataaa
atatatcttt tgatgctttg aaagaaggaa taattttaaa 2357 aataaaaaac
caataatccg agtcatactc atcagattat tggttgaaat taattatctt 2417
aagtcatcaa ttcttt 2433 6 689 PRT Staphylococcus aureus 6 Met Lys
Phe Lys Ser Leu Ile Thr Thr Thr Leu Ala Leu Gly Val Ile 1 5 10 15
Ala Ser Thr Gly Ala Asn Leu Asp Thr Asn Glu Ala Ser Ala Ala Ala 20
25 30 Lys Gln Ile Asp Lys Ser Ser Ser Ser Leu His His Gly Tyr Ser
Lys 35 40 45 Ile Gln Ile Pro Tyr Thr Ile Thr Val Asn Gly Thr Ser
Gln Asn Ile 50 55 60 Leu Ser Ser Leu Thr Phe Asn Lys Asn Gln Gln
Ile Ser Tyr Lys Asp 65 70 75 80 Ile Glu Asn Lys Val Lys Ser Val Leu
Tyr Phe Asn Arg Gly Ile Ser 85 90 95 Asp Ile Asp Leu Arg Leu Ser
Lys Gln Ala Lys Tyr Thr Val His Phe 100 105 110 Lys Asn Gly Thr Lys
Arg Val Val Asp Leu Lys Ala Gly Ile His Thr 115 120 125 Ala Asp Leu
Ile Asn Thr Ser Asp Ile Lys Ala Ile Ser Val Asn Val 130 135 140 Asp
Thr Lys Lys Gln Val Lys Asp Lys Glu Ala Lys Ala Asn Val Gln 145 150
155 160 Val Pro Tyr Thr Ile Thr Val Asn Gly Thr Ser Gln Asn Ile Leu
Ser 165 170 175 Asn Leu Thr Phe Lys Lys Asn Gln Gln Ile Ser Tyr Lys
Asp Leu Glu 180 185 190 Asn Asn Val Lys Ser Val Leu Lys Ser Asn Arg
Gly Ile Thr Asp Val 195 200 205 Asp Leu Arg Leu Ser Lys Gln Ala Lys
Phe Thr Val Asn Phe Lys Asn 210 215 220 Gly Thr Lys Lys Val Ile Asp
Leu Lys Ala Gly Ile Tyr Thr Ala Asn 225 230 235 240 Leu Ile Asn Thr
Gly Gly Ile Lys Asn Ile Asn Ile Asn Val Glu Thr 245 250 255 Lys Lys
Gln Ala Lys Asp Lys Glu Ala Lys Val Asn Asn Gln Val Pro 260 265 270
Tyr Ser Ile Asn Leu Asn Gly Thr Thr Thr Asn Ile Gln Ser Asn Leu 275
280 285 Ala Phe Ser Asn Lys Pro Trp Thr Asn Tyr Lys Asn Leu Thr Thr
Lys 290 295 300 Val Lys Ser Val Leu Lys Ser Asp Arg Gly Val Ser Glu
Arg Asp Leu 305 310 315 320 Lys His Ala Lys Lys Ala Tyr Tyr Thr Val
Tyr Phe Lys Asn Gly Gly 325 330 335 Lys Arg Val Ile His Leu Asn Ser
Asn Ile Tyr Thr Ala Asn Leu Val 340 345 350 His Ala Lys Asp Val Lys
Arg Ile Glu Val Thr Val Lys Thr Val Ser
355 360 365 Lys Val Lys Ala Glu Arg Tyr Val Pro Tyr Thr Ile Ala Val
Asn Gly 370 375 380 Ala Ser Asn Pro Thr Leu Ser Asp Leu Lys Phe Thr
Gly Asp Ser Arg 385 390 395 400 Val Ser Tyr Ser Asp Ile Lys Lys Lys
Val Lys Ser Val Leu Lys His 405 410 415 Asp Arg Gly Ile Gly Glu Arg
Glu Leu Lys Tyr Ala Glu Lys Ala Thr 420 425 430 Tyr Thr Val His Phe
Lys Asn Gly Thr Lys Lys Val Ile Asn Leu Asn 435 440 445 Ser Asn Ile
Ser Gln Leu Asn Leu Leu Tyr Val Lys Asp Ile Lys Asn 450 455 460 Ile
Asp Ile Asp Val Lys Thr Gly Ala Lys Ala Lys Val Tyr Ser Tyr 465 470
475 480 Val Pro Tyr Thr Ile Ala Val Asn Gly Thr Thr Thr Pro Ile Ala
Ser 485 490 495 Lys Leu Lys Leu Ser Asn Lys Gln Leu Ile Gly Tyr Gln
Asp Leu Asn 500 505 510 Lys Lys Val Lys Ser Val Leu Lys His Asp Arg
Gly Ile Asn Asp Ile 515 520 525 Glu Leu Lys Phe Ala Lys Gln Ala Lys
Tyr Thr Ile His Phe Lys Asn 530 535 540 Gly Lys Thr Gln Val Val Asp
Leu Lys Ser Asp Ile Phe Thr Arg Asn 545 550 555 560 Leu Phe Ser Val
Lys Asp Ile Lys Lys Ile Asp Ile Asn Val Lys Gln 565 570 575 Gln Ser
Lys Ser Asn Lys Ala Leu Asn Lys Val Thr Asn Lys Ala Thr 580 585 590
Lys Val Lys Phe Pro Val Thr Ile Asn Gly Phe Ser Asn Leu Val Ser 595
600 605 Asn Glu Phe Ala Phe Leu His Pro His Lys Ile Thr Thr Asn Asp
Leu 610 615 620 Asn Ala Lys Leu Arg Leu Ala Leu Arg Ser Asp Gln Gly
Ile Thr Lys 625 630 635 640 His Asp Ile Gly Leu Ser Glu Arg Thr Val
Tyr Lys Val Tyr Phe Lys 645 650 655 Asp Gly Ser Ser Lys Leu Glu Asp
Leu Lys Ala Ala Lys Gln Asp Ser 660 665 670 Lys Val Phe Lys Ala Thr
Asp Ile Lys Lys Val Asp Ile Glu Ile Lys 675 680 685 Phe
* * * * *