U.S. patent application number 14/535254 was filed with the patent office on 2015-06-18 for compositions and methods for the removal of biofilms.
The applicant listed for this patent is Research Institute at Nationwide Children's Hospital, University of Southern California. Invention is credited to Lauren O. Bakaletz, Steven D. Goodman.
Application Number | 20150166641 14/535254 |
Document ID | / |
Family ID | 44656747 |
Filed Date | 2015-06-18 |
United States Patent
Application |
20150166641 |
Kind Code |
A1 |
Goodman; Steven D. ; et
al. |
June 18, 2015 |
Compositions and Methods for the Removal of Biofilms
Abstract
This invention provides isolated or recombinant polypeptides
that are useful to vaccinate individuals suffering from
chronic/recurrent biofilm disease or as a therapeutic for those
with an existing infection. The individual's immune system will
then naturally generate antibodies which prevent or clear these
bacteria from the host by interfering with the construction and or
maintenance of a functional protective biofilm. Alternatively,
antibodies to the polypeptides can be administered to treat or
prevent infection. Bacteria that cannot form functional biofilms
are more readily cleared by the remainder of the host's immune
system.
Inventors: |
Goodman; Steven D.;
(Hilliard, OH) ; Bakaletz; Lauren O.; (Columbus,
OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Southern California
Research Institute at Nationwide Children's Hospital |
Los Angeles
Columbus |
CA
OH |
US
US |
|
|
Family ID: |
44656747 |
Appl. No.: |
14/535254 |
Filed: |
November 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13073782 |
Mar 28, 2011 |
8999291 |
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14535254 |
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61318743 |
Mar 29, 2010 |
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61347362 |
May 21, 2010 |
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61397891 |
Jun 16, 2010 |
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61454972 |
Mar 21, 2011 |
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Current U.S.
Class: |
424/139.1 ;
435/188; 435/331; 530/363; 530/387.3; 530/387.9; 530/391.1;
530/391.3; 530/391.7 |
Current CPC
Class: |
A61P 31/00 20180101;
A61K 2039/505 20130101; C07K 16/1217 20130101; C07K 16/1232
20130101; C07K 16/1275 20130101; G01N 2800/26 20130101; A61K
39/0258 20130101; A61P 31/04 20180101; A61P 31/12 20180101; C07K
16/1242 20130101; G01N 33/56911 20130101; C07K 2317/14 20130101;
Y02A 50/30 20180101; C07K 14/195 20130101; C07K 2317/34 20130101;
C07K 16/1203 20130101; C07K 16/1271 20130101; C07K 14/245 20130101;
C07K 2317/76 20130101; C07K 14/285 20130101; Y02A 50/474 20180101;
A61K 39/40 20130101; A61K 38/164 20130101; G01N 2800/52 20130101;
A61P 37/04 20180101; C07K 16/1214 20130101; C07K 14/21
20130101 |
International
Class: |
C07K 16/12 20060101
C07K016/12; A61K 39/40 20060101 A61K039/40 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under
Contract No. 5R01DE013230 awarded by the National Institute of
Dental and Craniofacial Research (NIDCR) at the National Institutes
of Health. The government has certain rights in the invention.
Claims
1-30. (canceled)
31. An antibody or antigen binding fragment that specifically
recognizes or binds an isolated or recombinant polypeptide selected
from the group of an amino acid sequence selected from: SEQ ID NO.
1 to 27; 42 to 336; or a DNA binding peptide identified in FIG. 6,
or an isolated or recombinant polypeptide comprising two or more of
any of SEQ ID NO. 1 to 27; 42 to 336; a DNA binding peptide
identified in FIG. 6; or a fragment or an equivalent of each
thereof.
32. The antibody of claim 31, wherein the antibody is selected from
the group of a polyclonal antibody, a monoclonal antibody, a
humanized antibody, a human antibody, an antibody derivative, a
veneered antibody, a diabody, an antibody derivative, a recombinant
human antibody, a chimeric antibody, or an antibody fragment.
33. The antibody or antigen binding fragment of claim 31, wherein
the antibody or antigen binding fragment further comprises
modification by one or more of the group: a conservative amino acid
mutation within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions;
by altering the number of cysteine residues or by conservative
amino acid mutations in the Fc hinge region; by chemical
modification; by pegylation; by conjugation to a serum protein; by
conjugation to human serum albumin; by conjugation to a detectable
label; by conjugation to a diagnostic agent; by conjugation to an
enzyme; by conjugation to a prosthetic group complex; by
conjugation to a fluorescent material; by conjugation to a
luminescent material; by conjugation to a bioluminescent material;
by conjugation to a radioactive material; by conjugation to a
therapeutic agent; by fusion to at least one additional functional
molecule; by fusion to a second antibody or antigen binding
fragment; or by conjugation to an antimicrobial agent.
34. A hybridoma cell line that produces the monoclonal antibody of
claim 32.
35-36. (canceled)
37. A composition comprising a carrier and an antibody of claim 31
or 49.
38. The composition of claim 37, further comprising one or more of
an adjuvant, an antigenic peptide or an antimicrobial.
39-48. (canceled)
49. The antibody or antigen binding fragment of claim 31, wherein
the isolated or recombinant polypeptide comprises an amino acid
sequence selected from SEQ ID NO. 6-9, 46, 70, and 93 or an
equivalent or a fragment of each thereof, or a DNA binding peptide
identified in FIG. 6 or an equivalent or a fragment of each
thereof; an isolated or recombinant polypeptide comprising two or
more of any of SEQ ID NO. 6-9, 46, 70, and 93 or an equivalent or a
fragment of each thereof, a DNA binding peptide identified in FIG.
6, or an equivalent or a fragment of each thereof.
50. The antibody or antigen binding fragment of claim 31 or 49,
wherein the isolated or recombinant polypeptide does not comprise a
polypeptide of the groups of: wild-type IHFalpha, IHFbeta, or SEQ
ID NOs.: 6 to 11.
51. The antibody or antigen binding fragment of claim 31 or 49,
wherein the isolated or recombinant polypeptide consists
essentially of the c-terminal half of the polypeptide.
52. The antibody of claim 50, wherein the antibody is a monoclonal
antibody.
53. The antibody of claim 51, wherein the antibody is a monoclonal
antibody.
54. A hybridoma cell line that produces the antibody of claim
52.
55. A hybridoma cell line that produces the antibody of claim
53.
56. A method for breaking down a biofilm comprising contacting the
biofilm with an effective amount of the antibody or antigen binding
fragment of claim 31 or 49.
57. A method for conferring passive immunity to a subject in need
thereof comprising administering an effective amount of the
antibody or antigen binding fragment of claim 31 or 49 to the
subject, thereby conferring passive immunity.
58. The method of claim 55, wherein the subject is a human
patient.
59. The method of claim 56, wherein the human patient is a
pediatric patient.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/073,782, filed Mar. 28, 2011, which claims the benefit
under 35 U.S.C. .sctn.119(e) of U.S. Provisional Application Nos.
61/318,743, filed Mar. 29, 2010; 61/347,362, filed May 21, 2010;
61/397,891, filed Jun. 16, 2010; and 61/454,972, filed Mar. 21,
2011; the entire content of each of which is incorporated by
reference into the present disclosure.
FIELD OF THE INVENTION
[0003] This invention generally relates to the methods and
compositions to lessen and/or cure clinical or industrial bacterial
biofilms.
BACKGROUND
[0004] Bacteria persisting in a biofilm in the mammalian body cause
about two-thirds of all chronic/recurrent diseases. These biofilms
are comprised of bacteria protected by an outer "slime" that is
often comprised primarily of DNA that prevents the innate and
adaptive immune systems, antibiotics and other antibacterial agents
from gaining access to the bacteria inside the biofilm, making it
extremely difficult to clear the infection from the body.
Furthermore, the biofilm can act as a reservoir for future acute
infections often with lethal consequences.
[0005] At least one protein from the DNABII family of proteins are
found in all known eubacteria and are naturally found outside of
the bacterial cell. While they elicit a strong innate immune
response, host subjects fail to naturally produce specific antibody
to family members as a result of infection. The major problem with
bacterial biofilms is the inability of the host immune system
and/or antibiotics and other antimicrobials to gain access to the
bacteria protected within the biofilm.
[0006] Biofilms are present in an industrial setting as well. For
example, biofilms are implicated in a wide range of petroleum
process problems, from the production field to the gas station
storage tank. In the field, sulfate reducing biofilm bacteria
produce hydrogen sulfide (soured oil). In the process pipelines,
biofilm activity develops slimes which impede filters and orifices.
Biofilm and biofilm organisms also cause corrosion of pipeline and
petroleum process equipment. These problems can be manifested
throughout an oil or gas production facility to the point where
fouling and corrosive biofilm organisms have even been found on the
surfaces of final product storage tanks.
[0007] In the home, biofilms are found in or on any surface that
supports microbial growth, e.g., in drains, on food preparation
surfaces, in toilets, and in swimming pools and spas.
[0008] Biofilms are implicated in a wide range of water processes,
both domestic and industrial. They can grow on the surface of
process equipment and impede the performance of the equipment, such
as degradation of heat transfer or plugging of filters and
membranes. Biofilms growing on a cooling tower fill can add enough
weight to cause collapse of the fill. Biofilms cause corrosion of
even highly specialized stainless steels. Biofilms in a water
process can degrade the value of a final product such as biofilm
contamination in a paper process or the attachment of even a single
cell on a silicon chip. Biofilms growing in drinking water
distribution systems can harbor potential pathogenic organisms,
corrosive organisms or bacteria that degrade the aesthetic quality
of the water.
[0009] Thus, a need exists to break through the protective barrier
of biofilms to treat or kill the associated bacterial infections
and clear them from surfaces and in water systems. This invention
satisfies this need and provides related advantages as well.
TABLE-US-00001 SEQ. ID NO. 1 A1-A2-A3-A4-A5-A6-A7-A8-A9
wherein:
[0010] A1 is V or I;
[0011] A2 is any one of K, Q, E, A, V or Y;
[0012] A3 is any one of K, L, T, V or F;
[0013] A4 is any one of S, I, R or V;
[0014] A5 is any one of G or S;
[0015] A6 is F;
[0016] A7 is G;
[0017] A8 is any one of N or S or T or K; and
[0018] A9 is F.
TABLE-US-00002 SEQ. ID NO. 2 is VKKSGFGNF SEQ ID NO. 3 is
B1-B2-B3-B4-B5-B5-B6-B7
wherein:
[0019] B1 is absent or any one of G or K;
[0020] B2 is absent or any one of R, I or K;
[0021] B3 is N or V;
[0022] B4 is P or I;
[0023] B5 is any one of K, Q, S or G;
[0024] B6 is any one of T, K or S; and
[0025] B7 is any one of G, K, Q or D.
TABLE-US-00003 Seq. ID NO. 4 is NP(K/Q)TG Seq. ID NO. 5 GRNP(K/Q)TG
Seq. ID NO. 6 Full Length Wild type (wt) 86-028NP Haemophilus
influenzae IhfA; Genbank accession No.: AAX88425.1, last accessed
March 21, 2011: MATITKLDIIEYLSDKYHLSK
QDTKNVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARR
VVTFKPGQKLRARVEKTK Seq. ID NO, 7 Full Length wt 86-028NP
Haemophilus influenzae HU, Genbank accession No.: YP_248142.1, last
accessed March 21, 2011: MRFVTIFINHAFNSSQVRLSFAQFLR
QIRKDTFKESNFLFNRRYKFMNKTDLIDAIANAAELNKKQAKAALEATLDAITASLK
EGEPVQLIGFGTFKVNERAARTGRNPQTGAEIQIAASKVPAFVSGKALKDAIK Seq. ID NO. 8
Full Length wt R2846 Haemophilus influenzae IhfA, Genbank accession
No.: .ADO96375, last accessed March 21, 2011:
MATITKLDIIEYLSDKYHLSKQDTKNVVENFL
EEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTFKPGQKLR ARVEKTK
Seq. ID NO. 9 Full Length wt Rd Haemophilus influenzae IhfA;
Genbank accession No.: AAC22959.1, last accessed March 21, 2011:
MATITKLDIIEYLSDKYHLSKQDTK
NVVENFLEEIRLSLESGQDVKLSGFGNFELRDKSSRPGRNPKTGDVVPVSARRVVTF
KPGQKLRARVEKTK; Seq. ID NO. 10 Full Length wt E. coli K12 IhfA;
Genbank accession No.: AAC74782.1, last accessed March 21, 2011:
MALTKAEMSEYLFDKLGLSKRDAKELVELFFE
EIRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITARRVVT
FRPGQKLKSRVENASPKDE; DNA Genbank No. NC_000913 Seq. ID NO, 11 Full
Length wt P. aeruginosa PA 01 IhfA; Genbank accession No.:
AAG06126.1, last accessed March 21, 2011: MGALTKAEIAERLYEELGLNKREA
KELVELFFEEIRQALEHNEQVKLSGFGNFDLRDKRQRPGRNPKTGEEIPITARRVVTF
RPGQKLKARVEAYAGTKS Seq ID NOS. 12 and 13: .beta.-3 and .alpha.-3
portions of (IHF .alpha.) SEQ ID NO. 12: TFRPGQ and SEQ ID NO. 13:
KLKSRVENASPKDE Seq ID NOS. 14 and 15: .beta.-3 and .alpha.-3
portions of (IHF .beta.) SEQ ID NO. 14: HFKPGK and SEQ ID NO. 15:
ELRDRANIYG Seq ID NOS. 16 and 17: .beta.-3 and .alpha.-3 portions
of SEQ ID NOS. 6, SEQ ID NOS. 16: TFKPGQ and SEQ ID NO. 17:
KLRARVEKTK SEQ ID NOS. 18 and 19: .beta.-3 and .alpha.-3 portions
of 2019 Haemophilus influenzae IhfA, SEQ ID Na 18: TFKPGQ and SEQ
ID NO. 19: KLRARVENTK SEQ ID NOS. 20 and 21: .beta.-3 and .alpha.-3
portions of SEQ ID NO. 8, SEQ ID NO. 20: TFKPGQ and SEQ ID NO, 21:
KLRARVEKTK SEQ ID NOS. 22 and 23: .beta.-3 and .alpha.-3 portions
of SEQ ID NO. 9, SEQ ID NO, 22: TFKPGQ and SEQ TD NO. 23:
KLRARVEKTK SEQ ID NOS. 24 and 25: .beta.-3 and .alpha.-3 portions
of SEQ ID NO. 10, SEQ ID NO, 24: TFRPGQ and SEQ ID NO. 25:
KLKSRVENASPKDE SEQ ID NOS. 26 and 27: .beta.-3 and .alpha.-3
portions of SEQ ID NO. 11, SEQ ID NO. 26: TFRPGQ and SEQ ID NO. 27:
KLKARVEAYAGTKS SEQ ID NO. 28: E. coli hupA, Genbank accession No.:
AP_003818, Last accessed March 21, 2011:
MNKTQLIDVIAEKAELSKTQAKAALESTLAAITESLKEGDAVQLVGFGTFK
VNHRAERTGRNPQTGKEIKIAAANVPAFVSGKALKDAVK SEQ ID NO. 29: E. coli
hupB, Genbank accession No.: AP_001090.1, Last accessed March 21,
2011: MNKSQLIDKIAAGADISKAAAGRALDAIIASVTESLKEGDDVALVGFG
TFAVKERAARTGRNPQTGKEITIAAAKVPSFRAGKALKDAVN SEQ ID NOS, 30 and 31:
.beta.-3 and .alpha.-3 portions of SEQ ID NO. 28, SEQ ID NO. 30:
AFVSGK and SEQ ID NO. 31: ALKDAVK SEQ ID NOS. 32 and 33: .beta.-3
and .alpha.-3 portions of SEQ ID NO. 29, SEQ ID NO. 32: SFRAGK and
SEQ ID NO. 33: ALKDAVN SEQ. ID NO. 34: C-terminal 20 amino acids of
IHF .alpha.: TFRPGQKLKSRVENASPKDE SEQ. ID NO. 35: C-terminal 20
amino acids of IHF .beta.: KYVPHFKPGKELRDRANIYG SEQ. ID NO. 36:
DNABII binding consensus sequence: WATCAANNNNTTR wherein W is A or
T, N is any base and R is a purine SEQ. ID NO. 337: E. coli
IHFalpha: GRNPKTGEDIPI SEQ. ID NO. 338: E. coli IHFbeta:
GRNPKTGDKVEL SEQ. ID NO. 339: E. coli HUalpha: GRNPQTGKEIKI SEQ. ID
NO. 340: E. coli HUbeta: GRNPQTGKEITI
Description of Tables
[0026] Tables 1-5 are the results of an in vitro bioassay of the
reversal of the biofilm in the indicated organism.
[0027] Table 6 is the scoring scheme of the relative amount of
biomass within the middle ear in the Chinchilla model of otitis
media (OM).
[0028] Table 7 is the results of an in vitro bioassay of the
reversal of the biofilm upon treatment with DNase.
[0029] Table 8 is a non-limited summary of DNA binding proteins
produced by gram (+) and gram (-) bacteria that can be used in the
methods provided herein.
[0030] Table 9A is a sequence alignment of relevant portions of the
DNA binding proteins of various embodiments of this invention. Bold
letters indicate an exact match to consensus, light gray lettering
indicates a conservative amino acid change, and lightly or darkly
shaded sequences are highly conserved across species. Gray shaded
undefined sequences at the amino and/or carboxy-terminal are
undefined amino acids that do not share consensus sequences. Table
9A is based on information previously published in Obeto et al.
(1994) Biochimie 76:901-908. Table 9B is a comparison of the 16
amino acid peptide motif to Liu et al. (2008) Cell Microbiol.
10(1):262-276.
[0031] Table 10 is a listing of .alpha., .beta., and C-terminal
portions of DNABII proteins from the indicated organism.
SUMMARY OF THE INVENTION
[0032] Within bacterial cells, the DNABII proteins are DNA binding
proteins that necessarily bend DNA substrates upon binding.
Similarly, DNA that is already in a bent conformation is a
preferred substrate as the energy required for bending is rendered
unnecessary.
[0033] The DNABII family of proteins is found outside of bacterial
cells in the biofilm state. Applicants have shown that these
proteins are in fact bound to the extracellular DNA at critical
branched junctions. In one aspect, Applicants have shown that by
immunizing the host with polypeptides and proteins that produce
specific antibodies, the DNA-based lattice is sufficiently altered
to now permit the host immune system to clear the biofilm.
[0034] Applicants have also demonstrated the removal of pre-formed
non-typeable Haemophilus influenza biofilms in the middle ear of
the chinchilla host by various modes of immunization with a DNABII
family member (E. coli integration host factor, IHF). This
chinchilla middle ear biofilm animal system has already been well
documented as an excellent model for human otitis media (or middle
ear infections).
[0035] The method for using this technology is straightforward. In
one embodiment, the polypeptides of this invention are used to
vaccinate individuals as a prophylactic to chronic/recurrent
biofilm disease or as a therapeutic for those with an existing
infection. The individual's immune system will then naturally
generate antibodies which prevent or clear these bacteria from the
host by interfering with the construction and or maintenance of a
functional protective biofilm. Alternatively, antibodies to the
polypeptides can be administered to treat or prevent infection.
Bacteria that cannot form functional biofilms are more readily
cleared by the remainder of the host's immune system.
[0036] Thus, in one aspect a method for inhibiting, competing or
titrating the binding of a DNABII polypeptide or protein to a
microbial DNA is provided by comprising, or alternatively
consisting essentially of, or yet further consisting of contacting
the DNABII polypeptide or protein or the microbial DNA with an
interfering agent, thereby inhibiting, competing or titrating the
binding of the DNABII protein or polypeptide to the microbial DNA.
The contacting can be performed in vitro or in vivo.
[0037] In another aspect, provided is a method for inhibiting,
preventing or breaking down a microbial biofilm, comprising, or
alternatively consisting essentially of, or yet further consisting
of contacting the biofilm with an interfering agent, thereby
inhibiting, preventing or breaking down the microbial biofilm. The
contacting can be performed in vitro or in vivo.
[0038] In a further aspect, provided is a method of inhibiting,
preventing or breaking down a biofilm in a subject, comprising, or
alternatively consisting essentially of, or yet further consisting
of administering to the subject an effective amount of an
interfering agent, thereby inhibiting, preventing or breaking down
the microbial biofilm.
[0039] In a yet further aspect, a method for inhibiting, preventing
or treating a microbial infection that produces a biofilm in a
subject is provided. The method comprises, or alternatively
consists essentially of, or yet further consists of administering
to the subject an effective amount of an interfering agent, thereby
inhibiting, preventing or treating a microbial infection that
produces the biofilm in the subject.
[0040] For the methods as described herein, any agent that
interferes or impedes the binding of the microbial DNA to the
DNABII protein or polypeptide is intended within the scope of this
invention. Non-limiting examples of interfering agents include:
[0041] (a) an isolated or recombinant integration host factor (IHF)
polypeptide or a fragment or an equivalent of each thereof;
[0042] (b) an isolated or recombinant histone-like protein from E.
coli strain U93 (HU) polypeptide or a fragment or an equivalent of
each thereof;
[0043] (c) an isolated or recombinant protein or polypeptide
identified in Table 8. Table 9A, Table 9B, Table 10 or a DNA
binding peptide identified in FIG. 6, or a fragment or an
equivalent of each thereof,
[0044] (d) an isolated or recombinant polypeptide of SEQ ID NO. 1
through 340, or a fragment or an equivalent of each thereof;
[0045] (e) an isolated or recombinant C-terminal polypeptide of SEQ
ID NO. 6 through 11, 28, 29, 42 through 100, Table 8 or those
C-terminal polypeptides identified in Table 10 or a fragment or an
equivalent of each thereof;
[0046] (f) a polypeptide or polynucleotide that competes with an
integration host factor on binding to a microbial DNA;
[0047] (g) a four-way junction polynucleotide resembling a Holliday
junction, a 3 way junction polynucleotide resembling a replication
fork, a polynucleotide that has inherent flexibility or bent
polynucleotide;
[0048] (h) an isolated or recombinant polynucleotide encoding any
one of (a) through (f) or an isolated or recombinant polynucleotide
of SEQ ID NO. 36 or an equivalent of each thereof, or a
polynucleotide that hybridizes under stringent conditions to the
polynucleotide its equivalent or its complement;
[0049] (i) an antibody or antigen binding fragment that
specifically recognizes or binds any one of (a) through (f), or an
equivalent or fragment of each antibody or antigen binding fragment
thereof;
[0050] (j) an isolated or recombinant polynucleotide encoding the
antibody or antigen binding fragment of (i) or its complement;
or
[0051] (k) a small molecule that competes with the binding of a
DNABII protein or polypeptide to a microbial DNA.
[0052] Also provided herein is a method for inducing an immune
response in or conferring passive immunity on a subject in need
thereof, comprising, or alternatively consisting essentially of, or
yet further consisting of, administering to the subject an
effective amount of one or more agents of the group:
[0053] (a) an isolated or recombinant integration host factor (IHF)
polypeptide, or a fragment or an equivalent of each thereof;
[0054] (b) an isolated or recombinant histone-like protein from E.
coli strain U93 (HU) polypeptide or a fragment or an equivalent of
each thereof;
[0055] (c) an isolated or recombinant protein polypeptide
identified in Table 8, Table 9A, Table 9B, Table 10 or an DNA
binding peptide identified in FIG. 6, or a fragment or an
equivalent of each thereof;
[0056] (d) an isolated or recombinant polypeptide of SEQ ID NO. 1
through 340, or a fragment or an equivalent thereof;
[0057] (e) an isolated or recombinant C-terminal polypeptide of SEQ
ID NO. 6 through 11, 28, 29, 42 through 100, Table 8 or those
C-terminal polypeptides identified in Table 10 or a fragment or an
equivalent of each thereof;
[0058] (f) an isolated or recombinant polynucleotide encoding any
one of (a) through (e) or an isolated or recombinant polynucleotide
of SEQ ID NO. 36 or an equivalent of each thereof, or a
polynucleotide that hybridizes under stringent conditions to the
polynucleotide, its equivalent or its complement;
[0059] (g) an antibody or antigen binding fragment that
specifically recognizes or binds any one of (a) through (e), or an
equivalent or fragment of each thereof
[0060] (h) an isolated or recombinant polynucleotide encoding the
antibody or antigen binding fragment of (g).
[0061] (i) an antigen presenting cell pulsed with any one of (a)
through (e); and
[0062] (j) an antigen presenting cell transfected with one or more
polynucleotides encoding any one of (a) through (e).
[0063] Subjects in need of such immune response include those at
risk of or suffering from an infection that produces a microbial
biofilm.
[0064] Also provided herein are compositions for use in the above
methods, non-limiting examples of which are discussed below.
[0065] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of
an amino acid sequence selected from SEQ ID NO. 1 to 5 or 12 to 27,
30 to 35, 101-340 or a DNA binding peptide identified in FIG.
6.
[0066] In another aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of, SEQ ID NO. 1 or 2, with the proviso
that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42
through 100.
[0067] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of, SEQ ID NO. 3, 4 or 5, with the
proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28, 29,
or 42 through 100.
[0068] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of, SEQ ID NO. 12, 14, 16, 18, 20, 22,
24, 26, 30 or 32, with the proviso that the polypeptide is none of
SEQ ID NO. 6 to 11, 28, 29, or 42 through 100.
[0069] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of SEQ ID NO. 13, 15, 17, 19, 21, 23, 25,
27, 31 or 33, with the proviso that the polypeptide is none of SEQ
ID NO. 6 to 11, 28, 29, or 42 through 100.
[0070] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of SEQ ID NO. 337, 338, 339, or 340, with
the proviso that the polypeptide is none of SEQ ID NO. 6 to 11, 28,
29, or 42 through 100.
[0071] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of, SEQ ID NO. 12 and 13 or 14 and 15 or
16 and 17 or 18 and 19 or 20 and 21 or 22 and 23 or 24 and 25, or
26 and 27 or 30 and 31 or 32 and 33, with the proviso that the
polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through
100.
[0072] In one aspect, provided is an isolated or recombinant
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of, the C-terminal region containing at
least 10, or alternatively at least 15, or alternatively at least
20, or alternatively at least 25, or alternatively at least 30,
C-terminal amino acids of a polypeptide of the group of a DNABII
polypeptide, an IHF polypeptide, an HU polypeptide, SEQ ID NO. 6
through 11, 28, 29 or those identified in Table 8, Table 10 or a
fragment or an equivalent of each thereof.
[0073] In one aspect, provided is an isolated or recombinant
polypeptide of the group of:
[0074] a polypeptide comprising SEQ ID NO. 12 and 13;
[0075] a polypeptide comprising SEQ ID NO. 14 and 15;
[0076] a polypeptide comprising SEQ ID NO. 16 and 17;
[0077] a polypeptide comprising SEQ ID NO. 18 and 19;
[0078] a polypeptide comprising SEQ ID NO. 20 and 21;
[0079] a polypeptide comprising SEQ ID NO. 23 and 24:
[0080] a polypeptide comprising SEQ ID NO. 25 and 26;
[0081] a polypeptide comprising SEQ ID NO. 30 and 31;
[0082] a polypeptide comprising SEQ ID NO. 32 and 33;
[0083] a polypeptide comprising SEQ ID NO. 34 and 35;
[0084] a polypeptide comprising SEQ ID NO. 337 and 338; or
[0085] a polypeptide comprising SEQ ID NO. 339 and 340;
[0086] with the proviso that the polypeptide is none of wild-type
of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or
42 through 100.
[0087] In one aspect, provided is an isolated or recombinant
polypeptide of the group of:
[0088] a polypeptide consisting essentially of SEQ ID NO. 12 and
13;
[0089] a polypeptide consisting essentially of SEQ ID NO. 14 and
15;
[0090] a polypeptide consisting essentially of SEQ ID NO. 16 and
17;
[0091] a polypeptide consisting essentially of SEQ ID NO. 18 and
19;
[0092] a polypeptide consisting essentially of SEQ ID NO. 20 and
21;
[0093] a polypeptide consisting essentially of SEQ ID NO. 23 and
24:
[0094] a polypeptide consisting essentially of SEQ ID NO. 25 and
26;
[0095] a polypeptide consisting essentially of SEQ ID NO. 30 and
31;
[0096] a polypeptide consisting essentially of SEQ ID NO. 32 and
33;
[0097] a polypeptide consisting essentially of SEQ ID NO. 34 and
35;
[0098] a polypeptide consisting essentially of SEQ ID NO. 337 and
338; or
[0099] a polypeptide consisting essentially of SEQ ID NO. 339 and
340;
[0100] with the proviso that the polypeptide is none of wild-type
of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or
42 through 100.
[0101] Also provided are isolated or recombinant polypeptides
comprising, or alternatively consisting essentially of, or yet
further consisting of, two or more, or three or more or four or
more, or multiples of the above-identified isolated polypeptides,
including fragments and equivalents thereof. Examples of such
include isolated polypeptides comprising SEQ ID NO. 1 through 4
and/or 12 through 29, and/or 30 through 33, and/or 30 through 35
e.g., SEQ ID NO. 1 and 2, or alternatively 1 and 3 or alternatively
1 and 4, or alternatively 2 and 3, or alternatively SEQ ID NO. 1, 2
and 3 or alternatively, 2, 3 and 4, or alternatively 1, 3 and 4 or
equivalent polypeptides, examples of which are shown in Table 9.
The polypeptides can be in any orientation, e.g., SEQ ID NO. 1, 2,
and 3 or SEQ ID NO. 3, 2 and 1 or alternatively SEQ ID NO. 2, 1 and
3, or alternatively, 3, 1 and 2, or alternatively 11 and 12, or
alternatively 1 and 12, or alternatively 2 and 12, or
alternatively, 1 and 12, or alternatively 2 and 13, or
alternatively 12, 16 and 1, or alternatively 1, 16 and 12.
[0102] In another aspect, this invention provides an isolated or
recombinant polypeptide comprising SEQ ID NO. 1 or 2 and 3 or 4 or
a polypeptide or recombinant polypeptide comprising, or
alternatively consisting essentially of, or yet further consisting
of an amino acid corresponding to fragments of a DNABII protein
such as the .beta.-3 and/or .alpha.-3 fragments of a Haemophilus
influenzae IHF.alpha. or IHF.beta. microorganism, non-limiting
examples of which include SEQ ID NO. 12 through 27, or a fragment
or an equivalent of each of the polypeptides, examples of which are
shown in Table 9. In one aspect, isolated wildtype polypeptides are
specifically excluded, e.g. that the polypeptide is none of SEQ ID
NO. 6 through 11 or a wildtype sequence identified in Table 8. In
this embodiment, SEQ ID NO. 1 or 2 or a polypeptide comprising, or
alternatively consisting essentially of, or yet further consisting
of an amino acid corresponding to the .beta.-3 and/or .alpha.-3
fragments of an IHF.alpha. or IHF.beta. microorganism, non-limiting
examples of which include SEQ ID NO. 12 through 27 and 30 through
33 or an equivalent of each thereof is located upstream or amino
terminus from SEQ ID NO. 3 or 4 or a fragment or an equivalent
thereof. In another aspect, the isolated polypeptide comprises SEQ
ID NO. 3 or 4 or a polypeptide comprising, or alternatively
consisting essentially of, or yet further consisting of an amino
acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of an
IHF.alpha. or IHF.beta. microorganism, non-limiting examples of
which include SEQ ID NO. 12 through 27, or an equivalent thereof
located upstream or amino terminus to SEQ ID. NO. 1 or 2 or an
equivalent thereof.
[0103] In any of the above embodiments, a peptide linker can be
added to the N-terminus or C-terminus of the polypeptide, fragment
or equivalent thereof. In one aspect, the linker joins the
polypeptides of this invention, e.g., SEQ ID NO. 1 to 4, 28, 29,
34, or 35 or 30 to 33, 34, or 35 or a polypeptide comprising, or
alternatively consisting essentially of, or yet further consisting
of an amino acid corresponding to the .beta.-3 and/or .alpha.-3
fragments of a Haemophilus influenza IHF.alpha. or IHF.beta.
microorganism, non-limiting examples of which include SEQ ID NO. 12
through 27 or an equivalent of each thereof. A "linker" or "peptide
linker" refers to a peptide sequence linked to either the
N-terminus or the C-terminus of a polypeptide sequence. In one
aspect, the linker is from about 1 to about 20 amino acid residues
long or alternatively 2 to about 10, about 3 to about 5 amino acid
residues long. An example of a peptide linker is
Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37).
[0104] Further provided is a fragment or an equivalent of the
isolated or recombinant polypeptide of any one of polypeptides
identified above as well as an isolated or recombinant polypeptide
comprising, or alternatively consisting essentially of or yet
further consisting of, two or more of the isolated or recombinant
polypeptides identified above.
[0105] Yet further provided is a polynucleotide that interferes
with the binding of the microbial DNA with a polypeptide or
fragment or equivalent thereof, e.g., SEQ ID 36, or a four-way
junction polynucleotide resembling a Holliday junction, a 3 way
junction polynucleotide resembling a replication fork, a
polynucleotide that has inherent flexibility or bent
polynucleotide; an isolated or recombinant polynucleotide encoding
a polypeptide described above or an antibody or fragment thereof,
which can be operatively linked to regulatory elements necessary
for the expression and/or replication of the polynucleotide. The
polynucleotide can be contained within a vector.
[0106] Also provided is an isolated host cell comprising, or
alternatively consisting essentially of, or yet further consisting
of an isolated or recombinant polypeptide described above, a
four-way junction polynucleotide resembling a Holliday junction, a
3 way junction polynucleotide resembling a replication fork, a
polynucleotide that has inherent flexibility or bent
polynucleotide; an isolated or recombinant polynucleotide as
described above, or a vector as described above.
[0107] In one aspect the cell is an isolated antigen presenting
cell comprising the isolated or recombinant polypeptide. In a
further aspect, the polypeptide is present on the surface of the
cell, such as a dendritic cell. In a further aspect, the antigen
presenting cell is transfected with one or more polynucleotides
encoding the polypeptide.
[0108] Yet further provided is an antibody or antigen binding
fragment that specifically recognizes and binds the isolated or
recombinant polypeptide as describe above, including a fragment or
an equivalent of the polypeptide. Non-limiting examples of
antibodies include a polyclonal antibody, a monoclonal antibody, a
humanized antibody, a human antibody, an antibody derivative, a
veneered antibody, a diabody, a chimeric antibody, an antibody
derivative, a recombinant human antibody, or an antibody fragment.
In a particular aspect, the antibody is a monoclonal antibody. Yet
further provided is a hybridoma cell line that produces the
monoclonal antibody.
[0109] This invention also provides isolated or recombinant
polynucleotides encoding one or more of the above-identified
isolated or recombinant polypeptides or antibodies or a fragment
thereof. Vectors comprising the isolated polynucleotides are
further provided. In one aspect where more than one isolated
polypeptide of this invention, the isolated polynucleotides can be
contained within a polycistronic vector.
[0110] Isolated host cells comprising one or more of isolated or
recombinant polypeptides or isolated or recombinant polynucleotides
or the vectors, described herein are further provided. In one
aspect the isolated host cell is a prokaryotic cell or eukaryotic
cell such as antigen presenting cell, e.g. a dendritic cell.
[0111] The polynucleotides, polypeptides, antibodies, antigen
binding fragment, vectors or host cells can further comprise a
detectable label.
[0112] Compositions comprising a carrier and one or more of an
isolated or recombinant polypeptide of the invention, an isolated
or recombinant polynucleotide of the invention, a vector of the
invention, an isolated host cell of the invention, or an antibody
of the embodiments are also provided. The carriers can be one or
more of a solid support, a medical device like a stent or dental
implant, or a liquid such as a pharmaceutically acceptable carrier.
The compositions can further comprise an adjuvant, an antimicrobial
or an antigenic peptide.
[0113] The compositions can further comprise additional
biologically active agents. A non-limiting example of such is a
antimicrobial agent such as other vaccine components (i.e.,
antigenic peptides) such as surface antigens, e.g. an OMP P5,
rsPilA, OMP 26, OMP P2, or Type IV Pilin protein (see Jurcisek and
Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, T
F, Bakaletz, L O and Smeesters, P R (2009) The Pediatric Infectious
Disease Journal, 28:S121-S126) and antimicrobial agents.
[0114] This invention also provides a method for producing an
antigenic peptide by growing or culturing a host cell comprising an
isolated polynucleotide encoding an antigenic peptide as described
above under conditions that favor the expression of the
polynucleotide. The polypeptide produced by this method can be
isolating for further in vitro or in vivo use.
[0115] A kit is also provided for diagnostic or therapeutic use
comprising a composition as described above and instructions for
use. A kit is also provided to perform screens for new drugs and/or
combination therapies as provided herein.
BRIEF DESCRIPTION OF THE FIGURES
[0116] FIG. 1A is a biofilm formed in the chinchilla middle ear by
NTHI strain 86-028NP and labeled for NTHI Tfp pilin protein
(appears as white or light gray speckles and small clusters in the
background of this image), as well as with DAPI for labeling of the
double stranded DNA (dsDNA) (appears as dark gray overlapping
strands and bundles of material with intermittent clumps in the
foreground of this image). This figure has been reproduced from
Jurcisek and Bakaletz (2007) J. of Bacteriology 189(10):3868-3875.
FIG. 1B is immunolabeling of bronchoalveolar lavage (BAL) from the
lung of a child with cystic fibrosis. The lung of a child with
cystic fibrosis was washed out via BAL and the particulate matter
from the wash was frozen and affixed to slides for immunolabeling.
The frozen particulate matter was immunolabeled with the anti-IHF
antibody. The presence of IHF positive foci in the biofilm of human
cystic fibrosis patients exemplifies that the etiology of human
cystic fibrosis includes a biofilm with IHF at the vertices of the
dsDNA. FIG. 1C shows secretions from human sinus collected at the
time of sinus surgery and embedded in OCT freezing medium (Optimal
Cutting Temperature medium, available commercially from Fisher
Scientific Cat. No. 14-373-65). 10 .mu.m frozen sections were cut
and labeled with anti-IHF (appearing as gray clusters). dsDNA
within the sample was stained with a fluorescent stain, DAPI
(4',6-diamidino-2-phenylindole, available commercially from
Invitrogen). The presence of IHF positive foci in the biofilm of
sinusitis patients exemplifies that the etiology of human sinusitis
includes a biofilm with IHF at the vertices of the dsDNA.
[0117] FIG. 2 is a immunohistochemical labeling of double stranded
DNA (appears as white strands in this image) within an NTHI biofilm
formed in the middle ear of chinchilla. Positive labeling for IHF
(as indicated by arrows pointing to punctuate foci in the middle
panel of this 3-panel image) was observed at nearly 100% of
vertices formed by dsDNA. Mean distance between vertices was
approximately 6 .mu.m, or approximately 18 kb between each vertex
if one assumes 0.34 nm per base of DNA for B-form DNA. To the best
of our knowledge, the only proteins that possess epitopes that
cross-react with anti-IHF are HU and IHF. Therefore, based on these
observations, it appeared that not only were there extracellular
DNABII proteins within the NTHI biofilm matrix, but more
importantly that these proteins appeared to be exclusively
positioned on eDNA strands that resembled cruciform structures in
conformation (see FIG. 1B, bottom section), thus strongly
suggesting their role in mediating the resulting bent conformation
of the eDNA.
[0118] FIGS. 3A-3E show that antibodies directed against IHF
reversed an established NTHI biofilm formed in a chamber slide.
FIG. 3A shows a biofilm treated with non specific antibody. FIG. 3B
shows a biofilm treated with naive rabbit serum. Note the robust
NTHI biofilm with abundant towers (appear as white to light gray
clustered areas) and water channels (black spaces). FIG. 3C is a
biofilm treated with anti-IHF. Note the eradication of biofilm
structure after treatment with an anti-IHF. Individual NTHI (appear
as small punctuate white to light gray spots) and sparse, short
towers (appear as denser white to light gray clustered areas)
remain. FIGS. 3D and 3E further depict that antibodies directed
against IHF reverse an established NTHI biofilm. As shown in FIG.
3E, the biofilm showed a dramatic loss of 3-dimensional structure
when compared to a biofilm incubated with naive serum (FIG. 3D).
Via COMSTAT analysis of multiple replicate assays, the measured
parameters of biofilm height, biomass and biofilm thickness were
all diminished by a mean of greater than 80% upon incubation with
anti-IHF.
[0119] FIGS. 4A and 4B are graphs showing treatment of an
established biofilm formed by NTHI with anti-IHF results in more
NTHI released into the supernatant. Sixteen (16) hour NTHI biofilms
grown in chamber slides were sham treated with sterile medium
(sBHI) or treated with naive rabbit serum or rabbit anti-IHF. Six
hours later (FIG. 4A) or 10 hours later (FIG. 4B), supernatants
were collected and analyzed. Note the greater number of NTHI in
supernatant after treatment with anti-IHF. Incubation with anti-IHF
resulted in a marked increase in planktonic bacteria available for
culture from the medium within the chamber slide within
approximately 6 hrs, and increasing notably at 10 hours of
incubation. These results suggested release of bacteria from the
biofilm matrix.
[0120] FIG. 5 shows the results of a transcutaneous immunization
with IHF reduced an established biofilm in the middle ear. Note
that bullae were blindly ranked onto a 0 to 4+ scale of relative
remaining biofilm mass.
[0121] FIG. 6A is a map indicating the amino acid residues of IHF
that interact with or bind to another IHF in an IHF-IHF dimmer
(indicated by triangles at the upper level) or interact with or
bind DNA (indicated by triangles at the lower level). The peptide
is divided by the short vertical bars into regions containing 3
amino acids.
[0122] FIG. 6B graphically depicts the interaction of microbial DNA
with an IHF.
[0123] FIG. 7 depicts the reduction of biofilms formed by S.
aureus, N. gonorrhoeae and P. aeruginosa upon incubation with
rabbit anti-IHF compared to incubation with naive rabbit serum.
[0124] FIGS. 8A-8F show the effect of incubation of biofilms formed
in vitro by E. coli with either naive serum or with anti-IHF serum.
Representative images of biofilms are shown in FIGS. 8A-8F with
height of individual biofilms shown to the right of each image,
whereas mean values in terms of percent reduction in biofilm
jheight, biomass and mean thickness as mediated by incubation with
anti-IHF are shown in the tables at the end of each row. FIGS. 8A
and 8B--parental strain MG1655; FIGS. 8C and 8D--HU-deficient hupA,
hubB double mutant; FIGS. 8E and 8F--IHF-deficient himD, himA
double mutant. Note the ability of anti-IHF serum to reduce biomass
induced by either the parental isolate or the HU-deficient mutant,
but not the IHF-deficient strain, as expected.
[0125] FIG. 9A demonstrates that immunization with IHF via a
trancutaneous delivery route induced the formation of antibodies
that significantly reduced the biomass of an NTHI-induced biofilm
resident within the middle ears of chinchillas (p<0.001).
[0126] FIG. 9B depicts representative images of biomasses that
remained in the ears of animals immunized with adjuvant alone
versus those immunized with IHF+adjuvant. Last column in FIG. 9B
shows images of biomasses at the extremes of the scoring system
used here. Top image is that of a middle ear that contains a
biomass that would receive a score of 4+, whereas lower image is a
healthy middle ear that would receive a score of 0, indicating no
biomass.
[0127] FIG. 10A shows H&E staining of a frozen section of a
biofilm recovered from the middle ear of a chinchilla that had been
immunized via TCI with adjuvant alone versus immunization with
IHF+adjuvant. Note condensed and collapsed appearance of biofilm
recovered from the animal immunized with IHF+adjuvant compared to
that immunized with adjuvant alone. Images shown in FIG. 10A are
shown at identical magnifications to illustrate the differences in
height and density between the two representative biomasses.
[0128] FIG. 10B demonstrates that there is a significant reduction
of bacterial load present in the middle ears of animals immunized
with adjuvant alone versus those immunized with IHF+adjuvant
(p<0.05).
[0129] FIG. 11 depicts an electrophoretic mobility shift assay
which demonstrated that IHF formed specific complexes with dsDNA
under conditions used to immunize chinchillas.
[0130] FIG. 12 graphically represents transcutaneous immunization
with native IHF+adjuvant induced the formation of antibodies that
significantly reduced the biomass resident within the middle ears
of chinchillas compared to receipt of either adjuvant alone
(p<0.017), dsDNA alone (p<0.003) or IHF to which dsDNA was
already bound+adjuvant (p<0.001). This outcome suggested that
binding of dsDNA to native IHF masked protective epitopes of this
DNABII family member.
[0131] FIG. 13 depicts the recognition of IHF (arrows) by antibody
in serum after subcutaneous immunization with IHF or with IHF to
which dsDNA was bound.
[0132] FIGS. 14A-14C are a demonstration of synergism between a
sub-optimal concentration of anti-IHF serum (1:200) and DNAseI
individually, then mixed (FIG. 14A); a sub-optimal concentration of
anti-IHF (1:100) and anti-outer membrane protein P5 serum (OMP P5)
individually, then mixed (FIG. 14B); or that of an effective
dilution of anti-IHF (in terms of debulking a biofilm but not
inducing bacterial cell death) and amoxicillin individually, then
mixed (FIG. 14C). In each of these situations, when any agent was
combined with anti-IHF, the biofilm debulking and/or killing effect
observed was greater than that noted when any single agent was used
alone. Note: biofilm height (in microns) is indicated under each
image.
[0133] FIG. 15 is a comparison of E. coli treated with either naive
rabbit serum or rabbit anti-IHF serum (top row of images); with
naive rat serum or rat anti-HNS serum (middle row of images); or
with naive mouse serum or with mouse anti-DPS serum (bottom row of
images). Note marked reduction in biomass following treatment with
anti-IHF serum, however neither anti-HNS nor anti-DPS serum induced
a reduction in biomass as used herein. Treatment with anti-IHF
resulted in a 48.2% reduction in the biofilm height, an 81%
reduction in the biofilm average thickness, and a 64.5% reduction
in the biomass. In contrast treatment with the anti-HNS or anti-DPS
resulted in a nominal height reduction of 0.7% and 4.2%,
respectively, a reduction in the average thickness of 5.8% and 6.4%
respectively, and a reduction in the biomass of 0.3% and -17.4%
respectively.
[0134] FIG. 16 shows in situ hybridization to demonstrate relative
spatial distribution of DNA from either a host organism or from the
bacterium when organized within a biofilm. In each circumstance,
DNA appears as brighter, whiter areas within the foreground of
these black and white images. DNA from the host is more densely
labeled within the upper right hand image, demonstrating its more
heavy distribution on the outer periphery of the biofilm, whereas
DNA from the bacterium is more densely labeled in the lower left
hand image of this 4-panel composite, thereby demonstrating its
more dense distribution within the inner reaches of the
biofilm.
DETAILED DESCRIPTION OF THE INVENTION
[0135] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
nucleotide sequences provided herein are presented in the 5' to 3'
direction. Although any methods and materials similar or equivalent
to those described herein can be used in the practice or testing of
the present invention, the preferred methods, devices, and
materials are now described. All technical and patent publications
cited herein are incorporated herein by reference in their
entirety. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
[0136] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of tissue culture,
immunology, molecular biology, microbiology, cell biology and
recombinant DNA, which are within the skill of the art. See, e.g.,
Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory
Manual, 3.sup.rd edition; the series Ausubel et al. eds. (2007)
Current Protocols in Molecular Biology; the series Methods in
Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991)
PCR 1: A Practical Approach (IRL Press at Oxford University Press);
MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and
Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005)
Culture of Animal Cells: A Manual of Basic Technique, 5.sup.th
edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.
4,683,195; Hames and Higgins eds. (1984) Nucleic Acid
Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames
and Higgins eds. (1984) Transcription and Translation; Immobilized
Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical
Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene
Transfer Vectors for Mammalian Cells (Cold Spring Harbor
Laboratory); Makrides ed. (2003) Gene Transfer and Expression in
Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical
Methods in Cell and Molecular Biology (Academic Press, London); and
Herzenberg et al. eds (1996) Weir's Handbook of Experimental
Immunology.
[0137] All numerical designations, e.g., pH, temperature, time,
concentration, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 1.0 or
0.1, as appropriate or alternatively by a variation of +/-15%, or
alternatively 10% or alternatively 5% or alternatively 2%. It is to
be understood, although not always explicitly stated, that all
numerical designations are preceded by the term "about". It also is
to be understood, although not always explicitly stated, that the
reagents described herein are merely exemplary and that equivalents
of such are known in the art.
[0138] As used in the specification and claims, the singular form
"a". "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a polypeptide"
includes a plurality of polypeptides, including mixtures
thereof
[0139] As used herein, the term "comprising" is intended to mean
that the compositions and methods include the recited elements, but
do not exclude others. "Consisting essentially of" when used to
define compositions and methods, shall mean excluding other
elements of any essential significance to the combination for the
intended use. Thus, a composition consisting essentially of the
elements as defined herein would not exclude trace contaminants
from the isolation and purification method and pharmaceutically
acceptable carriers, such as phosphate buffered saline,
preservatives, and the like. "Consisting of" shall mean excluding
more than trace elements of other ingredients and substantial
method steps for administering the compositions of this invention.
Embodiments defined by each of these transition terms are within
the scope of this invention.
[0140] A "biofilm" intends an organized community of microorganisms
that at times adhere to the surface of a structure, that may be
organic or inorganic, together with the polymers such as DNA that
they secrete and/or release. The biofilms are very resistant to
microbiotics and antimicrobial agents. They live on gingival
tissues, teeth and restorations, causing caries and periodontal
disease, also known as periodontal plaque disease. They also cause
chronic middle ear infections. Biofilms can also form on the
surface of dental implants, stents, catheter lines and contact
lenses. They grow on pacemakers, heart valve replacements,
artificial joints and other surgical implants. The Centers for
Disease Control) estimate that over 65% of nosocomial
(hospital-acquired) infections are caused by biofilms. They cause
chronic vaginal infections and lead to life-threatening systemic
infections in people with hobbled immune systems. Biofilms also are
involved in numerous diseases. For instance, cystic fibrosis
patients have Pseudomonas infections that often result in
antibiotic resistant biofilms.
[0141] The term "inhibiting, competing or titrating" intends a
reduction in the formation of the DNA/protein matrix (for example
as shown in FIG. 1) that is a component of a microbial biofilm.
[0142] A "DNABII polypeptide or protein" intends a DNA binding
protein or polypeptide that is composed of DNA-binding domains and
thus have a specific or general affinity for microbial DNA. In one
aspect, they bind DNA in the minor grove. Non-limiting examples of
DNABII proteins are an integration host factor (IHF) protein and a
histone-like protein from E. coli strain U93 (HU). Other DNA
binding proteins that may be associated with the biofilm include
DPS (Genbank Accession No.: CAA49169), H-NS (Genbank Accession No.:
CAA47740), Hfq (Genbank Accession No.: ACE63256), CbpA (Genbank
Accession No.: BAA03950) and CbpB (Genbank Accession No.:
NP.sub.--418813).
[0143] An "integration host factor" of "HF" protein is a bacterial
protein that is used by bacteriophages to incorporate their DNA
into the host bacteria. They also bind extracellular microbial DNA.
The genes that encode the IHF protein subunits in E. coli are himA
(Genbank accession No.: POA6X7.1) and himD (POA6Y1.1) genes.
Homologs for these genes are found in other organisms, and peptides
corresponding to these genes from other organisms can be found in
Table 10.
[0144] "HMGB1" is an high mobility group box (HMGB) 1 protein that
is reported to bind to and distort the minor groove of DNA and is
an example of an interfering agent. Recombinant or isolated protein
and polypeptide are commercially available from Atgenglobal,
ProSpecBio, Protein1 and Abnova.
[0145] "HU" or "histone-like protein from E. coli strain U93"
refers to a class of heterodimeric proteins typically associate
with E. coli. HU proteins are known to bind DNA junctions. Related
proteins have been isolated from other microorganisms. The complete
amino acid sequence of E. coli HU was reported by Laine et al.
(1980) Eur. J. Biochem. 103(3):447-481. Antibodies to the HU
protein are commercially available from Abcam. The genes that
encode the HU protein subunits in E. coli are hupA and hupB
corresponding to SEQ ID Nos: 28 and 29, respectively. Homologs for
these genes are found in other organisms, and peptides
corresponding to these genes from other organisms can be found in
Table 10.
[0146] The term "surface antigens" or "surface proteins" refers to
proteins or peptides on the surface of cells such as bacterial
cells. Examples of surface antigens are Outermembrane proteins such
as OMP P5 (Genbank accession No.: YP.sub.--004139079.1), OMP P2
(Genbank accession No.: ZZX87199.1), OMP P26 (Genbank Accession
No.: YP.sub.--665091.1), rsPilA or recombinant soluble PilA
(Genbank accession No.: EFU96734.1) and Type IV Pilin (Genbank
accession No.: YP.sub.--003864351.1).
[0147] The term "Haemophilus influenzae" refers to pathogenic
bacteria that can cause many different infections such as, for
example, ear infections, eye infections, and sinusitis. Many
different strains of Haemophilus influenzae have been isolated and
have an IhfA gene or protein. Some non-limiting examples of
different strains of Haemophilus influenzae include Rd KW20,
86-028NP, R2866, PittGG, PittEE, R2846, and 2019.
[0148] "Microbial DNA" intends single or double stranded DNA from a
microorganism that produces a biofilm.
[0149] "Inhibiting, preventing or breaking down" a biofilm intends
the prophylactic or therapeutic reduction in the structure of a
biofilm. An example of breaking down or reducing a biofilm is shown
in FIG. 5.
[0150] An "interfering agent" intends an agent that any one or more
of competes, inhibits, prevents, titrates a DNABII polypeptide such
as IHF to a microbial DNA or also breaks down a microbial biofilm.
It can be any one or more of a chemical or biological molecule. For
example, IHF can specifically bind, bend or distorted DNA
structures such as DNA containing four-way junctions, cis-platinum
adducts, DNA loop or base bulges. Examples of such agents, without
limitation, include (1) small molecules that inhibit the
DNA-binding activity of IHF, (2) small molecules such as polyamines
and spermine that compete with IHF in DNA binding, (3) polypeptides
such as peptide fragments of IHF that compete with IHF in DNA
binding, (4) antibodies or fragments thereof directed to IHF, or
(5) a four-way or bent polynucleotides or other types of
polynucleotides containing bent or distorted DNA structures that
compete in IHF-binding. A "small molecule that inhibits the binding
of an IHF to a nucleic acid" refers to (1) or (2) above and include
those that bind DNA in the minor grove, i.e., minor groove binding
molecules. A "four-way polynucleotide" intends a polynucleotide
that contains a four-way junction, also known as the Holliday
junction, between four strands of DNA.
[0151] A "bent polynucleotide" intends a double strand
polynucleotide that contains a small loop on one strand which does
not pair with the other strand. In some embodiments, the loop is
from 1 base to about 20 bases long, or alternatively from 2 bases
to about 15 bases long, or alternatively from about 3 bases to
about 12 bases long, or alternatively from about 4 bases to about
10 bases long, or alternatively has about 4, 5, or 6, or 7, or 8,
or 9, or 10 bases.
[0152] "Polypeptides that compete with IHF in DNA binding" intend
proteins or peptides that compete with IHF in binding bent or
distorted DNA structures but do not form a biofilm with the DNA.
Examples, without limitation, include fragments of IHF that include
one or more DNA binding domains of the IHF, or the biological
equivalents thereof. DNA binding domains are shown in FIG. 6.
[0153] A "subject" of diagnosis or treatment is a cell or an animal
such as a mammal, or a human. Non-human animals subject to
diagnosis or treatment and are those subject to infections or
animal models, for example, simians, murines, such as, rats, mice,
chinchilla, canine, such as dogs, leporids, such as rabbits,
livestock, sport animals, and pets.
[0154] The term "protein", "peptide" and "polypeptide" are used
interchangeably and in their broadest sense to refer to a compound
of two or more subunit amino acids, amino acid analogs or
peptidomimetics. The subunits may be linked by peptide bonds. In
another embodiment, the subunit may be linked by other bonds, e.g.,
ester, ether, etc. A protein or peptide must contain at least two
amino acids and no limitation is placed on the maximum number of
amino acids which may comprise a protein's or peptide's sequence.
As used herein the term "amino acid" refers to either natural
and/or unnatural or synthetic amino acids, including glycine and
both the D and L optical isomers, amino acid analogs and
peptidomimetics.
[0155] A "C-terminal polypeptide" intends at least the 10, or
alternatively at least the 15, or alternatively at least 20, or at
least the 25 C-terminal amino acids or alternatively half of a
polypeptide. In another aspect, for polypeptides containing 90
amino acids, the C-terminal polypeptide would comprise amino acids
46 through 90. In one aspect, the term intends the C-terminal 20
amino acids from the carboxy terminus.
[0156] The terms "polynucleotide" and "oligonucleotide" are used
interchangeably and refer to a polymeric form of nucleotides of any
length, either deoxyribonucleotides or ribonucleotides or analogs
thereof. Polynucleotides can have any three-dimensional structure
and may perform any function, known or unknown. The following are
non-limiting examples of polynucleotides: a gene or gene fragment
(for example, a probe, primer, EST or SAGE tag), exons, introns,
messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes and primers. A polynucleotide can
comprise modified nucleotides, such as methylated nucleotides and
nucleotide analogs. If present, modifications to the nucleotide
structure can be imparted before or after assembly of the
polynucleotide. The sequence of nucleotides can be interrupted by
non-nucleotide components. A polynucleotide can be further modified
after polymerization, such as by conjugation with a labeling
component. The term also refers to both double- and single-stranded
molecules. Unless otherwise specified or required, any embodiment
of this invention that is a polynucleotide encompasses both the
double-stranded form and each of two complementary single-stranded
forms known or predicted to make up the double-stranded form.
[0157] A polynucleotide is composed of a specific sequence of four
nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine
(T); and uracil (U) for thymine when the polynucleotide is RNA.
Thus, the term "polynucleotide sequence" is the alphabetical
representation of a polynucleotide molecule. This alphabetical
representation can be input into databases in a computer having a
central processing unit and used for bioinformatics applications
such as functional genomics and homology searching.
[0158] The term "isolated" or "recombinant" as used herein with
respect to nucleic acids, such as DNA or RNA, refers to molecules
separated from other DNAs or RNAs, respectively that are present in
the natural source of the macromolecule as well as polypeptides.
The term "isolated or recombinant nucleic acid" is meant to include
nucleic acid fragments which are not naturally occurring as
fragments and would not be found in the natural state. The term
"isolated" is also used herein to refer to polynucleotides,
polypeptides and proteins that are isolated from other cellular
proteins and is meant to encompass both purified and recombinant
polypeptides. In other embodiments, the term "isolated or
recombinant" means separated from constituents, cellular and
otherwise, in which the cell, tissue, polynucleotide, peptide,
polypeptide, protein, antibody or fragment(s) thereof, which are
normally associated in nature. For example, an isolated cell is a
cell that is separated from tissue or cells of dissimilar phenotype
or genotype. An isolated polynucleotide is separated from the 3'
and 5' contiguous nucleotides with which it is normally associated
in its native or natural environment, e.g., on the chromosome. As
is apparent to those of skill in the art, a non-naturally occurring
polynucleotide, peptide, polypeptide, protein, antibody or
fragment(s) thereof, does not require "isolation" to distinguish it
from its naturally occurring counterpart.
[0159] It is to be inferred without explicit recitation and unless
otherwise intended, that when the present invention relates to a
polypeptide, protein, polynucleotide or antibody, an equivalent or
a biologically equivalent of such is intended within the scope of
this invention. As used herein, the term "biological equivalent
thereof" is intended to be synonymous with "equivalent thereof"
when referring to a reference protein, antibody, fragment,
polypeptide or nucleic acid, intends those having minimal homology
while still maintaining desired structure or functionality. Unless
specifically recited herein, it is contemplated that any
polynucleotide, polypeptide or protein mentioned herein also
includes equivalents thereof. In one aspect, an equivalent
polynucleotide is one that hybridizes under stringent conditions to
the polynucleotide or complement of the polynucleotide as described
herein for use in the described methods. In another aspect, an
equivalent antibody or antigen binding polypeptide intends one that
binds with at least 70%, or alternatively at least 75%, or
alternatively at least 80%, or alternatively at least 85%, or
alternatively at least 90%, or alternatively at least 95% affinity
or higher affinity to a reference antibody or antigen binding
fragment. In another aspect, the equivalent thereof competes with
the binding of the antibody or antigen binding fragment to its
antigen under a competitive ELISA assay. In another aspect, an
equivalent intends at least about 80% homology or identity and
alternatively, at least about 85%, or alternatively at least about
90%, or alternatively at least about 95%, or alternatively 98%
percent homology or identity and exhibits substantially equivalent
biological activity to the reference protein, polypeptide or
nucleic acid. Examples of biologically equivalent polypeptides are
provided in Table 9 which identify conservative amino acid
substitutions to the preferred amino acid sequences.
[0160] A polynucleotide or polynucleotide region (or a polypeptide
or polypeptide region) having a certain percentage (for example,
80%, 85%, 90%, or 95%) of "sequence identity" to another sequence
means that, when aligned, that percentage of bases (or amino acids)
are the same in comparing the two sequences. The alignment and the
percent homology or sequence identity can be determined using
software programs known in the art, for example those described in
Current Protocols in Molecular Biology (Ausubel et al., eds. 1987)
Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default
parameters are used for alignment. A preferred alignment program is
BLAST, using default parameters. In particular, preferred programs
are BLASTN and BLASTP, using the following default parameters:
Genetic code=standard; filter=none; strand=both; cutoff=60;
expect=10: Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH
SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS
translations+SwissProtein+SPupdate+PIR. Details of these programs
can be found at the following Internet address:
ncbi.nlm.nih.gov/cgi-binBLAST. Sequence identity and percent
identity were determined by incorporating them into clustalW
(available at the web address: align.genome.jp, last accessed on
Mar. 7, 2011.
[0161] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or between two nucleic acid
molecules. Homology can be determined by comparing a position in
each sequence which may be aligned for purposes of comparison. When
a position in the compared sequence is occupied by the same base or
amino acid, then the molecules are homologous at that position. A
degree of homology between sequences is a function of the number of
matching or homologous positions shared by the sequences. An
"unrelated" or "non-homologous" sequence shares less than 40%
identity, or alternatively less than 25% identity, with one of the
sequences of the present invention.
[0162] "Homology" or "identity" or "similarity" can also refer to
two nucleic acid molecules that hybridize under stringent
conditions.
[0163] "Hybridization" refers to a reaction in which one or more
polynucleotides react to form a complex that is stabilized via
hydrogen bonding between the bases of the nucleotide residues. The
hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein
binding, or in any other sequence-specific manner. The complex may
comprise two strands forming a duplex structure, three or more
strands forming a multi-stranded complex, a single self-hybridizing
strand, or any combination of these. A hybridization reaction may
constitute a step in a more extensive process, such as the
initiation of a PCR reaction, or the enzymatic cleavage of a
polynucleotide by a ribozyme.
[0164] Examples of stringent hybridization conditions include:
incubation temperatures of about 25.degree. C. to about 37.degree.
C.; hybridization buffer concentrations of about 6.times.SSC to
about 10.times.SSC; formamide concentrations of about 0% to about
25%; and wash solutions from about 4.times.SSC to about
8.times.SSC. Examples of moderate hybridization conditions include:
incubation temperatures of about 40.degree. C. to about 50.degree.
C.; buffer concentrations of about 9.times.SSC to about
2.times.SSC; formamide concentrations of about 30% to about 50%;
and wash solutions of about 5.times.SSC to about 2.times.SSC.
Examples of high stringency conditions include: incubation
temperatures of about 55.degree. C. to about 68.degree. C.; buffer
concentrations of about 1x SSC to about 0.1x SSC; formamide
concentrations of about 55% to about 75%; and wash solutions of
about 1.times.SSC. 0.1.times.SSC, or deionized water. In general,
hybridization incubation times are from 5 minutes to 24 hours, with
1, 2, or more washing steps, and wash incubation times are about 1,
2, or 15 minutes. SSC is 0.15 M NaCl and 15 mM citrate buffer. It
is understood that equivalents of SSC using other buffer systems
can be employed.
[0165] As used herein, "expression" refers to the process by which
polynucleotides are transcribed into mRNA and/or the process by
which the transcribed mRNA is subsequently being translated into
peptides, polypeptides, or proteins. If the polynucleotide is
derived from genomic DNA, expression may include splicing of the
mRNA in an eukaryotic cell.
[0166] The term "encode" as it is applied to polynucleotides refers
to a polynucleotide which is said to "encode" a polypeptide if, in
its native state or when manipulated by methods well known to those
skilled in the art, it can be transcribed and/or translated to
produce the mRNA for the polypeptide and/or a fragment thereof. The
antisense strand is the complement of such a nucleic acid, and the
encoding sequence can be deduced therefrom.
[0167] As used herein, the terms "treating," "treatment" and the
like are used herein to mean obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disorder or sign or symptom
thereof, and/or may be therapeutic in terms of a partial or
complete cure for a disorder and/or adverse effect attributable to
the disorder.
[0168] To prevent intends to prevent a disorder or effect in vitro
or in vivo in a system or subject that is predisposed to the
disorder or effect. An example of such is preventing the formation
of a biofilm in a system that is infected with a microorganism
known to produce one.
[0169] A "composition" is intended to mean a combination of active
agent and another compound or composition, inert (for example, a
detectable agent or label) or active, such as an adjuvant.
[0170] A "pharmaceutical composition" is intended to include the
combination of an active agent with a carrier, inert or active,
making the composition suitable for diagnostic or therapeutic use
in vitro, in vivo or ex vivo.
[0171] "Pharmaceutically acceptable carriers" refers to any
diluents, excipients, or carriers that may be used in the
compositions of the invention. Pharmaceutically acceptable carriers
include ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances, such as
phosphates, glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol
and wool fat. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences, Mack Publishing Company, a
standard reference text in this field. They are preferably selected
with respect to the intended form of administration, that is, oral
tablets, capsules, elixirs, syrups and the like, and consistent
with conventional pharmaceutical practices.
[0172] A "biologically active agent" or an active agent of this
invention intends one or more of an isolated or recombinant
polypeptide, an isolated or recombinant polynucleotide, a vector,
an isolated host cell, or an antibody, as well as compositions
comprising one or more of same.
[0173] "Administration" can be effected in one dose, continuously
or intermittently throughout the course of treatment. Methods of
determining the most effective means and dosage of administration
are known to those of skill in the art and will vary with the
composition used for therapy, the purpose of the therapy, the
target cell being treated, and the subject being treated. Single or
multiple administrations can be carried out with the dose level and
pattern being selected by the treating physician. Suitable dosage
formulations and methods of administering the agents are known in
the art. Route of administration can also be determined and method
of determining the most effective route of administration are known
to those of skill in the art and will vary with the composition
used for treatment, the purpose of the treatment, the health
condition or disease stage of the subject being treated, and target
cell or tissue. Non-limiting examples of route of administration
include oral administration, nasal administration, injection, and
topical application.
[0174] An agent of the present invention can be administered for
therapy by any suitable route of administration. It will also be
appreciated that the preferred route will vary with the condition
and age of the recipient, and the disease being treated.
[0175] The term "effective amount" refers to a quantity sufficient
to achieve a desired effect. In the context of therapeutic or
prophylactic applications, the effective amount will depend on the
type and severity of the condition at issue and the characteristics
of the individual subject, such as general health, age, sex, body
weight, and tolerance to pharmaceutical compositions. In the
context of an immunogenic composition, in some embodiments the
effective amount is the amount sufficient to result in a protective
response against a pathogen. In other embodiments, the effective
amount of an immunogenic composition is the amount sufficient to
result in antibody generation against the antigen. In some
embodiments, the effective amount is the amount required to confer
passive immunity on a subject in need thereof. With respect to
immunogenic compositions, in some embodiments the effective amount
will depend on the intended use, the degree of immunogenicity of a
particular antigenic compound, and the health/responsiveness of the
subject's immune system, in addition to the factors described
above. The skilled artisan will be able to determine appropriate
amounts depending on these and other factors.
[0176] In the case of an in vitro application, in some embodiments
the effective amount will depend on the size and nature of the
application in question. It will also depend on the nature and
sensitivity of the in vitro target and the methods in use. The
skilled artisan will be able to determine the effective amount
based on these and other considerations. The effective amount may
comprise one or more administrations of a composition depending on
the embodiment.
[0177] The term "conjugated moiety" refers to a moiety that can be
added to an isolated chimeric polypeptide by forming a covalent
bond with a residue of chimeric polypeptide.
[0178] The moiety may bond directly to a residue of the chimeric
polypeptide or may form a covalent bond with a linker which in turn
forms a covalent bond with a residue of the chimeric
polypeptide.
[0179] A "peptide conjugate" refers to the association by covalent
or non-covalent bonding of one or more polypeptides and another
chemical or biological compound. In a non-limiting example, the
"conjugation" of a polypeptide with a chemical compound results in
improved stability or efficacy of the polypeptide for its intended
purpose. In one embodiment, a peptide is conjugated to a carrier,
wherein the carrier is a liposome, a micelle, or a pharmaceutically
acceptable polymer.
[0180] "Liposomes" are microscopic vesicles consisting of
concentric lipid bilayers. Structurally, liposomes range in size
and shape from long tubes to spheres, with dimensions from a few
hundred Angstroms to fractions of a millimeter. Vesicle-forming
lipids are selected to achieve a specified degree of fluidity or
rigidity of the final complex providing the lipid composition of
the outer layer. These are neutral (cholesterol) or bipolar and
include phospholipids, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylinositol (PI), and
sphingomyelin (SM) and other types of bipolar lipids including but
not limited to dioleoylphosphatidylethanolamine (DOPE), with a
hydrocarbon chain length in the range of 14-22, and saturated or
with one or more double C.dbd.C bonds. Examples of lipids capable
of producing a stable liposome, alone, or in combination with other
lipid components are phospholipids, such as hydrogenated soy
phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine,
lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebrosides,
distearoylphosphatidylethan-olamine (DSPE),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE) and
dioleoylphosphatidylethanolamine
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal).
Additional non-phosphorous containing lipids that can become
incorporated into liposomes include stearylamine, dodecylamine,
hexadecylamine, isopropyl myristate, triethanolamine-lauryl
sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol
ricinoleate, hexadecyl stereate, amphoteric acrylic polymers,
polyethyloxylated fatty acid amides, and the cationic lipids
mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA),
DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include
phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are
able to form vesicles. Typically, liposomes can be divided into
three categories based on their overall size and the nature of the
lamellar structure. The three classifications, as developed by the
New York Academy Sciences Meeting, "Liposomes and Their Use in
Biology and Medicine," December 1977, are multi-lamellar vesicles
(MLVs), small uni-lamellar vesicles (SUVs) and large uni-lamellar
vesicles (LUVs). The biological active agents can be encapsulated
in such for administration in accordance with the methods described
herein.
[0181] A "micelle" is an aggregate of surfactant molecules
dispersed in a liquid colloid. A typical micelle in aqueous
solution forms an aggregate with the hydrophilic "head" regions in
contact with surrounding solvent, sequestering the hydrophobic tail
regions in the micelle center. This type of micelle is known as a
normal phase micelle (oil-in-water micelle). Inverse micelles have
the head groups at the center with the tails extending out
(water-in-oil micelle). Micelles can be used to attach a
polynucleotide, polypeptide, antibody or composition described
herein to facilitate efficient delivery to the target cell or
tissue.
[0182] The phrase "pharmaceutically acceptable polymer" refers to
the group of compounds which can be conjugated to one or more
polypeptides described here. It is contemplated that the
conjugation of a polymer to the polypeptide is capable of extending
the half-life of the polypeptide in vivo and in vitro. Non-limiting
examples include polyethylene glycols, polyvinylpyrrolidones,
polyvinylalcohols, cellulose derivatives, polyacrylates,
polymethacrylates, sugars, polyols and mixtures thereof. The
biological active agents can be conjugated to a pharmaceutically
acceptable polymer for administration in accordance with the
methods described herein.
[0183] A "gene delivery vehicle" is defined as any molecule that
can carry inserted polynucleotides into a host cell. Examples of
gene delivery vehicles are liposomes, micelles biocompatible
polymers, including natural polymers and synthetic polymers;
lipoproteins; polypeptides; polysaccharides; lipopolysaccharides;
artificial viral envelopes; metal particles; and bacteria, or
viruses, such as baculovirus, adenovirus and retrovirus,
bacteriophage, cosmid, plasmid, fungal vectors and other
recombination vehicles typically used in the art which have been
described for expression in a variety of eukaryotic and prokaryotic
hosts, and may be used for gene therapy as well as for simple
protein expression.
[0184] A polynucleotide of this invention can be delivered to a
cell or tissue using a gene delivery vehicle. "Gene delivery,"
"gene transfer," "transducing," and the like as used herein, are
terms referring to the introduction of an exogenous polynucleotide
(sometimes referred to as a "transgene") into a host cell,
irrespective of the method used for the introduction. Such methods
include a variety of well-known techniques such as vector-mediated
gene transfer (by, e.g., viral infection/transfection, or various
other protein-based or lipid-based gene delivery complexes) as well
as techniques facilitating the delivery of "naked" polynucleotides
(such as electroporation, "gene gun" delivery and various other
techniques used for the introduction of polynucleotides). The
introduced polynucleotide may be stably or transiently maintained
in the host cell. Stable maintenance typically requires that the
introduced polynucleotide either contains an origin of replication
compatible with the host cell or integrates into a replicon of the
host cell such as an extrachromosomal replicon (e.g., a plasmid) or
a nuclear or mitochondrial chromosome. A number of vectors are
known to be capable of mediating transfer of genes to mammalian
cells, as is known in the art and described herein.
[0185] As used herein the term "eDNA" refers to extracellular DNA
found as a component to pathogenic biofilms.
[0186] A "plasmid" is an extra-chromosomal DNA molecule separate
from the chromosomal DNA which is capable of replicating
independently of the chromosomal DNA. In many cases, it is circular
and double-stranded. Plasmids provide a mechanism for horizontal
gene transfer within a population of microbes and typically provide
a selective advantage under a given environmental state. Plasmids
may carry genes that provide resistance to naturally occurring
antibiotics in a competitive environmental niche, or alternatively
the proteins produced may act as toxins under similar
circumstances.
[0187] "Plasmids" used in genetic engineering are called "plasmid
vectors". Many plasmids are commercially available for such uses.
The gene to be replicated is inserted into copies of a plasmid
containing genes that make cells resistant to particular
antibiotics and a multiple cloning site (MCS, or polylinker), which
is a short region containing several commonly used restriction
sites allowing the easy insertion of DNA fragments at this
location. Another major use of plasmids is to make large amounts of
proteins. In this case, researchers grow bacteria containing a
plasmid harboring the gene of interest. Just as the bacterium
produces proteins to confer its antibiotic resistance, it can also
be induced to produce large amounts of proteins from the inserted
gene. This is a cheap and easy way of mass-producing a gene or the
protein it then codes for.
[0188] A "yeast artificial chromosome" or "YAC" refers to a vector
used to clone large DNA fragments (larger than 100 kb and up to
3000 kb). It is an artificially constructed chromosome and contains
the telomeric, centromeric, and replication origin sequences needed
for replication and preservation in yeast cells. Built using an
initial circular plasmid, they are linearized by using restriction
enzymes, and then DNA ligase can add a sequence or gene of interest
within the linear molecule by the use of cohesive ends. Yeast
expression vectors, such as YACs, Ylps (yeast integrating plasmid),
and YEps (yeast episomal plasmid), are extremely useful as one can
get eukaryotic protein products with posttranslational
modifications as yeasts are themselves eukaryotic cells, however
YACs have been found to be more unstable than BACs, producing
chimeric effects.
[0189] A "viral vector" is defined as a recombinantly produced
virus or viral particle that comprises a polynucleotide to be
delivered into a host cell, either in vivo, ex vivo or in vitro.
Examples of viral vectors include retroviral vectors, adenovirus
vectors, adeno-associated virus vectors, alphavirus vectors and the
like. Infectious tobacco mosaic virus (TMV)-based vectors can be
used to manufacturer proteins and have been reported to express
Griffithsin in tobacco leaves (O'Keefe et al. (2009) Proc. Nat.
Acad. Sci. USA 106(15):6099-6104). Alphavirus vectors, such as
Semliki Forest virus-based vectors and Sindbis virus-based vectors,
have also been developed for use in gene therapy and immunotherapy.
See, Schlesinger & Dubensky (1999) Curr. Opin. Biotechnol.
5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In aspects
where gene transfer is mediated by a retroviral vector, a vector
construct refers to the polynucleotide comprising the retroviral
genome or part thereof, and a therapeutic gene.
[0190] As used herein, "retroviral mediated gene transfer" or
"retroviral transduction" carries the same meaning and refers to
the process by which a gene or nucleic acid sequences are stably
transferred into the host cell by virtue of the virus entering the
cell and integrating its genome into the host cell genome. The
virus can enter the host cell via its normal mechanism of infection
or be modified such that it binds to a different host cell surface
receptor or ligand to enter the cell. As used herein, retroviral
vector refers to a viral particle capable of introducing exogenous
nucleic acid into a cell through a viral or viral-like entry
mechanism.
[0191] Retroviruses carry their genetic information in the form of
RNA; however, once the virus infects a cell, the RNA is
reverse-transcribed into the DNA form which integrates into the
genomic DNA of the infected cell. The integrated DNA form is called
a provirus.
[0192] In aspects where gene transfer is mediated by a DNA viral
vector, such as an adenovirus (Ad) or adeno-associated virus (AAV),
a vector construct refers to the polynucleotide comprising the
viral genome or part thereof, and a transgene. Adenoviruses (Ads)
are a relatively well characterized, homogenous group of viruses,
including over 50 serotypes. See, e.g., International PCT
Application No. WO 95/27071. Ads do not require integration into
the host cell genome. Recombinant Ad derived vectors, particularly
those that reduce the potential for recombination and generation of
wild-type virus, have also been constructed. See, International PCT
Application Nos. WO 95/00655 and WO 95/11984. Wild-type AAV has
high infectivity and specificity integrating into the host cell's
genome. See, Hermonat & Muzyczka (1984) Proc. Natl. Acad. Sci.
USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol.
8:3988-3996.
[0193] Vectors that contain both a promoter and a cloning site into
which a polynucleotide can be operatively linked are well known in
the art. Such vectors are capable of transcribing RNA in vitro or
in vivo, and are commercially available from sources such as
Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).
In order to optimize expression and/or in vitro transcription, it
may be necessary to remove, add or alter 5' and/or 3' untranslated
portions of the clones to eliminate extra, potential inappropriate
alternative translation initiation codons or other sequences that
may interfere with or reduce expression, either at the level of
transcription or translation. Alternatively, consensus ribosome
binding sites can be inserted immediately 5' of the start codon to
enhance expression.
[0194] Gene delivery vehicles also include DNA/liposome complexes,
micelles and targeted viral protein-DNA complexes. Liposomes that
also comprise a targeting antibody or fragment thereof can be used
in the methods of this invention. In addition to the delivery of
polynucleotides to a cell or cell population, direct introduction
of the proteins described herein to the cell or cell population can
be done by the non-limiting technique of protein transfection,
alternatively culturing conditions that can enhance the expression
and/or promote the activity of the proteins of this invention are
other non-limiting techniques.
[0195] As used herein, the terms "antibody," "antibodies" and
"immunoglobulin" includes whole antibodies and any antigen binding
fragment or a single chain thereof. Thus the term "antibody"
includes any protein or peptide containing molecule that comprises
at least a portion of an immunoglobulin molecule. The terms
"antibody," "antibodies" and "immunoglobulin" also include
immunoglobulins of any isotype, fragments of antibodies which
retain specific binding to antigen, including, but not limited to,
Fab, Fab, F(ab).sub.2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL,
VhH, and V-NAR domains; minibodies, diabodies, triabodies,
tetrabodies and kappa bodies, multispecific antibody fragments
formed from antibody fragments and one or more isolated. Examples
of such include, but are not limited to a complementarity
determining region (CDR) of a heavy or light chain or a ligand
binding portion thereof, a heavy chain or light chain variable
region, a heavy chain or light chain constant region, a framework
(FR) region, or any portion thereof, at least one portion of a
binding protein, chimeric antibodies, humanized antibodies,
single-chain antibodies, and fusion proteins comprising an
antigen-binding portion of an antibody and a non-antibody protein.
The variable regions of the heavy and light chains of the
immunoglobulin molecule contain a binding domain that interacts
with an antigen. The constant regions of the antibodies (Abs) may
mediate the binding of the immunoglobulin to host tissues. The term
"anti-" when used before a protein name, anti-IHF, anti-HU,
anti-OMP P5, for example, refers to a monoclonal or polyclonal
antibody that binds and/or has an affinity to a particular protein.
For example, "anti-IHF" refers to an antibody that binds to the IHF
protein. The specific antibody may have affinity or bind to
proteins other than the protein it was raised against. For example,
anti-IHF, while specifically raised against the IHF protein, may
also bind other proteins that are related either through sequence
homology or through structure homology.
[0196] The antibodies can be polyclonal, monoclonal, multispecific
(e.g., bispecific antibodies), and antibody fragments, so long as
they exhibit the desired biological activity. Antibodies can be
isolated from any suitable biological source, e.g., murine, rat,
sheep and canine.
[0197] As used herein, "monoclonal antibody" refers to an antibody
obtained from a substantially homogeneous antibody population.
Monoclonal antibodies are highly specific, as each monoclonal
antibody is directed against a single determinant on the antigen.
The antibodies may be detectably labeled, e.g., with a
radioisotope, an enzyme which generates a detectable product, a
fluorescent protein, and the like. The antibodies may be further
conjugated to other moieties, such as members of specific binding
pairs, e.g., biotin (member of biotin-avidin specific binding
pair), and the like. The antibodies may also be bound to a solid
support, including, but not limited to, polystyrene plates or
beads, and the like.
[0198] Monoclonal antibodies may be generated using hybridoma
techniques or recombinant DNA methods known in the art. A hybridoma
is a cell that is produced in the laboratory from the fusion of an
antibody-producing lymphocyte and a non-antibody producing cancer
cell, usually a myeloma or lymphoma. A hyridoma proliferates and
produces a continuous syple of a specific monoclonal antibody.
Alternative techniques for generating or selecting antibodies
include in vitro exposure of lymphocytes to antigens of interest,
and screening of antibody display libraries in cells, phage, or
similar systems.
[0199] The term "human antibody" as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo). However, the term "human antibody" as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences.
Thus, as used herein, the term "human antibody" refers to an
antibody in which substantially every part of the protein (e.g.,
CDR, framework, C.sub.L, C.sub.H domains (e.g., C.sub.H1, C.sub.H2,
C.sub.H3), hinge, (VL, VH)) is substantially non-immunogenic in
humans, with only minor sequence changes or variations. Similarly,
antibodies designated primate (monkey, baboon, chimpanzee, etc.),
rodent (mouse, rat, rabbit, guinea pig, hamster, and the like) and
other mammals designate such species, sub-genus, genus, sub-family,
family specific antibodies. Further, chimeric antibodies include
any combination of the above. Such changes or variations optionally
and preferably retain or reduce the immunogenicity in humans or
other species relative to non-modified antibodies. Thus, a human
antibody is distinct from a chimeric or humanized antibody. It is
pointed out that a human antibody can be produced by a non-human
animal or prokaryotic or eukaryotic cell that is capable of
expressing functionally rearranged human immunoglobulin (e.g.,
heavy chain and/or light chain) genes. Further, when a human
antibody is a single chain antibody, it can comprise a linker
peptide that is not found in native human antibodies. For example,
an Fv can comprise a linker peptide, such as two to about eight
glycine or other amino acid residues, which connects the variable
region of the heavy chain and the variable region of the light
chain. Such linker peptides are considered to be of human
origin.
[0200] As used herein, a human antibody is "derived from" a
particular germline sequence if the antibody is obtained from a
system using human immunoglobulin sequences, e.g., by immunizing a
transgenic mouse carrying human immunoglobulin genes or by
screening a human immunoglobulin gene library. A human antibody
that is "derived from" a human germline immunoglobulin sequence can
be identified as such by comparing the amino acid sequence of the
human antibody to the amino acid sequence of human germline
immunoglobulins. A selected human antibody typically is at least
90% identical in amino acids sequence to an amino acid sequence
encoded by a human germline immunoglobulin gene and contains amino
acid residues that identify the human antibody as being human when
compared to the germline immunoglobulin amino acid sequences of
other species (e.g., murine germline sequences). In certain cases,
a human antibody may be at least 95%, or even at least 96%, 97%,
98%, or 99% identical in amino acid sequence to the amino acid
sequence encoded by the germline immunoglobulin gene. Typically, a
human antibody derived from a particular human germline sequence
will display no more than 10 amino acid differences from the amino
acid sequence encoded by the human germline immunoglobulin gene. In
certain cases, the human antibody may display no more than 5, or
even no more than 4, 3, 2, or 1 amino acid difference from the
amino acid sequence encoded by the germline immunoglobulin
gene.
[0201] A "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable and
constant regions derived from human germline immunoglobulin
sequences. The term also intends recombinant human antibodies.
Methods to making these antibodies are described herein.
[0202] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
an animal (e.g., a mouse) that is transgenic or transchromosomal
for human immunoglobulin genes or a hybridoma prepared therefrom,
antibodies isolated from a host cell transformed to express the
antibody, e.g., from a transfectoma, antibodies isolated from a
recombinant, combinatorial human antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of human immunoglobulin gene sequences to other
DNA sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies can be subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. Methods to making these antibodies are described
herein.
[0203] As used herein, chimeric antibodies are antibodies whose
light and heavy chain genes have been constructed, typically by
genetic engineering, from antibody variable and constant region
genes belonging to different species.
[0204] As used herein, the term "humanized antibody" or "humanized
immunoglobulin" refers to a human/non-human chimeric antibody that
contains a minimal sequence derived from non-human immunoglobulin.
For the most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a variable region of
the recipient are replaced by residues from a variable region of a
non-human species (donor antibody) such as mouse, rat, rabbit, or
non-human primate having the desired specificity, affinity and
capacity. Humanized antibodies may comprise residues that are not
found in the recipient antibody or in the donor antibody. The
humanized antibody can optionally also comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a
human immunoglobulin. a non-human antibody containing one or more
amino acids in a framework region, a constant region or a CDR, that
have been substituted with a correspondingly positioned amino acid
from a human antibody. In general, humanized antibodies are
expected to produce a reduced immune response in a human host, as
compared to a non-humanized version of the same antibody. The
humanized antibodies may have conservative amino acid substitutions
which have substantially no effect on antigen binding or other
antibody functions. Conservative substitutions groupings
include:glycine-alanine, valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine,
serine-threonine and asparagine-glutamine.
[0205] The terms "polyclonal antibody" or "polyclonal antibody
composition" as used herein refer to a preparation of antibodies
that are derived from different B-cell lines. They are a mixture of
immunoglobulin molecules secreted against a specific antigen, each
recognizing a different epitope.
[0206] As used herein, the term "antibody derivative", comprises a
full-length antibody or a fragment of an antibody, wherein one or
more of the amino acids are chemically modified by alkylation,
pegylation, acylation, ester formation or amide formation or the
like, e.g., for linking the antibody to a second molecule. This
includes, but is not limited to, pegylated antibodies,
cysteine-pegylated antibodies, and variants thereof.
[0207] As used herein, the term "label" intends a directly or
indirectly detectable compound or composition that is conjugated
directly or indirectly to the composition to be detected, e.g.,
N-terminal histadine tags (N-His), magnetically active isotopes,
e.g., .sup.115Sn, .sup.117Sn and .sup.119Sn, a non-radioactive
isotopes such as .sup.13C and .sup.15N, polynucleotide or protein
such as an antibody so as to generate a "labeled" composition. The
term also includes sequences conjugated to the polynucleotide that
will provide a signal upon expression of the inserted sequences,
such as green fluorescent protein (GFP) and the like. The label may
be detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition which is
detectable. The labels can be suitable for small scale detection or
more suitable for high-throughput screening. As such, suitable
labels include, but are not limited to magnetically active
isotopes, non-radioactive isotopes, radioisotopes, fluorochromes,
chemiluminescent compounds, dyes, and proteins, including enzymes.
The label may be simply detected or it may be quantified. A
response that is simply detected generally comprises a response
whose existence merely is confirmed, whereas a response that is
quantified generally comprises a response having a quantifiable
(e.g., numerically reportable) value such as an intensity,
polarization, and/or other property. In luminescence or
fluorescence assays, the detectable response may be generated
directly using a luminophore or fluorophore associated with an
assay component actually involved in binding, or indirectly using a
luminophore or fluorophore associated with another (e.g., reporter
or indicator) component. Examples of luminescent labels that
produce signals include, but are not limited to bioluminescence and
chemiluminescence. Detectable luminescence response generally
comprises a change in, or an occurrence of, a luminescence signal.
Suitable methods and luminophores for luminescently labeling assay
components are known in the art and described for example in
Haugland, Richard P. (1996) Handbook of Fluorescent Probes and
Research Chemicals (6.sup.th ed.). Examples of luminescent probes
include, but are not limited to, aequorin and luciferases.
[0208] As used herein, the term "immunoconjugate" comprises an
antibody or an antibody derivative associated with or linked to a
second agent, such as a cytotoxic agent, a detectable agent, a
radioactive agent, a targeting agent, a human antibody, a humanized
antibody, a chimeric antibody, a synthetic antibody, a
semisynthetic antibody, or a multispecific antibody.
[0209] Examples of suitable fluorescent labels include, but are not
limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin,
erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green,
stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other
suitable optical dyes are described in the Haugland, Richard P.
(1996) Handbook of Fluorescent Probes and Research Chemicals
(6.sup.th ed.).
[0210] In another aspect, the fluorescent label is functionalized
to facilitate covalent attachment to a cellular component present
in or on the surface of the cell or tissue such as a cell surface
marker. Suitable functional groups, including, but not are limited
to, isothiocyanate groups, amino groups, haloacetyl groups,
maleimides, succinimidyl esters, and sulfonyl halides, all of which
may be used to attach the fluorescent label to a second molecule.
The choice of the functional group of the fluorescent label will
depend on the site of attachment to either a linker, the agent, the
marker, or the second labeling agent.
[0211] "Eukaryotic cells" comprise all of the life kingdoms except
monera. They can be easily distinguished through a membrane-bound
nucleus. Animals, plants, fungi, and protists are eukaryotes or
organisms whose cells are organized into complex structures by
internal membranes and a cytoskeleton. The most characteristic
membrane-bound structure is the nucleus. Unless specifically
recited, the term "host" includes a eukaryotic host, including, for
example, yeast, higher plant, insect and mammalian cells.
Non-limiting examples of eukaryotic cells or hosts include simian,
bovine, porcine, murine, rat, avian, reptilian and human.
[0212] "Prokaryotic cells" that usually lack a nucleus or any other
membrane-bound organelles and are divided into two domains,
bacteria and archaea. In addition to chromosomal DNA, these cells
can also contain genetic information in a circular loop called an
episome. Bacterial cells are very small, roughly the size of an
animal mitochondrion (about 1-2 .mu.m in diameter and 10 .mu.m
long). Prokaryotic cells feature three major shapes: rod shaped,
spherical, and spiral. Instead of going through elaborate
replication processes like eukaryotes, bacterial cells divide by
binary fission. Examples include but are not limited to Bacillus
bacteria, E. coli bacterium, and Salmonella bacterium.
[0213] A "native" or "natural" antigen is a polypeptide, protein or
a fragment which contains an epitope, which has been isolated from
a natural biological source, and which can specifically bind to an
antigen receptor, in particular a T cell antigen receptor (TCR), in
a subject.
[0214] The terms "antigen" and "antigenic" refer to molecules with
the capacity to be recognized by an antibody or otherwise act as a
member of an antibody-ligand pair. "Specific binding" refers to the
interaction of an antigen with the variable regions of
immunoglobulin heavy and light chains. Antibody-antigen binding may
occur in vivo or in vitro. The skilled artisan will understand that
macromolecules, including proteins, nucleic acids, fatty acids,
lipids, lipopolysaccharides and polysaccharides have the potential
to act as an antigen. The skilled artisan will further understand
that nucleic acids encoding a protein with the potential to act as
an antibody ligand necessarily encode an antigen. The artisan will
further understand that antigens are not limited to full-length
molecules, but can also include partial molecules. The term
"antigenic" is an adjectival reference to molecules having the
properties of an antigen. The term encompasses substances which are
immunogenic, i.e., immunogens, as well as substances which induce
immunological unresponsiveness, or anergy, i.e., anergens.
[0215] An "altered antigen" is one having a primary sequence that
is different from that of the corresponding wild-type antigen.
Altered antigens can be made by synthetic or recombinant methods
and include, but are not limited to, antigenic peptides that are
differentially modified during or after translation, e.g., by
phosphorylation, glycosylation, cross-linking, acylation,
proteolytic cleavage, linkage to an antibody molecule, membrane
molecule or other ligand. (Ferguson et al. (1988) Ann. Rev.
Biochem. 57:285-320). A synthetic or altered antigen of the
invention is intended to bind to the same TCR as the natural
epitope.
[0216] A "self-antigen" also referred to herein as a native or
wild-type antigen is an antigenic peptide that induces little or no
immune response in the subject due to self-tolerance to the
antigen. An example of a self-antigen is the melanoma specific
antigen gp100.
[0217] The terms "major histocompatibility complex" or "MHC" refers
to a complex of genes encoding cell-surface molecules that are
required for antigen presentation to T cells and for rapid graft
rejection. In humans, the MHC is also known as the "human leukocyte
antigen" or "HLA" complex. The proteins encoded by the MHC are
known as "MHC molecules" and are classified into class I and class
II MHC molecules. Class I MHC includes membrane heterodimeric
proteins made up of an a chain encoded in the MHC noncovalently
linked with the .beta. 2-microglobulin. Class I MHC molecules are
expressed by nearly all nucleated cells and have been shown to
function in antigen presentation to CD8.sup.+ T cells. Class I
molecules include HLA-A, B, and C in humans. Class II MHC molecules
also include membrane heterodimeric proteins consisting of
noncovalently associated .alpha. and .beta. chains. Class II MHC
molecules are known to function in CD4.sup.+ T cells and, in
humans, include HLA-DP, -DQ, and DR. In a preferred embodiment,
invention compositions and ligands can complex with MHC molecules
of any HLA type. Those of skill in the art are familiar with the
serotypes and genotypes of the HLA. See:
bimas.dcrt.nih.gov/cgi-bin/molbio/hla coefficient viewing page.
Rammensee H. G., Bachmann J., and Stevanovic S. MHC Ligands and
Peptide Motifs (1997) Chapman & Hall Publishers; Schreuder G.
M. Th. et al. The HLA dictionary (1999) Tissue Antigens
54:409-437.
[0218] The term "antigen-presenting matrix", as used herein,
intends a molecule or molecules which can present antigen in such a
way that the antigen can be bound by a T-cell antigen receptor on
the surface of a T cell. An antigen-presenting matrix can be on the
surface of an antigen-presenting cell (APC), on a vesicle
preparation of an APC, or can be in the form of a synthetic matrix
on a solid support such as a bead or a plate. An example of a
synthetic antigen-presenting matrix is purified MHC class I
molecules complexed to P2-microglobulin, multimers of such purified
MHC class I molecules, purified MHC Class II molecules, or
functional portions thereof, attached to a solid support.
[0219] The term "antigen presenting cells (APC)" refers to a class
of cells capable of presenting one or more antigens in the form of
antigen-MHC complex recognizable by specific effector cells of the
immune system, and thereby inducing an effective cellular immune
response against the antigen or antigens being presented. While
many types of cells may be capable of presenting antigens on their
cell surface for T-cell recognition, only professional APCs have
the capacity to present antigens in an efficient amount and further
to activate T-cells for cytotoxic T-lymphocyte (CTL) responses.
APCs can be intact whole cells such as macrophages, B-cells and
dendritic cells; or other molecules, naturally occurring or
synthetic, such as purified MHC class I molecules complexed to
.beta.2-microglobulin.
[0220] The term "dendritic cells (DCs)" refers to a diverse
population of morphologically similar cell types found in a variety
of lymphoid and non-lymphoid tissues (Steinman (1991) Ann. Rev.
Immunol. 9:271-296). Dendritic cells constitute the most potent and
preferred of mammalian APCs. A subset, if not all, of DCs are
derived from bone marrow progenitor cells, circulate in small
numbers in the peripheral blood and appear either as immature
Langerhans' cells or terminally differentiated mature cells. While
DCs can be derived from monocytes, they possess distinct
phenotypes. For example, a particular differentiating marker, CD14
antigen, is not found in dendritic cells but is expressed at very
high levels in monocytes by monocytes. See for example Jersmann et
al., (2005) Immunol. Cell Biol. 83:462.
[0221] Also, mature dendritic cells are not phagocytic, whereas the
monocytes are strongly phagocytosing cells. Mature monocytes and
DCs endocytose material through different mechanisms. Monocytes
engulf by means of phagocytosis, whereas DCs utilize
macropinocytosis. Thus, DCs generally engulf cargo of a smaller
size than monocytes (See for example Conner and Schmid, (2003)
Nature 433:37-44. It has been shown that DCs are as endocytically
active as other antigen presenting cells, and provide all the
signals necessary for T cell activation and proliferation (See,
e.g. Levine and Chain, (1992) ONAS 89(17):8342.
[0222] The term "antigen presenting cell recruitment factors" or
"APC recruitment factors" include both intact, whole cells as well
as other molecules that are capable of recruiting antigen
presenting cells. Examples of suitable APC recruitment factors
include molecules such as interleukin 4 (IL4), granulocyte
macrophage colony stimulating factor (GM-CSF), Sepragel and
macrophage inflammatory protein 3 alpha (MIP3.alpha.). These are
available from Immunex, Schering-Plough and R&D Systems
(Minneapolis, Minn.). They also can be recombinantly produced using
the methods disclosed in Current Protocols In Molecular Biology (F.
M. Ausubel et al., eds. (1987)). Peptides, proteins and compounds
having the same biological activity as the above-noted factors are
included within the scope of this invention.
[0223] The term "immune effector cells" refers to cells capable of
binding an antigen and which mediate an immune response. These
cells include, but are not limited to, T cells, B cells, monocytes,
macrophages, NK cells and cytotoxic T lymphocytes (CTLs), for
example CTL lines, CTL clones, and CTLs from tumor, inflammatory,
or other infiltrates. Certain diseased tissue expresses specific
antigens and CTLs specific for these antigens have been
identified.
[0224] The term "immune effector molecule" as used herein, refers
to molecules capable of antigen-specific binding, and includes
antibodies, T cell antigen receptors, B cell antigen receptors, and
MHC Class I and Class II molecules.
[0225] A "naive" immune effector cell is an immune effector cell
that has never been exposed to an antigen capable of activating
that cell. Activation of naive immune effector T cells requires
both recognition of the antigen:MHC complex and the simultaneous
delivery of a costimulatory signal by a professional APC in order
to proliferate and differentiate into antigen-specific armed
effector T cells. Activated T cells can then activate specific B
cells through immunological synapses by providing a co-stimulation
signal. Activated B cells subsequently produce antibodies directed
to a specific antigen. Naive B cells can also be activated by T
cell-independent mechanisms. This occurs when antigens are capable
of binding to the B cell receptor and producing a co-stimulation
signal.
[0226] "Immune response" broadly refers to the antigen-specific
responses of lymphocytes to foreign substances. The terms
"immunogen" and "immunogenic" refer to molecules with the capacity
to elicit an immune response. All immunogens are antigens, however,
not all antigens are immunogenic. An immune response of this
invention can be humoral (via antibody activity) or cell-mediated
(via T cell activation). The response may occur in vivo or in
vitro. The skilled artisan will understand that a variety of
macromolecules, including proteins, nucleic acids, fatty acids,
lipids, lipopolysaccharides and polysaccharides have the potential
to be immunogenic. The skilled artisan will further understand that
nucleic acids encoding a molecule capable of eliciting an immune
response necessarily encode an immunogen. The artisan will further
understand that immunogens are not limited to full-length
molecules, but may include partial molecules.
[0227] The term "passive immunity" refers to the transfer of
immunity from one subject to another through the transfer of
antibodies. Passive immunity may occur naturally, as when maternal
antibodies are transferred to a fetus. Passive immunity may also
occur artificially as when antibody compositions are administered
to non-immune subjects. Antibody donors and recipients may be human
or non-human subjects. Antibodies may be polyclonal or monoclonal,
may be generated in vitro or in vivo, and may be purified,
partially purified, or unpurified depending on the embodiment. In
some embodiments described herein, passive immunity is conferred on
a subject in need thereof through the administration of antibodies
or antigen binding fragments that specifically recognize or bind to
a particular antigen. In some embodiments, passive immunity is
conferred through the administration of an isolated or recombinant
polynucleotide encoding an antibody or antigen binding fragment
that specifically recognizes or binds to a particular antigen.
[0228] In the context of this invention, a "ligand" is a
polypeptide. In one aspect, the term "ligand" as used herein refers
to any molecule that binds to a specific site on another molecule.
In other words, the ligand confers the specificity of the protein
in a reaction with an immune effector cell or an antibody to a
protein or DNA to a protein. In one aspect it is the ligand site
within the protein that combines directly with the complementary
binding site on the immune effector cell.
[0229] In one aspect, a peptide or ligand of the invention binds to
an antigenic determinant or epitope on an immune effector cell,
such as an antibody or a T cell receptor (TCR). A ligand may be an
antigen, peptide, protein or epitope of the invention.
[0230] In another aspect, ligands may bind to a receptor on an
antibody. In one embodiment, the ligand of the invention is about 4
to about 8 amino acids in length.
[0231] In a further aspect, ligands may bind to a receptor on an
MHC class I molecule. In one embodiment, the ligand of the
invention is about 7 to about 11 amino acids in length.
[0232] In a yet further aspect, ligands may bind to a receptor on
an MHC class II molecule. In one embodiment, the ligand of the
invention is about 10 to about 20 amino acids long.
[0233] As used herein, the term "educated, antigen-specific immune
effector cell", is an immune effector cell as defined above, which
has previously encountered an antigen. In contrast with its naive
counterpart, activation of an educated, antigen-specific immune
effector cell does not require a costimulatory signal. Recognition
of the peptide:MHC complex is sufficient.
[0234] "Activated", when used in reference to a T cell, implies
that the cell is no longer in G.sub.0 phase, and begins to produce
one or more of cytotoxins, cytokines, and other related
membrane-associated proteins characteristic of the cell type (e.g.,
CD8.sup.+ or CD4.sup.+), is capable of recognizing and binding any
target cell that displays the particular antigen on its surface,
and releasing its effector molecules.
[0235] The term "cross-reactive" is used to describe compounds of
the invention which are functionally overlapping. More
particularly, the immunogenic properties of a native ligand and/or
immune effector cells activated thereby are shared to a certain
extent by the altered ligand such that the altered ligand is
"cross-reactive" with the native ligand and/or the immune effector
cells activated thereby. For purposes of this invention,
cross-reactivity is manifested at multiple levels: (i) at the
ligand level, e.g., the altered ligands can bind the TCR of and
activate native ligand CTLs; (ii) at the T cell level, i.e.,
altered ligands of the invention bind the TCR of and activate a
population of T cells (distinct from the population of native
ligand CTLs) which can effectively target and lyse cells displaying
the native ligand; and (iii) at the antibody level, e.g.,
"anti"-altered ligand antibodies can detect, recognize and bind the
native ligand and initiate effector mechanisms in an immune
response which ultimately result in elimination of the native
ligand from the host.
[0236] As used herein, the term "inducing an immune response in a
subject" is a term well understood in the art and intends that an
increase of at least about 2-fold, more preferably at least about
5-fold, more preferably at least about 10-fold, more preferably at
least about 100-fold, even more preferably at least about 500-fold,
even more preferably at least about 1000-fold or more in an immune
response to an antigen (or epitope) can be detected or measured,
after introducing the antigen (or epitope) into the subject,
relative to the immune response (if any) before introduction of the
antigen (or epitope) into the subject. An immune response to an
antigen (or epitope), includes, but is not limited to, production
of an antigen-specific (or epitope-specific) antibody, and
production of an immune cell expressing on its surface a molecule
which specifically binds to an antigen (or epitope). Methods of
determining whether an immune response to a given antigen (or
epitope) has been induced are well known in the art. For example,
antigen-specific antibody can be detected using any of a variety of
immunoassays known in the art, including, but not limited to,
ELISA, wherein, for example, binding of an antibody in a sample to
an immobilized antigen (or epitope) is detected with a
detectably-labeled second antibody (e.g., enzyme-labeled mouse
anti-human Ig antibody).
[0237] "Co-stimulatory molecules" are involved in the interaction
between receptor-ligand pairs expressed on the surface of antigen
presenting cells and T cells. Research accumulated over the past
several years has demonstrated convincingly that resting T cells
require at least two signals for induction of cytokine gene
expression and proliferation (Schwartz (1990) Science 248:1349-1356
and Jenkins (1992) Immunol. Today 13:69-73). One signal, the one
that confers specificity, can be produced by interaction of the
TCR/CD3 complex with an appropriate MHC/peptide complex. The second
signal is not antigen specific and is termed the "co-stimulatory"
signal. This signal was originally defined as an activity provided
by bone-marrow-derived accessory cells such as macrophages and
dendritic cells, the so called "professional" APCs. Several
molecules have been shown to enhance co-stimulatory activity. These
are heat stable antigen (HSA) (Liu et al. (1992) J. Exp. Med.
175:437-445), chondroitin sulfate-modified MHC invariant chain
(Ii-CS) (Naujokas et al. (1993) Cell 74:257-268), intracellular
adhesion molecule 1 (ICAM-1) (Van (1992) Cell 71:1065-1068). These
molecules each appear to assist co-stimulation by interacting with
their cognate ligands on the T cells. Co-stimulatory molecules
mediate co-stimulatory signal(s), which are necessary, under normal
physiological conditions, to achieve full activation of naive T
cells. One exemplary receptor-ligand pair is the B7 co-stimulatory
molecule on the surface of APCs and its counter-receptor CD28 or
CTLA-4 on T cells (Freeman et al. (1993) Science 262:909-911; Young
et al. (1992) J. Clin. Invest. 90:229 and Nabavi et al. (1992)
Nature 360:266-268). Other important co-stimulatory molecules are
CD40, CD54, CD80, and CD86. The term "co-stimulator) molecule"
encompasses any single molecule or combination of molecules which,
when acting together with a peptide/MHC complex bound by a TCR on
the surface of a T cell, provides a co-stimulatory effect which
achieves activation of the T cell that binds the peptide. The term
thus encompasses B7, or other co-stimulatory molecule(s) on an
antigen-presenting matrix such as an APC, fragments thereof (alone,
complexed with another molecule(s), or as part of a fusion protein)
which, together with peptide/MHC complex, binds to a cognate ligand
and results in activation of the T cell when the TCR on the surface
of the T cell specifically binds the peptide. Co-stimulatory
molecules are commercially available from a variety of sources,
including, for example, Beckman Coulter. Inc. (Fullerton. Calif.).
It is intended, although not always explicitly stated, that
molecules having similar biological activity as wild-type or
purified co-stimulatory molecules (e.g., recombinantly produced or
muteins thereof) are intended to be used within the spirit and
scope of the invention.
[0238] As used herein, "solid phase support" or "solid support",
used interchangeably, is not limited to a specific type of support.
Rather a large number of supports are available and are known to
one of ordinary skill in the art. Solid phase supports include
silica gels, resins, derivatized plastic films, glass beads,
cotton, plastic beads, alumina gels. As used herein, "solid
support" also includes synthetic antigen-presenting matrices,
cells, and liposomes. A suitable solid phase support may be
selected on the basis of desired end use and suitability for
various protocols. For example, for peptide synthesis, solid phase
support may refer to resins such as polystyrene (e.g., PAM-resin
obtained from Bachem Inc., Peninsula Laboratories, etc.),
POLYHIPE.RTM. resin (obtained from Aminotech, Canada), polyamide
resin (obtained from Peninsula Laboratories), polystyrene resin
grafted with polyethylene glycol (TentaGel.RTM., Rapp Polymere,
Tubingen, Germany) or polydimethylacrylamide resin (obtained from
Milligen/Biosearch, Calif.).
[0239] An example of a solid phase support include glass,
polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,
natural and modified celluloses, polyacrylamides, gabbros, and
magnetite. The nature of the carrier can be either soluble to some
extent or insoluble. The support material may have virtually any
possible structural configuration so long as the coupled molecule
is capable of binding to a polynucleotide, polypeptide or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. or alternatively polystyrene
beads. Those skilled in the art will know many other suitable
carriers for binding antibody or antigen, or will be able to
ascertain the same by use of routine experimentation.
[0240] The term "immunomodulatory agent", as used herein, is a
molecule, a macromolecular complex, or a cell that modulates an
immune response and encompasses a synthetic antigenic peptide of
the invention alone or in any of a variety of formulations
described herein; a polypeptide comprising a synthetic antigenic
peptide of the invention; a polynucleotide encoding a peptide or
polypeptide of the invention; a synthetic antigenic peptide of the
invention bound to a Class I or a Class II MHC molecule on an
antigen-presenting matrix, including an APC and a synthetic
antigen-presenting matrix (in the presence or absence of
co-stimulatory molecule(s)); a synthetic antigenic peptide of the
invention covalently or non-covalently complexed to another
molecule(s) or macromolecular structure; and an educated,
antigen-specific immune effector cell which is specific for a
peptide of the invention.
[0241] The term "modulate an immune response" includes inducing
(increasing, eliciting) an immune response; and reducing
(suppressing) an immune response. An immunomodulatory method (or
protocol) is one that modulates an immune response in a
subject.
[0242] As used herein, the term "cytokine" refers to any one of the
numerous factors that exert a variety of effects on cells, for
example, inducing growth or proliferation. Non-limiting examples of
cytokines which may be used alone or in combination in the practice
of the present invention include, interleukin-2 (IL-2), stem cell
factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6),
interleukin 12 (IL-12), G-CSF, granulocyte macrophage-colony
stimulating factor (GM-CSF), interleukin-1 alpha (IL-1.alpha.),
interleukin-11 (IL-11), MIP-11, leukemia inhibitory factor (LIF),
c-kit ligand, thrombopoietin (TPO) and flt3 ligand. The present
invention also includes culture conditions in which one or more
cytokine is specifically excluded from the medium. Cytokines are
commercially available from several vendors such as, for example,
Genzyme (Framingham, Mass.), Genentech (South San Francisco.
Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems
(Minneapolis, Minn.) and Immunex (Seattle, Wash.). It is intended,
although not always explicitly stated, that molecules having
similar biological activity as wild-type or purified cytokines
(e.g., recombinantly produced or muteins thereof) are intended to
be used within the spirit and scope of the invention.
Diagnostic and Therapeutic Methods
[0243] A method is provided for inhibiting, competing or titrating
the binding of a DNABII polypeptide or protein to a microbial DNA,
by contacting the DNABII polypeptide or protein or the microbial
DNA with an interfering agent, thereby inhibiting, competing or
titrating the binding of the DNABII protein or polypeptide to the
microbial DNA. In a further aspect, the DNABII polypeptide and the
microbial DNA are detectably labeled, for example with luminescent
molecules that will emit a signal when brought into close contact
with each other. The contacting can be performed in vitro or in
vivo.
[0244] In another aspect, a method for inhibiting, preventing or
breaking down a microbial biofilm is provided by contacting the
biofilm with an interfering agent, thereby inhibiting, preventing
or breaking down the microbial biofilm. In a further aspect, the
DNABII polypeptide and the microbial DNA are detectably labeled,
for example with luminescent molecules that will emit a signal when
brought into close contact with each other. The contacting can be
performed in vitro or in vivo.
[0245] When practiced in vitro, the methods are useful to screen
for or confirm interfering agents having the same, similar or
opposite ability as the polypeptides, polynucleotides, antibodies,
host cells, small molecules and compositions of this invention.
Alternatively, they can be used to identify which interfering agent
is best suited to treat a microbial infection. For example, one can
screen for new agents or combination therapies by having two
samples containing for example, the DNABII polypeptide and
microbial DNA and the agent to be tested. The second sample
contains the DNABII polypeptide and microbial DNA and an agent
known to active, e.g., an anti-IHF antibody or a small molecule to
serve as a positive control. In a further aspect, several samples
are provided and the interfering agents are added to the system in
increasing dilutions to determine the optimal dose that would
likely be effective in treating a subject in the clinical setting.
As is apparent to those of skill in the art, a negative control
containing the DNABII polypeptide and the microbial DNA can be
provided. In a further aspect, the DNABII polypeptide and the
microbial DNA are detectably labeled, for example with luminescent
molecules that will emit a signal when brought into close contact
with each other. The samples are contained under similar conditions
for an effective amount of time for the agent to inhibit, compete
or titrate the interaction between the DNABII polypeptide and
microbial DNA and then the sample is assayed for emission of signal
from the luminescent molecules. If the sample emits a signal, then
the agent is not effective to inhibit binding.
[0246] In another aspect, the in vitro method is practiced in a
miniaturized chamber slide system wherein the microbial (such as a
bacterial) isolate causing an infection could be isolated from the
human/animal then cultured to allow it to grow as a biofilm in
vitro, see for example Experiment No. 1 below. The interfering
agent (such as anti-IHF antibody) or potential interfering agent
biofilm is added alone or in combination with another agent to the
culture with or without increasing dilutions of the potential
interfering agent or interfering agent such as an anti-IHF (or
other antibody, small molecule, agent etc.) to find the optimal
dose that would likely be effective at treating that patient when
delivered to the subject where the infection existed. As apparent
to those of skill in the art, a positive and negative control can
be performed simultaneously.
[0247] In a further aspect, the method is practiced in a high
throughput platform with the interfering agent (such as anti-IHF
antibody) and/or potential interfering agent (alone or in
combination with another agent) in a flow cell. The interfering
agent (such as anti-IHF antibody) or potential interfering agent
biofilm is added alone or in combination with another agent to the
culture with or without increasing dilutions of the potential
interfering agent or interfering agent such as an anti-IHF (or
other antibody, small molecule, agent etc.) to find the optimal
dose that would likely be effective at treating that patient when
delivered to the subject where the infection existed. Biofilm
isolates are sonicated to separate biofilm bacteria from DNABII
polypeptide such as IHF bound to microbial DNA. The DNABII
polypeptide--DNA complexes are isolated by virtue of the anti-IHF
antibody on the platform. The microbial DNA is then be released
with e.g. a salt wash, and used to identify the biofilm bacteria
added. The freed DNA is then identified, e.g., by PCR sequenced. If
DNA is not freed, then the interfering agent(s) successfully
performed or bound the microbial DNA. If DNA is found in the
sample, then the agent did not interfere with DNABII
polypeptide-microbial DNA binding. As is apparent to those of skill
in the art, a positive and/or negative control can be
simultaneously performed.
[0248] The above methods also can be used as a diagnostic test
since it is possible that a given bacterial species will respond
better to reversal of its biofilm by one agent more than another,
this rapid high throughput assay system could allow one skilled in
the art to assay a panel of possible anti-IHF-like agents to
identify the most efficacious of the group.
[0249] The advantage of these methods is that most clinical
microbiology labs in hospitals are already equipped to perform
these sorts of assays (i.e. determination of MIC, MBC values) using
bacteria that are growing in liquid culture (or planktonically). As
is apparent to those of skill in the art, bacteria generally do not
grow planktonically when they are causing diseases. Instead they
are growing as a stable biofilm and these biofilms are
significantly more resistant to treatment by antibiotics,
antibodies or other therapeutics. This resistance is why most
MIC/MBC values fail to accurately predict efficacy in vivo. Thus,
by determining what "dose" of agent could reverse a bacterial
biofilm in vitro (as described above) Applicants' pre-clinical
assay would be a more reliable predictor of clinical efficacy, even
as an application of personalized medicine.
[0250] In addition to the clinical setting, the methods can be used
to identify the microbe causing the infection and/or confirm
effective interfering agents in an industrial setting.
[0251] In a further aspect of the above methods, an antibiotic or
antimicrobial known to inhibit growth of the underlying infection
is added sequentially or concurrently, to determine if the
infection can be inhibited. It is also possible to add the
interfering agent to the microbial DNA or DNABII polypeptide before
adding the missing complex to assay for biofilm inhibition.
[0252] When practiced in vivo in non-human animal such as a
chinchilla, the method provides a pre-clinical screen to identify
interfering agents that can be used alone or in combination with
other agents to break down biofilms. Examples of this method are
shown in Experiment Nos. 2 through 7, below.
[0253] In another aspect, provided herein is a method of
inhibiting, preventing or breaking down a biofilm in a subject by
administering to the subject an effective amount of an interfering
agent, thereby inhibiting, preventing or breaking down the
microbial biofilm. Examples of this method are shown in Experiment
Nos. 2 through 7, below.
[0254] For the purpose of the above noted in vitro and in vivo
methods, the interfering agent is of the group of:
[0255] (a) an isolated or recombinant integration host factor (IHF)
polypeptide or a fragment or an equivalent of each thereof;
[0256] (b) an isolated or recombinant histone-like protein from E.
coli strain U93 (HU) polypeptide or a fragment or an equivalent of
each thereof;
[0257] (c) an isolated or recombinant protein polypeptide
identified in Table 8, Table 9A, Table 9B, Table 10 or a DNA
binding peptide identified in FIG. 6, or a fragment or an
equivalent of each thereof;
[0258] (d) an isolated or recombinant polypeptide of SEQ ID NO. 1
through 340, or a fragment or an equivalent of each thereof;
[0259] (e) an isolated or recombinant C-terminal polypeptide of SEQ
ID NO. 6 through 11, 28, 29, 42 through 100, Table 8 or those
C-terminal polypeptides identified in Table 10 or a fragment or an
equivalent of each thereof;
[0260] (f) a polypeptide that competes with an integration host
factor on binding to a microbial DNA;
[0261] (g) a four-way junction polynucleotide resembling a Holliday
junction, a 3 way junction polynucleotide resembling a replication
fork, a polynucleotide that has inherent flexibility or bent
polynucleotide;
[0262] (h) an isolated or recombinant polynucleotide encoding any
one of (a) through (f) or an isolated or recombinant polynucleotide
of SEQ ID NO. 36 or an equivalent of each thereof, or a
polynucleotide that hybridizes under stringent conditions to the
polynucleotide its equivalent or its complement;
[0263] (i) an antibody or antigen binding fragment that
specifically recognizes or binds any one of (a) through (f), or an
equivalent or fragment of each antibody or antigen binding fragment
thereof;
[0264] (j) isolated or recombinant polynucleotide encoding the
antibody or antigen binding fragment of (i) or its complement;
or
[0265] (k) a small molecule that competes with the binding of a
DNABII protein or polypeptide to a microbial DNA.
[0266] Also provided herein is a method for inducing an immune
response in or conferring passive immunity on subject in need
thereof, comprising, or alternatively consisting essentially of, or
yet further consisting of, administering to the subject an
effective amount of one or more of the group:
[0267] (a) an isolated or recombinant integration host factor (IHF)
polypeptide, or a fragment or an equivalent of each thereof;
[0268] (b) an isolated or recombinant histone-like protein from E.
coli strain U93 (HU) polypeptide or a fragment or an equivalent of
each thereof;
[0269] (c) an isolated or recombinant protein polypeptide
identified in Table 8, Table 9A, Table 9B. Table 10 or an DNA
binding peptide identified in FIG. 6, or a fragment or an
equivalent of each thereof;
[0270] (d) an isolated or recombinant polypeptide of SEQ ID NO. 1
through 340, or a fragment or an equivalent thereof;
[0271] (e) an isolated or recombinant C-terminal polypeptide of SEQ
ID NO. 6 through 11, 28, 29, 42 through 100, Table 8 or those
C-terminal polypeptides identified in Table 10 or a fragment or an
equivalent of each thereof;
[0272] (f) an isolated or recombinant polynucleotide encoding any
one of (a) through (e) or an isolated or recombinant polynucleotide
of SEQ ID NO. 36 or an equivalent of each thereof, or a
polynucleotide that hybridizes under stringent conditions to the
polynucleotide, its equivalent or its complement;
[0273] (g) an antibody or antigen binding fragment that
specifically recognizes or binds any one of (a) through (e), or an
equivalent or fragment of each thereof;
[0274] (h) an isolated or recombinant polynucleotide encoding the
antibody or antigen binding fragment of (g);
[0275] (i) and antigen presenting cell pulsed with any one of (a)
through (e); and
[0276] (j) and antigen presenting cell transfected with one or more
polynucleotides encoding any one of (a) through (e).
[0277] In one particular aspect, the interfering agent is an
isolated or recombinant integration host factor (IHF) polypeptide
or a fragment thereof, a C-terminal fragment of an IHF polypeptide
of an equivalent of each thereof. In another particular aspect, the
interfering agent is an isolated or recombinant HU polypeptide or a
fragment thereof, a C-terminal fragment of HU polypeptide, or an
equivalent of each thereof. Non-limiting examples of such are an
IHF or HU alpha or beta polypeptide; an IHF polypeptide; Moraxella
catarrhalis HU; E. coli HupA, HupB, himA, himD; E. faecalis HU
(such as V583), HMGB1 (High Mobility Group Box 1, a protein with
similar DNA binding and DNA substrate specificities but not in
primary amino acid sequence to the DNABII family of proteins; a
functional orthologue) and those identified in Table 8 or Table
10.
[0278] In a further aspect, the methods further comprise, or
alternatively consist essentially of, or yet further consist of
administering to the subject an effective amount of one or more of
an antimicrobial, an antigenic peptide or an adjuvant.
[0279] A non-limiting example of an antimicrobial agent is another
vaccine component such as a surface antigen, e.g. an OMP P5,
rsPilA, OMP 26, OMP P2, or Type IV Pilin protein (see Jurcisek and
Bakaletz (2007) J. of Bacteriology 189(10):3868-3875 and Murphy, T
F, Bakaletz, L O and Smeesters, P R (2009) The Pediatric Infectious
Disease Journal, 28:S121-S126).
[0280] The agents and compositions of this invention can be
concurrently or sequentially administered with other antimicrobial
agents and/or surface antigens. In one particular aspect,
administration is locally to the site of the infection by direct
injection or by inhalation for example. Other non-limiting examples
of administration include by one or more method comprising
transdermally, urethrally, sublingually, rectally, vaginally,
ocularly, subcutaneous, intramuscularly, intraperitoneally,
intranasally, by inhalation or orally.
[0281] Microbial infections and disease that can be treated by the
methods of this invention include infection by the organisms
identified in Experiment No. 1 and Table 8, e.g., Streptococcus
agalactiae, Neisseria meningitidis, Treponemes, denticola,
pallidum, Burkholderia cepacia, or Burkholderia pseudomallei. In
one aspect, the microbial infection is one or more of Haemophilus
influenzae (nontypeable), Moraxella catarrhalis, Streptococcus
pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa,
Mycobacterium tuberculosis. These microbial infections may be
present in the upper, mid and lower airway (otitis, sinusitis,
bronchitis but also exacerbations of chronic obstructive pulmonary
disease (COPD), chronic cough, complications of and/or primary
cause of cystic fibrosis (CF) and community acquired pneumonia
(CAP). Thus, by practicing the in vivo methods of this invention,
these diseases and complications from these infections can also be
prevented or treated.
[0282] Infections might also occur in the oral cavity (caries,
periodontitis) and caused by Streptococcus mutans, Porphyromonas
gingivalis, Aggregatibacter actinomyctemcomitans. Infections might
also be localized to the skin (abscesses, `staph` infections,
impetigo, secondary infection of burns, Lyme disease) and caused by
Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas
aeruginosa and Borrelia burdorferi. Infections of the urinary tract
(UTI) can also be treated and are typically caused by Escherichia
coli. Infections of the gastrointestinal tract (GI) (diarrhea,
cholera, gall stones, gastric ulcers) are typically caused by
Salmonella enterica serovar, Vibrio cholerae and Helicobacter
pylori. Infections of the genital tract include and are typically
caused by Neisseria gonorrhoeae. Infections can be of the bladder
or of an indwelling device caused by Enterococcus faecalis.
Infections associated with implanted prosthetic devices, such as
artificial hip or knee replacements, or dental implants, or medical
devices such as pumps, catheters, stents, or monitoring systems,
typically caused by a variety of bacteria, can be treated by the
methods of this invention. These devices can be coated or
conjugated to an agent as described herein. Thus, by practicing the
in vivo methods of this invention, these diseases and complications
from these infections can also be prevented or treated.
[0283] Infections caused by Streptococcus agalactiae can also be
treated by the methods of this invention and it is the major cause
of bacterial septicemia in newborns. Infections caused by Neisseria
meningitidis which can cause meningitis can also be treated.
[0284] Thus, routes of administration applicable to the methods of
the invention include intranasal, intramuscular, urethrally,
intratracheal, subcutaneous, intradermal, topical application,
intravenous, rectal, nasal, oral, inhalation, and other enteral and
parenteral routes of administration. Routes of administration may
be combined, if desired, or adjusted depending upon the agent
and/or the desired effect. An active agent can be administered in a
single dose or in multiple doses. Embodiments of these methods and
routes suitable for delivery, include systemic or localized routes.
In general, routes of administration suitable for the methods of
the invention include, but are not limited to, direct injection,
enteral, parenteral, or inhalational routes.
[0285] Parenteral routes of administration other than inhalation
administration include, but are not limited to, topical,
transdermal, subcutaneous, intramuscular, intraorbital,
intracapsular, intraspinal, intrasternal, and intravenous routes,
i.e., any route of administration other than through the alimentary
canal. Parenteral administration can be conducted to effect
systemic or local delivery of the inhibiting agent. Where systemic
delivery is desired, administration typically involves invasive or
systemically absorbed topical or mucosal administration of
pharmaceutical preparations.
[0286] The interfering agents of the invention can also be
delivered to the subject by enteral administration. Enteral routes
of administration include, but are not limited to, oral and rectal
(e.g., using a suppository) delivery.
[0287] Methods of administration of the active through the skin or
mucosa include, but are not limited to, topical application of a
suitable pharmaceutical preparation, transcutaneous transmission,
transdermal transmission, injection and epidermal administration.
For transdermal transmission, absorption promoters or iontophoresis
are suitable methods. Iontophoretic transmission may be
accomplished using commercially available "patches" that deliver
their product continuously via electric pulses through unbroken
skin for periods of several days or more.
[0288] In various embodiments of the methods of the invention, the
interfering agent will be administered by inhalation, injection or
orally on a continuous, daily basis, at least once per day (QD),
and in various embodiments two (BID), three (TID), or even four
times a day. Typically, the therapeutically effective daily dose
will be at least about 1 mg, or at least about 10 mg, or at least
about 100 mg, or about 200-about 500 mg, and sometimes, depending
on the compound, up to as much as about 1 g to about 2.5 g.
[0289] Dosing of can be accomplished in accordance with the methods
of the invention using capsules, tablets, oral suspension,
suspension for intra-muscular injection, suspension for intravenous
infusion, gel or cream for topical application, or suspension for
intra-articular injection.
[0290] Dosage, toxicity and therapeutic efficacy of compositions
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, for example,
to determine the LD50 (the dose lethal to 50% of the population)
and the ED50 (the dose therapeutically effective in 50% of the
population). The dose ratio between toxic and therapeutic effects
is the therapeutic index and it can be expressed as the ratio
LD50/ED50. Compositions which exhibit high therapeutic indices are
preferred. While compounds that exhibit toxic side effects may be
used, care should be taken to design a delivery system that targets
such compounds to the site of affected tissue in order to minimize
potential damage to uninfected cells and, thereby, reduce side
effects.
[0291] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the methods, the therapeutically effective
dose can be estimated initially from cell culture assays. A dose
can be formulated in animal models to achieve a circulating plasma
concentration range that includes the IC50 (i.e., the concentration
of the test compound which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0292] In some embodiments, an effective amount of a composition
sufficient for achieving a therapeutic or prophylactic effect,
ranges from about 0.000001 mg per kilogram body weight per
administration to about 10,000 mg per kilogram body weight per
administration. Suitably, the dosage ranges are from about 0.0001
mg per kilogram body weight per administration to about 100 mg per
kilogram body weight per administration. Administration can be
provided as an initial dose, followed by one or more "booster"
doses. Booster doses can be provided a day, two days, three days, a
week, two weeks, three weeks, one, two, three, six or twelve months
after an initial dose. In some embodiments, a booster dose is
administered after an evaluation of the subject's response to prior
administrations.
[0293] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to, the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of the therapeutic
compositions described herein can include a single treatment or a
series of treatments.
Polypeptides
[0294] Also provided herein are interfering agents and compositions
for use in the methods described herein, wherein the interfering
agent is of the group:
[0295] (a) an isolated or recombinant integration host factor (IHF)
polypeptide or a fragment or an equivalent of each thereof;
[0296] (b) an isolated or recombinant histone-like protein from E.
coli strain U93 (HU) polypeptide or a fragment or an equivalent of
each thereof;
[0297] (c) an isolated or recombinant protein polypeptide
identified in Table 8, Table 9A, Table 9B, Table 10 or a DNA
binding peptide identified in FIG. 6, or a fragment or an
equivalent of each thereof,
[0298] (d) an isolated or recombinant polypeptide of SEQ ID NO. 1
through 340, or a fragment or an equivalent thereof;
[0299] (e) an isolated or recombinant C-terminal polypeptide of SEQ
ID NO. 6 through 11, 28, 29, 42 through 100, Table 8 or those
C-terminal polypeptides identified in Table 10 or a fragment or an
equivalent of each thereof or
[0300] (f) a polypeptide or polynucleotide that competes with an
integration host factor on binding to a microbial DNA.
In one particular aspect, the interfering agent is an isolated or
recombinant DNABII polypeptide or a fragment or an equivalent of
each thereof. Non-limiting examples of such are an IHF or HU alpha
or beta polypeptide; an IHF a polypeptide; Moraxella catarrhalis
HU; E. coli HupA, HupB, himA, himD; E. faecalis HU (such as V583),
HMGB1 and those identified in Table 8.
[0301] In another aspect, the interfering agent is an isolated or
recombinant polypeptide consisting essentially of an amino acid
sequence selected from SEQ ID NO. 1 to 5 or 12 to 27, 30 to 35,
101-340 or a DNA binding peptide identified in FIG. 6.
[0302] In another aspect, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of SEQ ID NO. 1 or 2, with the proviso that the
polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through
100.
[0303] In another aspect, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of SEQ ID NO. 3, 4 or 5, with the proviso that the
polypeptide is none of SEQ ID NO. 6 to 11, 28, 29, or 42 through
100.
[0304] In another aspect, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of SEQ ID NO. 12, 14, 16, 18, 20, 22, 24, 26, 30 or 32,
with the proviso that the polypeptide is none of SEQ ID NO. 6 to
11, 28, 29, or 42 through 100.
[0305] In another aspect, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of SEQ ID NO. 13, 15, 17, 19, 21, 23, 25, 27, 31 33, 34,
or 35 with the proviso that the polypeptide is none of SEQ ID NO. 6
to 11, 28, 29, or 42 through 100.
[0306] In another aspect, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of an isolated or recombinant polypeptide of the group
of:
[0307] a polypeptide comprising SEQ ID NO. 12 and 13;
[0308] a polypeptide comprising SEQ ID NO. 14 and 15;
[0309] a polypeptide comprising SEQ ID NO. 16 and 17;
[0310] a polypeptide comprising SEQ ID NO. 18 and 19:
[0311] a polypeptide comprising SEQ ID NO. 20 and 21:
[0312] a polypeptide comprising SEQ ID NO. 23 and 24;
[0313] a polypeptide comprising SEQ ID NO. 25 and 26;
[0314] a polypeptide comprising SEQ ID NO. 30 and 31;
[0315] a polypeptide comprising SEQ ID NO. 32 and 33;
[0316] a polypeptide comprising SEQ ID NO. 34 and 35;
[0317] a polypeptide comprising SEQ ID NO. 337 and 338; or
[0318] a polypeptide comprising SEQ ID NO. 339 and 340;
[0319] with the proviso that the polypeptide is none of wild-type
of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or
42 through 100.
[0320] In another aspect, the isolated or recombinant polypeptide
is of the group:
[0321] a polypeptide consisting essentially of SEQ ID NO. 12 and
13;
[0322] a polypeptide consisting essentially of SEQ ID NO. 14 and
15;
[0323] a polypeptide consisting essentially of SEQ ID NO. 16 and
17;
[0324] a polypeptide consisting essentially of SEQ ID NO. 18 and
19;
[0325] a polypeptide consisting essentially of SEQ ID NO. 20 and
21;
[0326] a polypeptide consisting essentially of SEQ ID NO. 23 and
24;
[0327] a polypeptide consisting essentially of SEQ ID NO. 25 and
26;
[0328] a polypeptide consisting essentially of SEQ ID NO. 30 and
31;
[0329] a polypeptide consisting essentially of SEQ ID NO. 32 and
33;
[0330] a polypeptide consisting essentially of SEQ ID NO. 34 and
35;
[0331] a polypeptide consisting essentially of SEQ ID NO. 337 and
338; or
[0332] a polypeptide consisting essentially of SEQ ID NO. 339 and
340;
[0333] with the proviso that the polypeptide is none of wild-type
of any one of IHF alpha, IHF beta or SEQ ID NO. 6 to 11, 28, 29, or
42 through 100.
[0334] Further provided as agents for use in the methods of this
invention are fragments or an equivalent of the isolated or
recombinant polypeptides described above. An example of a fragment
is a C-terminal polypeptide. In a further aspect, the isolated or
recombinant polypeptide comprises, or alternatively consists
essentially of, or yet further consists of two or more of the
isolated or recombinant polypeptides described above.
[0335] For example, the isolated or recombinant polypeptide
comprises, or alternatively consists essentially of, or yet further
consists of any one of SEQ ID. NO. 1 to 6, 12 to 27 or 30 to 33, or
a fragment or an equivalent polypeptide, examples of which are
identified in Table 8 or shown in Table 9A, Table 9B or Table 10.
In one aspect, isolated wild-type polypeptides are excluded, i.e.,
that the polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or
a wildtype sequence identified in Table 8 or shown in Table 9A.
[0336] In one aspect, this invention provides an isolated or
recombinant polypeptide consisting essentially of an amino acid
sequence of the group SEQ ID. NO. 1 to 6, 12 to 27 or 30 to 35, 1
to 6 and 13 to 35, or a polypeptide comprising, or alternatively
consisting essentially of, or yet further consisting of an amino
acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of a
Haemophilus influenzae IHF.alpha. or IHF.beta., non-limiting
examples of which include SEQ ID NO. 12 through 27, or a fragment
or equivalent thereof of each thereof. In another aspect, the
invention provides an isolated or recombinant polypeptide
comprising, or alternatively consisting essentially of, or yet
further consisting of an amino acid sequence of the group SEQ ID
NO. 1 to 4, or a fragment or an equivalent of each thereof, or a
polypeptide comprising, or alternatively consisting essentially of,
or yet further consisting of an amino acid corresponding to the
.beta.-3 and/or .alpha.-3 fragments of a Haemophilus influenzae
IHF.alpha. or IHF.beta., non-limiting examples of which include SEQ
ID NO. 12 through 27 or a fragment or a biological equivalent
thereof which further comprises independently at least 2, or
alternatively al least 3, or alternatively at least 4, or
alternatively at least 5, or at least 6, or alternatively at least
7, or alternatively at least 8, or alternatively at least 9 or
alternatively at least 10 amino acids at the amino and/or carboxyl
terminus of the polypeptide. In one aspect, isolated wildtype DNA
binding polypeptides are excluded, i.e., that the polypeptide is
none of SEQ ID NO. 6 through 11, 28, 29, or 42 through 100 or an
isolated wildtype polypeptide sequence listed in Table 8 or shown
in Table 9A.
[0337] In another aspect, this invention provides an isolated or
recombinant polypeptide comprising, or alternatively consisting
essentially of, or yet further consisting of, SEQ ID. NO 1 or 2
alone or in combination with a polypeptide comprising, or
alternatively consisting essentially of, or yet further consisting
of an amino acid corresponding to the 3-3 and/or .alpha.-3
fragments of a Haemophilus influenzae IHF.alpha. or IHF.beta.,
non-limiting examples of which include SEQ ID Nos. 12 through 27 or
a fragment or a biological equivalent of each thereof. In one
aspect, isolated wildtype DNA binding polypeptides are excluded,
i.e., that the polypeptide is none of SEQ ID NO. 6 through 11, 28,
29, or 42 through 100 or an isolated wildtype polypeptide sequence
listed in Table 8 or shown in Table 9A.
[0338] In a yet further aspect, this invention provides an isolated
or recombinant polypeptide comprising or alternatively consisting
essentially of or yet further consisting of, SEQ ID. NO. 3 or 4 or
a fragment or an equivalent of each thereof, alone or in
combination with a polypeptide comprising, or alternatively
consisting essentially of, or yet further consisting of an amino
acid corresponding to the .beta.-3 and/or .alpha.-3 fragments of a
Haemophilus influenzae IHF.alpha. or IHF.beta., non-limiting
examples of which include SEQ ID NO. 12 through 27, and 34-35 or a
biological equivalent of each thereof. In one aspect, isolated
wildtype DNA binding polypeptides are excluded, i.e., that the
polypeptide is none of SEQ ID NO. 6 through 11, 28, 29, or 42
through 100 or an isolated wildtype polypeptide sequence listed in
Table 8 or shown in Table 9A.
[0339] This invention also provides isolated or recombinant
polypeptides comprising or alternatively consisting essentially of,
or yet further consisting of, two or more, or three or more, four
or more, five or more, six or more, seven or more, eight or more,
nine or more, ten or more, eleven or more, twelve or more, thirteen
or more of all fourteen of the isolated polypeptides or a fragment
or an equivalent of each thereof. Examples of such include isolated
or recombinant polypeptides comprising SEQ ID NO. I through 4,
e.g., SEQ ID NO. 1 and 2, or alternatively 1 and 3 or alternatively
1 and 4, or alternatively 2 and 3, or alternatively SEQ ID NO. 1, 2
and 3 or alternatively, 2, 3 and 4, or alternatively 1, 3 and 4.
The polypeptides can be in any orientation, e.g., SEQ ID NO. 1, 2,
and 3 or SEQ ID NO. 3, 2 and 1 or alternatively 2, 1 and 3, or
alternatively, 3, 1 and 2. Biological equivalents of these
polypeptides are further included in this invention with the
proviso that the sequences do not include isolated wildtype
sequences such as those identified in Tables 8 and 9.
[0340] In another aspect, this invention provides an isolated or
recombinant polypeptide comprising or alternatively consisting
essentially of, or yet further consisting of, SEQ ID NO. 1 or 2 and
3 or 4, or a fragment or an equivalent of each thereof, with the
proviso that the polypeptide is none of SEQ ID NO. 5 through 10,
and they may further comprise any one or more of SEQ ID Nos. 11
through 26, e.g., 11 and 12, or alternatively 1 and 11, or
alternatively 2 and 11, or alternatively, 1 and 12, or
alternatively 2 and 12, or alternatively 11, 12 and 1, or
alternatively 2, 11 and 12. In this embodiment, SEQ ID NO. 1 or 2
is located upstream or amino terminus from SEQ ID NO. 3 or 4, with
the proviso that the amino acid sequence is not an isolated
wildtype polypeptide, e.g., none of SEQ ID NO. 6 through I 1, 28
and 29. In another aspect, the isolated polypeptide comprises SEQ
ID NO. 3 or 4 located upstream or amino terminus to SEQ ID. NO. 1
or 2. Fragments Biological equivalents of these polypeptides are
further included in this invention with the proviso that the
sequence do not include isolated wildtype polypeptides.
[0341] In one embodiment, any polypeptide or protein having
sequence identity to the wildtype polypeptides or those disclosed
in Pedulla et al. (1996) PNAS 93:15411-15416 is excluded from this
invention.
[0342] In any of the above embodiments, a peptide linker can be
added to the N-terminus or C-terminus of the polypeptide. A
"linker" or "peptide linker" refers to a peptide sequence linked to
either the N-terminus or the C-terminus of a polypeptide sequence.
In one aspect, the linker is from about 1 to about 20 amino acid
residues long or alternatively 2 to about 10, about 3 to about 5
amino acid residues long. An example of a peptide linker is
Gly-Pro-Ser-Leu-Lys-Leu (SEQ ID NO: 37). Other examples include
Gly-Gly-Gly: Gly-Pro-Ser-Leu (SEQ ID NO: 38); Gly-Pro-Ser;
Pro-Ser-Leu-Lys (SEQ ID NO: 39); Gly-Pro-Ser-Leu-Lys (SEQ ID NO:
40) and Ser-Leu-Lys-Leu (SEQ ID NO: 41).
[0343] The isolated polypeptides of this invention are intended to
include isolated wildtype and recombinantly produced polypeptides
and proteins from prokaryotic and eukaryotic host cells, as well as
muteins, analogs and fragments thereof, examples of such cells are
described above. In some embodiments, the term also includes
antibodies and anti-idiotypic antibodies as described herein. Such
polypeptides can be isolated or produced using the methods known in
the art and briefly described herein.
[0344] It is understood that functional equivalents or variants of
the wild type polypeptide or protein also are within the scope of
this invention, for example, those having conservative amino acid
substitutions of the amino acids, see for example, Table 9. Other
analogs include fusion proteins comprising a protein or polypeptide
of this invention which can include a polypeptide joined to an
antigen presenting matrix.
[0345] In a further aspect, the polypeptides are conjugated or
linked to a detectable label. Suitable labels are known in the art
and described herein.
[0346] In a yet further aspect, the polypeptides with or without a
detectable label can be contained or expressed on the surface of a
host prokaryotic or eukaryotic host cell, such as a dendritic
cell.
[0347] The proteins and polypeptides are obtainable by a number of
processes known to those of skill in the art, which include
purification, chemical synthesis and recombinant methods.
Polypeptides can be isolated from preparations such as host cell
systems by methods such as immunoprecipitation with antibody, and
standard techniques such as gel filtration, ion-exchange,
reversed-phase, and affinity chromatography. For such methodology,
see for example Deutscher et al. (1999) Guide To Protein
Purification: Methods In Enzymology (Vol. 182, Academic Press).
Accordingly, this invention also provides the processes for
obtaining these polypeptides as well as the products obtainable and
obtained by these processes.
[0348] The polypeptides also can be obtained by chemical synthesis
using a commercially available automated peptide synthesizer such
as those manufactured by Perkin/Elmer/Applied Biosystems, Inc.,
Model 430A or 431A, Foster City, Calif., USA. The synthesized
polypeptide can be precipitated and further purified, for example
by high performance liquid chromatography (HPLC). Accordingly, this
invention also provides a process for chemically synthesizing the
proteins of this invention by providing the sequence of the protein
and reagents, such as amino acids and enzymes and linking together
the amino acids in the proper orientation and linear sequence.
[0349] Alternatively, the proteins and polypeptides can be obtained
by well-known recombinant methods as described, for example, in
Sambrook et al. (1989) supra, using a host cell and vector systems
described herein.
[0350] Also provided by this application are the polypeptides
described herein conjugated to a detectable agent for use in the
diagnostic methods. For example, detectably labeled polypeptides
can be bound to a column and used for the detection and
purification of antibodies. They also are useful as immunogens for
the production of antibodies as described below. The polypeptides
of this invention are useful in an in vitro assay system to screen
for agents or drugs, which modulate cellular processes.
[0351] It is well know to those skilled in the art that
modifications can be made to the peptides of the invention to
provide them with altered properties. As used herein the term
"amino acid" refers to either natural and/or unnatural or synthetic
amino acids, including glycine and both the D or L optical isomers,
and amino acid analogs and peptidomimetics. A peptide of three or
more amino acids is commonly called an oligopeptide if the peptide
chain is short. If the peptide chain is long, the peptide is
commonly called a polypeptide or a protein.
[0352] Peptides of the invention can be modified to include
unnatural amino acids. Thus, the peptides may comprise D-amino
acids, a combination of D- and L-amino acids, and various
"designer" amino acids (e.g., .beta.-methyl amino acids,
C-.alpha.-methyl amino acids, and N-.alpha.-methyl amino acids,
etc.) to convey special properties to peptides. Additionally, by
assigning specific amino acids at specific coupling steps, peptides
with .alpha.-helices .beta. turns, .beta. sheets, .gamma.-turns,
and cyclic peptides can be generated. Generally, it is believed
that .alpha.-helical secondary structure or random secondary
structure is preferred.
[0353] The polypeptides of this invention also can be combined with
various solid phase carriers, such as an implant, a stent, a paste,
a gel, a dental implant, or a medical implant or liquid phase
carriers, such as beads, sterile or aqueous solutions,
pharmaceutically acceptable carriers, pharmaceutically acceptable
polymers, liposomes, micelles, suspensions and emulsions. Examples
of non-aqueous solvents include propyl ethylene glycol,
polyethylene glycol and vegetable oils. When used to prepare
antibodies or induce an immune response in vivo, the carriers also
can include an adjuvant that is useful to non-specifically augment
a specific immune response. A skilled artisan can easily determine
whether an adjuvant is required and select one. However, for the
purpose of illustration only, suitable adjuvants include, but are
not limited to Freund's Complete and Incomplete, mineral salts and
polynucleotides. Other suitable adjuvants include monophosphoryl
lipid A (MPL), mutant derivatives of the heat labile enterotoxin of
E. coli, mutant derivatives of cholera toxin, CPG oligonucleotides,
and adjuvants derived from squalene.
[0354] This invention also provides a pharmaceutical composition
comprising or alternatively consisting essentially of, or yet
further consisting of, any of a polypeptide, analog, mutein, or
fragment of this invention, alone or in combination with each other
or other agents, such an antibiotic and an acceptable carrier or
solid support. These compositions are useful for various diagnostic
and therapeutic methods as described herein.
Polynucleotides
[0355] This invention also provides isolated or recombinant
polynucleotides encoding one or more of the above-identified
isolated or recombinant polypeptides and their respective
complementary strands. Vectors comprising the isolated or
recombinant polynucleotides are further provided examples of which
are known in the art and briefly described herein. In one aspect
where more than one isolated or recombinant polynucleotide is to be
expressed as a single unit, the isolated or recombinant
polynucleotides can be contained within a polycistronic vector. The
polynucleotides can be DNA, RNA, mRNA or interfering RNA, such as
siRNA, miRNA or dsRNA.
[0356] In another aspect, this invention provides an interfering
agent that is a polynucleotide that interferes with the binding of
the DNA to a polypeptide or protein in a microbial biofilm, or a
four-way junction polynucleotide resembling a Holliday junction, a
3 way junction polynucleotide resembling a replication fork, a
polynucleotide that has inherent flexibility or bent polynucleotide
which can treat or inhibit DNABII polynucleotide from binding to
microbial DNA as well treat, prevent or inhibit biofilm formation
and associated infections and disorders. One of skill in the art
can make such polynucleotides using the information provided herein
and knowledge of those of skill in the art. See Goodman and Kay
(1999) J. Biological Chem. 274(52):37004-37011 and Kamashev and
Rouviere-Yaniv (2000) EMBO J. 19(23):6527-6535.
[0357] The invention further provides the isolated or recombinant
polynucleotide operatively linked to a promoter of RNA
transcription, as well as other regulatory sequences for
replication and/or transient or stable expression of the DNA or
RNA. As used herein, the term "operatively linked" means positioned
in such a manner that the promoter will direct transcription of RNA
off the DNA molecule. Examples of such promoters are SP6, T4 and
T7. In certain embodiments, cell-specific promoters are used for
cell-specific expression of the inserted polynucleotide. Vectors
which contain a promoter or a promoter/enhancer, with termination
codons and selectable marker sequences, as well as a cloning site
into which an inserted piece of DNA can be operatively linked to
that promoter are known in the art and commercially available. For
general methodology and cloning strategies, see Gene Expression
Technology (Goeddel ed., Academic Press, Inc. (1991)) and
references cited therein and Vectors: Essential Data Series (Gacesa
and Ramji, eds., John Wiley & Sons, N.Y. (1994)) which contains
maps, functional properties, commercial suppliers and a reference
to GenEMBL accession numbers for various suitable vectors.
[0358] In one embodiment, polynucleotides derived from the
polynucleotides of the invention encode polypeptides or proteins
having diagnostic and therapeutic utilities as described herein as
well as probes to identify transcripts of the protein that may or
may not be present. These nucleic acid fragments can by prepared,
for example, by restriction enzyme digestion of larger
polynucleotides and then labeled with a detectable marker.
Alternatively, random fragments can be generated using nick
translation of the molecule. For methodology for the preparation
and labeling of such fragments, see Sambrook, et al. (1989)
supra.
[0359] Expression vectors containing these nucleic acids are useful
to obtain host vector systems to produce proteins and polypeptides.
It is implied that these expression vectors must be replicable in
the host organisms either as episomes or as an integral part of the
chromosomal DNA. Non-limiting examples of suitable expression
vectors include plasmids, yeast vectors, viral vectors and
liposomes. Adenoviral vectors are particularly useful for
introducing genes into tissues in vivo because of their high levels
of expression and efficient transformation of cells both in vitro
and in vivo. When a nucleic acid is inserted into a suitable host
cell, e.g., a prokaryotic or a eukaryotic cell and the host cell
replicates, the protein can be recombinantly produced. Suitable
host cells will depend on the vector and can include mammalian
cells, animal cells, human cells, simian cells, insect cells, yeast
cells, and bacterial cells constructed using known methods. See
Sambrook, et al. (1989) supra. In addition to the use of viral
vector for insertion of exogenous nucleic acid into cells, the
nucleic acid can be inserted into the host cell by methods known in
the art such as transformation for bacterial cells; transfection
using calcium phosphate precipitation for mammalian cells; or
DEAE-dextran; electroporation; or microinjection. See, Sambrook et
al. (1989) supra, for methodology. Thus, this invention also
provides a host cell, e.g. a mammalian cell, an animal cell (rat or
mouse), a human cell, or a prokaryotic cell such as a bacterial
cell, containing a polynucleotide encoding a protein or polypeptide
or antibody.
[0360] A polynucleotide can comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs. If present,
modifications to the nucleotide structure can be imparted before or
after assembly of the polynucleotide. The sequence of nucleotides
can be interrupted by non-nucleotide components. A polynucleotide
can be further modified after polymerization, such as by
conjugation with a labeling component. The term also refers to both
double- and single-stranded molecules. Unless otherwise specified
or required, any embodiment of this invention that is a
polynucleotide encompasses both the double-stranded form and each
of two complementary single-stranded forms known or predicted to
make up the double-stranded form.
[0361] When the vectors are used for gene therapy in vivo or ex
vivo, a pharmaceutically acceptable vector is preferred, such as a
replication-incompetent retroviral or adenoviral vector.
Pharmaceutically acceptable vectors containing the nucleic acids of
this invention can be further modified for transient or stable
expression of the inserted polynucleotide. As used herein, the term
"pharmaceutically acceptable vector" includes, but is not limited
to, a vector or delivery vehicle having the ability to selectively
target and introduce the nucleic acid into dividing cells. An
example of such a vector is a "replication-incompetent" vector
defined by its inability to produce viral proteins, precluding
spread of the vector in the infected host cell. An example of a
replication-incompetent retroviral vector is LNL6 (Miller et al.
(1989) BioTechniques 7:980-990). The methodology of using
replication-incompetent retroviruses for retroviral-mediated gene
transfer of gene markers has been established. (Bordignon (1989)
PNAS USA 86:8912-8952; Culver (1991) PNAS USA 88:3155; and Rill
(1991) Blood 79(10):2694-2700).
[0362] This invention also provides genetically modified cells that
contain and/or express the polynucleotides of this invention. The
genetically modified cells can be produced by insertion of upstream
regulatory sequences such as promoters or gene activators (see,
U.S. Pat. No. 5,733,761).
[0363] The polynucleotides can be conjugated to a detectable
marker, e.g., an enzymatic label or a radioisotope for detection of
nucleic acid and/or expression of the gene in a cell. A wide
variety of appropriate detectable markers are known in the art,
including fluorescent, radioactive, enzymatic or other ligands,
such as avidin/biotin, which are capable of giving a detectable
signal. In one aspect, one will likely desire to employ a
fluorescent label or an enzyme tag, such as urease, alkaline
phosphatase or peroxidase, instead of radioactive or other
environmentally undesirable reagents. In the case of enzyme tags,
calorimetric indicator substrates can be employed to provide a
means visible to the human eye or spectrophotometrically, to
identify specific hybridization with complementary nucleic
acid-containing samples. Thus, this invention further provides a
method for detecting a single-stranded polynucleotide or its
complement, by contacting target single-stranded polynucleotide
with a labeled, single-stranded polynucleotide (a probe) which is a
portion of the polynucleotide of this invention under conditions
permitting hybridization (preferably moderately stringent
hybridization conditions) of complementary single-stranded
polynucleotides, or more preferably, under highly stringent
hybridization conditions. Hybridized polynucleotide pairs are
separated from un-hybridized, single-stranded polynucleotides. The
hybridized polynucleotide pairs are detected using methods known to
those of skill in the art and set forth, for example, in Sambrook
et al. (1989) supra.
[0364] The polynucleotide embodied in this invention can be
obtained using chemical synthesis, recombinant cloning methods,
PCR, or any combination thereof. Methods of chemical polynucleotide
synthesis are known in the art and need not be described in detail
herein. One of skill in the art can use the sequence data provided
herein to obtain a desired polynucleotide by employing a DNA
synthesizer or ordering from a commercial service.
[0365] The polynucleotides of this invention can be isolated or
replicated using PCR. The PCR technology is the subject matter of
U.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 and
described in PCR: The Polymerase Chain Reaction (Mullis et al.
eds., Birkhauser Press, Boston (1994)) or MacPherson et al. (1991)
and (1995) supra, and references cited therein. Alternatively, one
of skill in the art can use the sequences provided herein and a
commercial DNA synthesizer to replicate the DNA. Accordingly, this
invention also provides a process for obtaining the polynucleotides
of this invention by providing the linear sequence of the
polynucleotide, nucleotides, appropriate primer molecules,
chemicals such as enzymes and instructions for their replication
and chemically replicating or linking the nucleotides in the proper
orientation to obtain the polynucleotides. In a separate
embodiment, these polynucleotides are further isolated. Still
further, one of skill in the art can insert the polynucleotide into
a suitable replication vector and insert the vector into a suitable
host cell (prokaryotic or eukaryotic) for replication and
amplification. The DNA so amplified can be isolated from the cell
by methods known to those of skill in the art. A process for
obtaining polynucleotides by this method is further provided herein
as well as the polynucleotides so obtained.
[0366] RNA can be obtained by first inserting a DNA polynucleotide
into a suitable host cell. The DNA can be delivered by any
appropriate method, e.g., by the use of an appropriate gene
delivery vehicle (e.g., liposome, plasmid or vector) or by
electroporation. When the cell replicates and the DNA is
transcribed into RNA; the RNA can then be isolated using methods
known to those of skill in the art, for example, as set forth in
Sambrook et al. (1989) supra. For instance, mRNA can be isolated
using various lytic enzymes or chemical solutions according to the
procedures set forth in Sambrook et al. (1989) supra, or extracted
by nucleic-acid-binding resins following the accompanying
instructions provided by manufactures.
[0367] Polynucleotides exhibiting sequence complementarity or
homology to a polynucleotide of this invention are useful as
hybridization probes or as an equivalent of the specific
polynucleotides identified herein. Since the full coding sequence
of the transcript is known, any portion of this sequence or
homologous sequences, can be used in the methods of this
invention.
[0368] It is known in the art that a "perfectly matched" probe is
not needed for a specific hybridization. Minor changes in probe
sequence achieved by substitution, deletion or insertion of a small
number of bases do not affect the hybridization specificity. In
general, as much as 20% base-pair mismatch (when optimally aligned)
can be tolerated. Preferably, a probe useful for detecting the
aforementioned mRNA is at least about 80% identical to the
homologous region. More preferably, the probe is 85% identical to
the corresponding gene sequence after alignment of the homologous
region; even more preferably, it exhibits 90% identity.
[0369] These probes can be used in radioassays (e.g. Southern and
Northern blot analysis) to detect, prognose, diagnose or monitor
various cells or tissues containing these cells. The probes also
can be attached to a solid support or an array such as a chip for
use in high throughput screening assays for the detection of
expression of the gene corresponding a polynucleotide of this
invention. Accordingly, this invention also provides a probe
comprising or corresponding to a polynucleotide of this invention,
or its equivalent, or its complement, or a fragment thereof,
attached to a solid support for use in high throughput screens.
[0370] The total size of fragment, as well as the size of the
complementary stretches, will depend on the intended use or
application of the particular nucleic acid segment. Smaller
fragments will generally find use in hybridization embodiments,
wherein the length of the complementary region may be varied, such
as between at least 5 to 10 to about 100 nucleotides, or even full
length according to the complementary sequences one wishes to
detect.
[0371] Nucleotide probes having complementary sequences over
stretches greater than 5 to 10 nucleotides in length are generally
preferred, so as to increase stability and selectivity of the
hybrid, and thereby improving the specificity of particular hybrid
molecules obtained. More preferably, one can design polynucleotides
having gene-complementary stretches of 10 or more or more than 50
nucleotides in length, or even longer where desired. Such fragments
may be readily prepared by, for example, directly synthesizing the
fragment by chemical means, by application of nucleic acid
reproduction technology, such as the PCR technology with two
priming oligonucleotides as described in U.S. Pat. No. 4,603,102 or
by introducing selected sequences into recombinant vectors for
recombinant production. In one aspect, a probe is about 50-75 or
more alternatively, 50-100, nucleotides in length.
[0372] The polynucleotides of the present invention can serve as
primers for the detection of genes or gene transcripts that are
expressed in cells described herein. In this context, amplification
means any method employing a primer-dependent polymerase capable of
replicating a target sequence with reasonable fidelity.
Amplification may be carried out by natural or recombinant
DNA-polymerases such as T7 DNA polymerase, Klenow fragment of E.
coli DNA polymerase, and reverse transcriptase. For illustration
purposes only, a primer is the same length as that identified for
probes.
[0373] One method to amplify polynucleotides is PCR and kits for
PCR amplification are commercially available. After amplification,
the resulting DNA fragments can be detected by any appropriate
method known in the art, e.g., by agarose gel electrophoresis
followed by visualization with ethidium bromide staining and
ultraviolet illumination.
[0374] Methods for administering an effective amount of a gene
delivery vector or vehicle to a cell have been developed and are
known to those skilled in the art and described herein. Methods for
detecting gene expression in a cell are known in the art and
include techniques such as in hybridization to DNA microarrays, in
situ hybridization, PCR, RNase protection assays and Northern blot
analysis. Such methods are useful to detect and quantify expression
of the gene in a cell. Alternatively expression of the encoded
polypeptide can be detected by various methods. In particular it is
useful to prepare polyclonal or monoclonal antibodies that are
specifically reactive with the target polypeptide. Such antibodies
are useful for visualizing cells that express the polypeptide using
techniques such as immunohistology, ELISA, and Western blotting.
These techniques can be used to determine expression level of the
expressed polynucleotide.
Antibodies and Derivatives Thereof
[0375] This invention also provides an antibody that binds and/or
specifically recognizes and binds an isolated polypeptide for use
in the methods of the invention. The antibody can be any of the
various antibodies described herein, non-limiting examples of such
include a polyclonal antibody, a monoclonal antibody, a chimeric
antibody, a human antibody, a veneered antibody, a diabody, a
humanized antibody, an antibody derivative, a recombinant humanized
antibody, or a derivative or fragment of each thereof. In one
aspect, the fragment comprises, or alternatively consists
essentially of, or yet further consists of the CDR of the antibody.
In one aspect, the antibody is detectably labeled or further
comprises a detectable label conjugated to it. Also provided is a
hybridoma cell line that produces a monoclonal antibody of this
invention. Compositions comprising or alternatively consisting
essentially of or yet further, consisting of one or more of the
above embodiments are further provided herein. Further provided are
polynucleotides that encode the amino acid sequence of the
antibodies and fragments as well as methods to produce
recombinantly or chemically synthesize the antibody polypeptides
and fragments thereof. The antibody polypeptides can be produced in
a eukaryotic or prokaryotic cell, or by other methods known in the
art and described herein.
[0376] Antibodies can be generated using conventional techniques
known in the art and are well-described in the literature. Several
methodologies exist for production of polyclonal antibodies. For
example, polyclonal antibodies are typically produced by
immunization of a suitable mammal such as, but not limited to,
chickens, goats, guinea pigs, hamsters, horses, mice, rats, and
rabbits. An antigen is injected into the mammal, which induces the
B-lymphocytes to produce immunoglobulins specific for the antigen.
Immunoglobulins may be purified from the mammal's serum. Antibodies
specific to IHF.alpha. and IHF.beta. can be generated by injection
of polypeptides corresponding to different epitopes of IHF.alpha.
and IHF.beta.. For example, antibodies can be generated using the
20 amino acids of each subunit such as TFRPGQKLKSRVENASPKDE (SEQ ID
NO.34) for IHF.alpha. and KYVPHFKPGKELRDRANIYG (SEQ ID No. 35) for
IHF.beta.. Variations of this methodology include modification of
adjuvants, routes and site of administration, injection volumes per
site and the number of sites per animal for optimal production and
humane treatment of the animal. For example, adjuvants typically
are used to improve or enhance an immune response to antigens. Most
adjuvants provide for an injection site antigen depot, which allows
for a slow release of antigen into draining lymph nodes. Other
adjuvants include surfactants which promote concentration of
protein antigen molecules over a large surface area and
immunostimulatory molecules. Non-limiting examples of adjuvants for
polyclonal antibody generation include Freund's adjuvants, Ribi
adjuvant system, and Titermax. Polyclonal antibodies can be
generated using methods known in the art some of which are
described in U.S. Pat. Nos. 7,279,559; 7,119,179; 7,060,800;
6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.
[0377] Monoclonal antibodies can be generated using conventional
hybridoma techniques known in the art and well-described in the
literature. For example, a hybridoma is produced by fusing a
suitable immortal cell line (e.g., a myeloma cell line such as, but
not limited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5,
P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144,
ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3,
HL-60, MLA 144, NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like,
or heteromyelomas, fusion products thereof, or any cell or fusion
cell derived there from, or any other suitable cell line as known
in the art (see, those at the following web addresses e.g.,
atcc.org, lifetech.com., last accessed on Nov. 26, 2007), with
antibody producing cells, such as, but not limited to, isolated or
cloned spleen, peripheral blood, lymph, tonsil, or other immune or
B cell containing cells, or any other cells expressing heavy or
light chain constant or variable or framework or CDR sequences,
either as endogenous or heterologous nucleic acid, as recombinant
or endogenous, viral, bacterial, algal, prokaryotic, amphibian,
insect, reptilian, fish, mammalian, rodent, equine, ovine, goat,
sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial
DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single,
double or triple stranded, hybridized, and the like or any
combination thereof. Antibody producing cells can also be obtained
from the peripheral blood or, preferably the spleen or lymph nodes,
of humans or other suitable animals that have been immunized with
the antigen of interest. Any other suitable host cell can also be
used for expressing-heterologous or endogenous nucleic acid
encoding an antibody, specified fragment or variant thereof, of the
present invention. The fused cells (hybridomas) or recombinant
cells can be isolated using selective culture conditions or other
suitable known methods, and cloned by limiting dilution or cell
sorting, or other known methods.
[0378] Other suitable methods of producing or isolating antibodies
of the requisite specificity can be used, including, but not
limited to, methods that select recombinant antibody from a peptide
or protein library (e.g., but not limited to, a bacteriophage,
ribosome, oligonucleotide, RNA, cDNA, or the like, display library;
e.g., as available from various commercial vendors such as
MorphoSys (Martinsreid/Planegg. Del.), BioInvent (Lund, Sweden),
Affitech (Oslo, Norway) using methods known in the art. Art known
methods are described in the patent literature some of which
include U.S. Pat. Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476;
5,817,483; 5,824,514; 5,976,862. Alternative methods rely upon
immunization of transgenic animals (e.g., SCID mice, Nguyen et al.
(1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996)
Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.
93:154-161 that are capable of producing a repertoire of human
antibodies, as known in the art and/or as described herein. Such
techniques, include, but are not limited to, ribosome display
(Hanes et al. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942;
Hanes et al. (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135);
single cell antibody producing technologies (e.g., selected
lymphocyte antibody method ("SLAM") (U.S. Pat. No. 5,627,052, Wen
et al. (1987) J. Immunol. 17:887-892; Babcook et al. (1996) Proc.
Natl. Acad. Sci. USA 93:7843-7848); gel microdroplet and flow
cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell
Systems, (Cambridge, Mass.).; Gray et al. (1995) J. Imm. Meth.
182:155-163; and Kenny et al. (1995) Bio. Technol. 13:787-790);
B-cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports
19:125-134).
[0379] Antibody derivatives of the present invention can also be
prepared by delivering a polynucleotide encoding an antibody of
this invention to a suitable host such as to provide transgenic
animals or mammals, such as goats, cows, horses, sheep, and the
like, that produce such antibodies in their milk. These methods are
known in the art and are described for example in U.S. Pat. Nos.
5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;
and 5,304,489.
[0380] The term "antibody derivative" includes post-translational
modification to linear polypeptide sequence of the antibody or
fragment. For example, U.S. Pat. No. 6,602,684 B1 describes a
method for the generation of modified glycol-forms of antibodies,
including whole antibody molecules, antibody fragments, or fusion
proteins that include a region equivalent to the Fc region of an
immunoglobulin, having enhanced Fc-mediated cellular toxicity, and
glycoproteins so generated.
[0381] The antibodies of the invention also include derivatives
that are modified by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from generating an anti-idiotypic response.
Antibody derivatives include, but are not limited to, antibodies
that have been modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Additionally, the
derivatives may contain one or more non-classical amino acids.
[0382] Antibody derivatives also can be prepared by delivering a
polynucleotide of this invention to provide transgenic plants and
cultured plant cells (e.g., but not limited to tobacco, maize, and
duckweed) that produce such antibodies, specified portions or
variants in the plant parts or in cells cultured therefrom. For
example, Cramer et al. (1999) Curr. Top. Microbol. Immunol.
240:95-118 and references cited therein, describe the production of
transgenic tobacco leaves expressing large amounts of recombinant
proteins, e.g., using an inducible promoter. Transgenic maize have
been used to express mammalian proteins at commercial production
levels, with biological activities equivalent to those produced in
other recombinant systems or purified from natural sources. See,
e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and
references cited therein. Antibody derivatives have also been
produced in large amounts from transgenic plant seeds including
antibody fragments, such as single chain antibodies (scFv's),
including tobacco seeds and potato tubers. See, e.g., Conrad et al.
(1998) Plant Mol. Biol. 38:101-109 and references cited therein.
Thus, antibodies can also be produced using transgenic plants,
according to know methods.
[0383] Antibody derivatives also can be produced, for example, by
adding exogenous sequences to modify immunogenicity or reduce,
enhance or modify binding, affinity, on-rate, off-rate, avidity,
specificity, half-life, or any other suitable characteristic.
Generally part or all of the non-human or human CDR sequences are
maintained while the non-human sequences of the variable and
constant regions are replaced with human or other amino acids.
[0384] In general, the CDR residues are directly and most
substantially involved in influencing antigen binding. Humanization
or engineering of antibodies can be performed using any known
method such as, but not limited to, those described in U.S. Pat.
Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;
5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;
5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.
[0385] Chimeric, humanized or primatized antibodies of the present
invention can be prepared based on the sequence of a reference
monoclonal antibody prepared using standard molecular biology
techniques. DNA encoding the heavy and light chain immunoglobulins
can be obtained from the hybridoma of interest and engineered to
contain non-reference (e.g., human) immunoglobulin sequences using
standard molecular biology techniques. For example, to create a
chimeric antibody, the murine variable regions can be linked to
human constant regions using methods known in the art (U.S. Pat.
No. 4,816,567). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (U.S. Pat. No. 5,225,539 and U.S. Pat. Nos. 5,530,101;
5,585,089; 5,693,762 and 6,180,370). Similarly, to create a
primatized antibody the murine CDR regions can be inserted into a
primate framework using methods known in the art (WO 93/02108 and
WO 99/55369).
[0386] Techniques for making partially to fully human antibodies
are known in the art and any such techniques can be used. According
to one embodiment, fully human antibody sequences are made in a
transgenic mouse which has been engineered to express human heavy
and light chain antibody genes. Multiple strains of such transgenic
mice have been made which can produce different classes of
antibodies. B cells from transgenic mice which are producing a
desirable antibody can be fused to make hybridoma cell lines for
continuous production of the desired antibody. (See for example,
Russel et al. (2000) Infection and Immunity April 2000:1820-1826;
Gallo et al. (2000) European J. of Immun. 30:534-540; Green (1999)
J. of Immun. Methods 231:11-23; Yang et al. (1999A) J. of Leukocyte
Biology 66:401-410; Yang (1999B) Cancer Research 59(6):1236-1243;
Jakobovits (1998) Advanced Drug Delivery Reviews 31:33-42; Green
and Jakobovits (1998) J. Exp. Med. 188(3):483-495; Jakobovits
(1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al. (1997)
Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News
17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits
(1996) Weir's Handbook of Experimental Immunology, The Integrated
Immune System Vol. IV, 194.1-194.7; Jakobovits (1995) Current
Opinion in Biotechnology 6:561-566; Mendez et al. (1995) Genomics
26:294-307; Jakobovits (1994) Current Biology 4(8):761-763; Arbones
et al. (1994) Immunity 1(4):247-260; Jakobovits (1993) Nature
362(6417):255-258; Jakobovits et al. (1993) Proc. Natl. Acad. Sci.
USA 90(6):2551-2555; and U.S. Pat. No. 6,075,181.)
[0387] The antibodies of this invention also can be modified to
create chimeric antibodies. Chimeric antibodies are those in which
the various domains of the antibodies' heavy and light chains are
coded for by DNA from more than one species. See, e.g., U.S. Pat.
No. 4,816,567.
[0388] Alternatively, the antibodies of this invention can also be
modified to create veneered antibodies. Veneered antibodies are
those in which the exterior amino acid residues of the antibody of
one species are judiciously replaced or "veneered" with those of a
second species so that the antibodies of the first species will not
be immunogenic in the second species thereby reducing the
immunogenicity of the antibody. Since the antigenicity of a protein
is primarily dependent on the nature of its surface, the
immunogenicity of an antibody could be reduced by replacing the
exposed residues which differ from those usually found in another
mammalian species antibodies. This judicious replacement of
exterior residues should have little, or no, effect on the interior
domains, or on the interdomain contacts. Thus, ligand binding
properties should be unaffected as a consequence of alterations
which are limited to the variable region framework residues. The
process is referred to as "veneering" since only the outer surface
or skin of the antibody is altered, the supporting residues remain
undisturbed.
[0389] The procedure for "veneering" makes use of the available
sequence data for human antibody variable domains compiled by Kabat
et al. (1987) Sequences of Proteins of Immunological Interest, 4th
ed., Bethesda, Md., National Institutes of Health, updates to this
database, and other accessible U.S. and foreign databases (both
nucleic acid and protein). Non-limiting examples of the methods
used to generate veneered antibodies include EP 519596; U.S. Pat.
No. 6,797,492; and described in Padlan et al. (1991) Mol. Immunol.
28(4-5):489-498.
[0390] The term "antibody derivative" also includes "diabodies"
which are small antibody fragments with two antigen-binding sites,
wherein fragments comprise a heavy chain variable domain (VH)
connected to a light chain variable domain (VL) in the same
polypeptide chain. (See for example, EP 404,097; WO 93/11161; and
Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.)
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. (See also, U.S. Pat. No. 6,632,926 to Chen
et al. which discloses antibody variants that have one or more
amino acids inserted into a hypervariable region of the parent
antibody and a binding affinity for a target antigen which is at
least about two fold stronger than the binding affinity of the
parent antibody for the antigen).
[0391] The term "antibody derivative" further includes engineered
antibody molecules, fragments and single domains such as scFv,
dAbs, nanobodies, minibodies, Unibodies, and Affibodies (Holliger
& Hudson (2005) Nature Biotech 23(9):1126-36; U.S. Patent
Publication US 2006/0211088; PCT Publication WO2007/059782; U.S.
Pat. No. 5,831,012).
[0392] The term "antibody derivative" further includes "linear
antibodies". The procedure for making linear antibodies is known in
the art and described in Zapata et al. (1995) Protein Eng. 8(10):
1057-1062. Briefly, these antibodies comprise a pair of tandem Fd
segments (V.sub.H-C.sub.H 1-VH-C.sub.H 1) which form a pair of
antigen binding regions. Linear antibodies can be bispecific or
monospecific.
[0393] The antibodies of this invention can be recovered and
purified from recombinant cell cultures by known methods including,
but not limited to, protein A purification, ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. High
performance liquid chromatography ("HPLC") can also be used for
purification.
[0394] Antibodies of the present invention include naturally
purified products, products of chemical synthetic procedures, and
products produced by recombinant techniques from a eukaryotic host,
including, for example, yeast, higher plant, insect and mammalian
cells, or alternatively from a prokaryotic host as described above.
A number of antibody production systems are described in Birch
& Radner (2006) Adv. Drug Delivery Rev. 58: 671-685.
[0395] If an antibody being tested binds with protein or
polypeptide, then the antibody being tested and the antibodies
provided by this invention are equivalent. It also is possible to
determine without undue experimentation, whether an antibody has
the same specificity as the antibody of this invention by
determining whether the antibody being tested prevents an antibody
of this invention from binding the protein or polypeptide with
which the antibody is normally reactive. If the antibody being
tested competes with the antibody of the invention as shown by a
decrease in binding by the monoclonal antibody of this invention,
then it is likely that the two antibodies bind to the same or a
closely related epitope. Alternatively, one can pre-incubate the
antibody of this invention with a protein with which it is normally
reactive, and determine if the antibody being tested is inhibited
in its ability to bind the antigen. If the antibody being tested is
inhibited then, in all likelihood, it has the same, or a closely
related, epitopic specificity as the antibody of this
invention.
[0396] The term "antibody" also is intended to include antibodies
of all immunoglobulin isotypes and subclasses. Particular isotypes
of a monoclonal antibody can be prepared either directly by
selecting from an initial fusion, or prepared secondarily, from a
parental hybridoma secreting a monoclonal antibody of different
isotype by using the sib selection technique to isolate class
switch variants using the procedure described in Steplewski et al.
(1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J.
Immunol. Methods 74:307. Alternatively, recombinant DNA techniques
may be used.
[0397] The isolation of other monoclonal antibodies with the
specificity of the monoclonal antibodies described herein can also
be accomplished by one of ordinary skill in the art by producing
anti-idiotypic antibodies. Herlyn et al. (1986) Science 232:100. An
anti-idiotypic antibody is an antibody which recognizes unique
determinants present on the monoclonal antibody of interest.
[0398] In some aspects of this invention, it will be useful to
detectably or therapeutically label the antibody. Suitable labels
are described supra. Methods for conjugating antibodies to these
agents are known in the art. For the purpose of illustration only,
antibodies can be labeled with a detectable moiety such as a
radioactive atom, a chromophore, a fluorophore, or the like. Such
labeled antibodies can be used for diagnostic techniques, either in
vivo, or in an isolated test sample.
[0399] The coupling of antibodies to low molecular weight haptens
can increase the sensitivity of the antibody in an assay. The
haptens can then be specifically detected by means of a second
reaction. For example, it is common to use haptens such as biotin,
which reacts avidin, or dinitrophenol, pyridoxal, and fluorescein,
which can react with specific anti-hapten antibodies. See, Harlow
and Lane (1988) supra.
[0400] The variable region of the antibodies of the present
invention can be modified by mutating amino acid residues within
the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one
or more binding properties (e.g., affinity) of the antibody.
Mutations may be introduced by site-directed mutagenesis or
PCR-mediated mutagenesis and the effect on antibody binding, or
other functional property of interest, can be evaluated in
appropriate in vitro or in vivo assays. Preferably conservative
modifications are introduced and typically no more than one, two,
three, four or five residues within a CDR region are altered. The
mutations may be amino acid substitutions, additions or
deletions.
[0401] Framework modifications can be made to the antibodies to
decrease immunogenicity, for example, by "backmutating" one or more
framework residues to the corresponding germline sequence.
[0402] In addition, the antibodies of the invention may be
engineered to include modifications within the Fe region to alter
one or more functional properties of the antibody, such as serum
half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Such modifications
include, but are not limited to, alterations of the number of
cysteine residues in the hinge region to facilitate assembly of the
light and heavy chains or to increase or decrease the stability of
the antibody (U.S. Pat. No. 5,677,425); and amino acid mutations in
the Fc hinge region to decrease the biological half life of the
antibody (U.S. Pat. No. 6,165,745).
[0403] Additionally, the antibodies of the invention may be
chemically modified. Glycosylation of an antibody can be altered,
for example, by modifying one or more sites of glycosylation within
the antibody sequence to increase the affinity of the antibody for
antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861). Alternatively, to
increase antibody-dependent cell-mediated cytotoxicity, a
hypofucosylated antibody having reduced amounts of fucosyl residues
or an antibody having increased bisecting GlcNac structures can be
obtained by expressing the antibody in a host cell with altered
glycosylation mechanism (Shields, R. L. et al., 2002 J. Biol. Chem.
277:26733-26740; Umana et al., 1999 Nat. Biotech. 17:176-180).
[0404] The antibodies of the invention can be pegylated to increase
biological half-life by reacting the antibody or fragment thereof
with polyethylene glycol (PEG) or a reactive ester or aldehyde
derivative of PEG, under conditions in which one or more PEG groups
become attached to the antibody or antibody fragment. Antibody
pegylation may be carried out by an acylation reaction or an
alkylation reaction with a reactive PEG molecule (or an analogous
reactive water-soluble polymer). As used herein, the term
"polyethylene glycol" is intended to encompass any of the forms of
PEG that have been used to derivatize other proteins, such as mono
(C1-C10) alkoxy- or aryloxy-polyethylene glycol or polyethylene
glycol-maleimide. The antibody to be pegylated can be an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the invention
(EP 0 154 316 and EP 0 401 384).
[0405] Additionally, antibodies may be chemically modified by
conjugating or fusing the antigen-binding region of the antibody to
serum protein, such as human serum albumin, to increase half-life
of the resulting molecule. Such approach is for example described
in EP 0322094 and EP 0 486 525.
[0406] The antibodies or fragments thereof of the present invention
may be conjugated to a diagnostic agent and used diagnostically,
for example, to monitor the development or progression of a disease
and determine the efficacy of a given treatment regimen. Examples
of diagnostic agents include enzymes, prosthetic groups,
fluorescent materials, luminescent materials, bioluminescent
materials, radioactive materials, positron emitting metals using
various positron emission tomographies, and nonradioactive
paramagnetic metal ions. The detectable substance may be coupled or
conjugated either directly to the antibody or fragment thereof, or
indirectly, through a linker using techniques known in the art.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
Examples of suitable prosthetic group complexes include
streptavidin/biotin and avidin/biotin. Examples of suitable
fluorescent materials include umbelliferone, fluorescein,
fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine
fluorescein, dansyl chloride or phycoerythrin. An example of a
luminescent material includes luminol. Examples of bioluminescent
materials include luciferase, luciferin, and aequorin. Examples of
suitable radioactive material include .sup.125I, .sup.131I,
Indium-111, Lutetium-171, Bismuth-212, Bismuth-213. Astatine-211,
Copper-62, Copper-64, Copper-67, Yttrium-90. Iodine-125,
Iodine-131, Phosphorus-32, Phosphorus-33, Scandium-47. Silver-111,
Gallium-67, Praseodymium-142, Samarium-153, Terbium-161,
Dysprosium-166, Holmium-166, Rhenium-186, Rhenium-188, Rhenium-189,
Lead-212, Radium-223, Actinium-225, Iron-59, Selenium-75,
Arsenic-77, Strontium-89, Molybdenum-99, Rhodium-105,
Palladium-109, Praseodymium-143, Promethium-149, Erbium-169,
Iridium-194, Gold-198, Gold-199, and Lead-211. Monoclonal
antibodies may be indirectly conjugated with radiometal ions
through the use of bifunctional chelating agents that are
covalently linked to the antibodies. Chelating agents may be
attached through amines (Meares et al., 1984 Anal. Biochem. 142:
68-78); sulfhydral groups (Koyama 1994 Chem. Abstr. 120: 217262t)
of amino acid residues and carbohydrate groups (Rodwell et al. 1986
PNAS USA 83: 2632-2636: Quadri et al. 1993 Nucl. Med. Biol. 20:
559-570).
[0407] Further, the antibodies or fragments thereof of the present
invention may be conjugated to a therapeutic agent. Suitable
therapeutic agents include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin, antimetabolites (such as
methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase,
gemcitabine, cladribine), alkylating agents (such as
mechlorethamine, thioepa, chlorambucil, melphalan, carmustine
(BSNU), lomustine (CCNU), cyclophosphamide, busulfan,
dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine,
mitomycin C, cisplatin and other platinum derivatives, such as
carboplatin), antibiotics (such as dactinomycin (formerly
actinomycin), bleomycin, daunorubicin (formerly daunomycin),
doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone,
plicamycin, anthramycin (AMC)), diphtheria toxin and related
molecules (such as diphtheria A chain and active fragments thereof
and hybrid molecules), ricin toxin (such as ricin A or a
deglycosylated ricin A chain toxin), cholera toxin, a Shiga-like
toxin (SLT-1, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin,
pertussis toxin, tetanus toxin, soybean Bowman-Birk protease
inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin,
gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, enomycin toxins and mixed toxins.
[0408] Additional suitable conjugated molecules include
ribonuclease (RNase), DNase I, an antisense nucleic acid, an
inhibitory RNA molecule such as a siRNA molecule, an
immunostimulatory nucleic acid, aptamers, ribozymes, triplex
forming molecules, and external guide sequences. Aptamers are small
nucleic acids ranging from 15-50 bases in length that fold into
defined secondary and tertiary structures, such as stem-loops or
G-quartets, and can bind small molecules, such as ATP (U.S. Pat.
No. 5,631,146) and theophiline (U.S. Pat. No. 5,580,737), as well
as large molecules, such as reverse transcriptase (U.S. Pat. No.
5,786,462) and thrombin (U.S. Pat. No. 5,543,293). Ribozymes are
nucleic acid molecules that are capable of catalyzing a chemical
reaction, either intramolecularly or intermolecularly. Ribozymes
typically cleave nucleic acid substrates through recognition and
binding of the target substrate with subsequent cleavage. Triplex
forming function nucleic acid molecules can interact with
double-stranded or single-stranded nucleic acid by forming a
triplex, in which three strands of DNA form a complex dependant on
both Watson-Crick and Hoogsteen base-pairing. Triplex molecules can
bind target regions with high affinity and specificity.
[0409] The functional nucleic acid molecules may act as effectors,
inhibitors, modulators, and stimulators of a specific activity
possessed by a target molecule, or the functional nucleic acid
molecules may possess a de novo activity independent of any other
molecules.
[0410] The therapeutic agents can be linked to the antibody
directly or indirectly, using any of a large number of available
methods. For example, an agent can be attached at the hinge region
of the reduced antibody component via disulfide bond formation,
using cross-linkers such as N-succinyl
3-(2-pyridyldithio)proprionate (SPDP), or via a carbohydrate moiety
in the Fc region of the antibody (Yu et al. 1994 Int. J. Cancer 56:
244; Upeslacis et al., "Modification of Antibodies by Chemical
Methods," in Monoclonal antibodies: principles and applications,
Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc. 1995); Price,
"Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal antibodies: Production, engineering and
clinical application, Ritter et al. (eds.), pages 60-84 (Cambridge
University Press 1995)).
[0411] Techniques for conjugating therapeutic agents to antibodies
are well known (Amon et al., "Monoclonal Antibodies For
Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal
Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56
(Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al.
(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody
Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications,
Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And
Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody
in Cancer Therapy", in Monoclonal Antibodies For Cancer Detection
And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press
1985), and Thorpe et al., "The Preparation And Cytotoxic Properties
Of Antibody-Toxin Conjugates" 1982 Immunol. Rev. 62:119-58).
[0412] The antibodies of the invention or antigen-binding regions
thereof can be linked to another functional molecule such as
another antibody or ligand for a receptor to generate a bi-specific
or multi-specific molecule that binds to at least two or more
different binding sites or target molecules. Linking of the
antibody to one or more other binding molecules, such as another
antibody, antibody fragment, peptide or binding mimetic, can be
done, for example, by chemical coupling, genetic fusion, or
noncovalent association. Multi-specific molecules can further
include a third binding specificity, in addition to the first and
second target epitope.
[0413] Bi-specific and multi-specific molecules can be prepared
using methods known in the art. For example, each binding unit of
the bi-specific molecule can be generated separately and then
conjugated to one another. When the binding molecules are proteins
or peptides, a variety of coupling or cross-linking agents can be
used for covalent conjugation. Examples of cross-linking agents
include protein A, carbodiimide,
N-succinimidyl-S-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-I-carboxylate
(sulfo-SMCC) (Karpovsky et al., 1984 J. Exp. Med. 160:1686; Liu et
al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). When the binding
molecules are antibodies, they can be conjugated by sulfhydryl
bonding of the C-terminus hinge regions of the two heavy
chains.
[0414] The antibodies or fragments thereof of the present invention
may be linked to a moiety that is toxic to a cell to which the
antibody is bound to form "depleting" antibodies. These antibodies
are particularly useful in applications where it is desired to
deplete an NK cell.
[0415] The antibodies of the invention may also be attached to
solid supports, which are particularly useful for immunoassays or
purification of the target antigen. Such solid supports include,
but are not limited to, glass, cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene.
[0416] The antibodies also can be bound to many different carriers.
Thus, this invention also provides compositions containing the
antibodies and another substance, active or inert. Examples of
well-known carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylase, natural and modified
cellulose, polyacrylamide, agarose, and magnetite. The nature of
the carrier can be either soluble or insoluble for purposes of the
invention. Those skilled in the art will know of other suitable
carriers for binding monoclonal antibodies, or will be able to
ascertain such, using routine experimentation.
Isolation, Culturing and Expansion of APCs, Including Dendritic
Cells
[0417] This invention also provides isolated host cells comprising
one or more of an isolated polypeptides or isolated polynucleotides
or the vectors of this invention. In one aspect the isolated host
cell is a eukaryotic cell such as antigen presenting cell (APC),
e.g. a dendritic cell. In another aspect, the isolated host cell is
a prokaryotic cell. In one aspect, the invention is an isolated
host cell that is cultured under conditions that promote expression
of the polynucleotide. This invention also provides the host cell,
expression system and polypeptide produced by the expression
system.
[0418] The following is a brief description of two fundamental
approaches for the isolation of APC. These approaches involve (1)
isolating bone marrow precursor cells (CD34.sup.+) from blood and
stimulating them to differentiate into APC; or (2) collecting the
precommitted APCs from peripheral blood. In the first approach, the
patient must be treated with cytokines such as GM-CSF to boost the
number of circulating CD34.sup.+ stem cells in the peripheral
blood.
[0419] The second approach for isolating APCs is to collect the
relatively large numbers of precommitted APCs already circulating
in the blood. Previous techniques for isolating committed APCs from
human peripheral blood have involved combinations of physical
procedures such as metrizamide gradients and adherence/nonadherence
steps (Freudenthal et al. (1990) PNAS 87:7698-7702); Percoll
gradient separations (Mehta-Damani et al. (1994) J. Immunol.
153:996-1003); and fluorescence activated cell sorting techniques
(Thomas et al. (1993) J. Immunol. 151:6840-52).
[0420] One technique for separating large numbers of cells from one
another is known as countercurrent centrifugal elutriation (CCE).
Cell samples are placed in a special elutriation rotor. The rotor
is then spun at a constant speed of, for example, 3000 rpm. Once
the rotor has reached the desired speed, pressurized air is used to
control the flow rate of cells. Cells in the elutriator are
subjected to simultaneous centrifugation and a washout stream of
buffer which is constantly increasing in flow rate. This results in
fractional cell separations based largely but not exclusively on
differences in cell size.
[0421] In one aspect of the invention, the APC are precommitted or
mature dendritic cells which can be isolated from the white blood
cell fraction of a mammal, such as a murine, simian or a human
(See, e.g., WO 96/23060). The white blood cell fraction can be from
the peripheral blood of the mammal. This method includes the
following steps: (a) providing a white blood cell fraction obtained
from a mammalian source by methods known in the art such as
leukapheresis; (b) separating the white blood cell fraction of step
(a) into four or more subfractions by countercurrent centrifugal
elutriation, (c) stimulating conversion of monocytes in one or more
fractions from step (b) to dendritic cells by contacting the cells
with calcium ionophore. GM-CSF and IL-13 or GM-CSF and IL-4, (d)
identifying the dendritic cell-enriched fraction from step (c), and
(e) collecting the enriched fraction of step (d), preferably at
about 4.degree. C. One way to identify the dendritic cell-enriched
fraction is by fluorescence-activated cell sorting. The white blood
cell fraction can be treated with calcium ionophore in the presence
of other cytokines, such as recombinant (rh) rhTL-12, rhGM-CSF, or
rhIL-4. The cells of the white blood cell fraction can be washed in
buffer and suspended in Ca.sup.++/Mg.sup.++ free media prior to the
separating step. The white blood cell fraction can be obtained by
leukapheresis. The dendritic cells can be identified by the
presence of at least one of the following markers: HLA-DR, HLA-DQ,
or B7.2, and the simultaneous absence of the following markers:
CD3, CD16, CD56, CD57, and CD19, CD20. Monoclonal antibodies
specific to these cell surface markers are commercially
available.
[0422] More specifically, the method requires collecting an
enriched collection of white cells and platelets from leukapheresis
that is then further fractionated by countercurrent centrifugal
elutriation (CCE) (Abrahamsen et al. (1991) J. Clin. Apheresis.
6:48-53). In this technique, cells are subject to simultaneous
centrifugation and a washout stream of buffer which is constantly
increasing in flow rate. The constantly increasing countercurrent
flow of buffer leads to fractional cell separations that are
largely based on cell size.
[0423] Quality control of APC and more specifically DC collection
and confirmation of their successful activation in culture is
dependent upon a simultaneous multi-color FACS analysis technique
which monitors both monocytes and the dendritic cell subpopulation
as well as possible contaminant T lymphocytes. Cell sorting is
based on differential expression of cell surface markers including
CD3 (T cells), CD16/CD56/CD57 (NK/LAK cells), and CD19/CD20 (B
cells). DCs are distinguishable from monocytes based in part on
levels of CD14, which is expressed at very high levels in monocytes
compared to DCs. DCs show high levels of expression of HLA-DR,
significant HLA-DQ and B7.2 (but little or no B7.1) at the time
they are circulating in the blood (in addition they express Leu M7
and M9, myeloid markers which are also expressed by monocytes and
neutrophils).
[0424] When combined with a third color reagent for analysis of
dead cells, propidium iodide (PI), it is possible to make positive
identification of all cell subpopulations
[0425] The goal of FACS analysis at the time of collection is to
confirm that the DCs are enriched in the expected fractions, to
monitor neutrophil contamination, and to make sure that appropriate
markers are expressed. This rapid bulk collection of enriched DCs
from human peripheral blood, suitable for clinical applications, is
absolutely dependent on the analytic FACS technique described above
for quality control. If need be, mature DCs can be immediately
separated from monocytes at this point by fluorescent sorting for
"cocktail negative" cells. It may not be necessary to routinely
separate DCs from monocytes because, the monocytes themselves are
still capable of differentiating into DCs or functional DC-like
cells in culture.
[0426] Once collected, the DC rich/monocyte APC fractions (usually
150 through 190) can be pooled and cryopreserved for future use, or
immediately placed in short term culture.
[0427] Alternatively, others have reported that a method for
upregulating (activating) dendritic cells and converting monocytes
to an activated dendritic cell phenotype. This method involves the
addition of calcium ionophore to the culture media convert
monocytes into activated dendritic cells. Adding the calcium
ionophore A23187, for example, at the beginning of a 24-48 hour
culture period resulted in uniform activation and dendritic cell
phenotypic conversion of the pooled "monocyte plus DC" fractions:
characteristically, the activated population becomes uniformly CD14
(Leu M3) negative, and upregulates HLA-DR, HLA-DQ, ICAM-1, B7.1,
and B7.2. Furthermore this activated bulk population functions as
well on a small numbers basis as a further purified.
[0428] Specific combination(s) of cytokines have been used
successfully to amplify (or partially substitute) for the
activation/conversion achieved with calcium ionophore: these
cytokines include but are not limited to purified or recombinant
("rh") rhGM-CSF, rhIL-2, and rhIL-4. Each cytokine when given alone
is inadequate for optimal upregulation.
Presentation of Antigen to the APC
[0429] For purposes of immunization, the polypeptides (e.g., SEQ ID
NO. 1 through 33) can be delivered to antigen-presenting cells as
protein/peptide or in the form of cDNA encoding the
protein/peptide. Antigen-presenting cells (APCs) can consist of
dendritic cells (DCs), monocytes/macrophages, B lymphocytes or
other cell type(s) expressing the necessary MHC/co-stimulatory
molecules. The methods described below focus primarily on DCs which
are the most potent, preferred APCs.
[0430] Pulsing is accomplished in vitro/ex vivo by exposing APCs to
the antigenic protein or polypeptide(s) of this invention. The
protein or peptide(s) are added to APCs at a concentration of 1-10
.mu.m for approximately 3 hours. Transfection of APCs with
polynucleotides encoding antigens or antigenic polypeptides is
accomplished by exposing APCs to the nucleic acids in the presence
of transfection agents known in the art, including but not limited
to cationic lipids. Transfected or pulsed APCs can subsequently be
administered to the host via an intravenous, subcutaneous,
intranasal, intramuscular or intraperitoneal route of delivery.
[0431] Protein/peptide antigen can also be delivered in vivo with
adjuvant via the intravenous, subcutaneous, intranasal,
intramuscular or intraperitoneal route of delivery.
Foster Antigen Presenting Cells
[0432] Foster antigen presenting cells are particularly useful as a
target cell. Foster APCs are derived from the human cell line 174X
CEM.T2, referred to as T2, which contains a mutation in its antigen
processing pathway that restricts the association of endogenous
peptides with cell surface MHC class I molecules (Zweerink et al.
(1993) J. Immunol. 150:1763-1771). This is due to a large
homozygous deletion in the MHC class II region encompassing the
genes TAP 1, TAP2, LMP 1, and LMP2, which are required for antigen
presentation to MHC class 1-restricted CD8.sup.+ CTLs. In effect,
only "empty" MHC class I molecules are presented on the surface of
these cells. Exogenous peptide added to the culture medium binds to
these MHC molecules provided that the peptide contains the
allele-specific binding motif. These T2 cells are referred to
herein as "foster" APCs. They can be used in conjunction with this
invention to present antigen(s).
[0433] Transduction of T2 cells with specific recombinant MHC
alleles allows for redirection of the MHC restriction profile.
Libraries tailored to the recombinant allele will be preferentially
presented by them because the anchor residues will prevent
efficient binding to the endogenous allele.
[0434] High level expression of MHC molecules makes the APC more
visible to the CTLs. Expressing the MHC allele of interest in T2
cells using a powerful transcriptional promoter (e.g., the CMV
promoter) results in a more reactive APC (most likely due to a
higher concentration of reactive MHC-peptide complexes on the cell
surface).
Expansion of Immune Effector Cells
[0435] The present invention makes use of these APCs to stimulate
production of an enriched population of antigen-specific immune
effector cells. The antigen-specific immune effector cells are
expanded at the expense of the APCs, which die in the culture. The
process by which naive immune effector cells become educated by
other cells is described essentially in Coulie (1997) Molec. Med.
Today 3:261-268.
[0436] The APCs prepared as described above are mixed with naive
immune effector cells. Preferably, the cells may be cultured in the
presence of a cytokine, for example IL2. Because dendritic cells
secrete potent immunostimulatory cytokines, such as IL12, it may
not be necessary to add supplemental cytokines during the first and
successive rounds of expansion. In any event, the culture
conditions are such that the antigen-specific immune effector cells
expand (i.e. proliferate) at a much higher rate than the APCs.
Multiple infusions of APCs and optional cytokines can be performed
to further expand the population of antigen-specific cells.
[0437] In one embodiment, the immune effector cells are T cells. In
a separate embodiment, the immune effector cells can be genetically
modified by transduction with a transgene coding for example, L-2,
IL-11 or IL-13. Methods for introducing transgenes in vitro, ex
vivo and in vivo are known in the art.
Functional Analysis with Antibodies
[0438] Antibodies of this invention can be used to purify the
polypeptides of this invention and to identify biological
equivalent polypeptide and/or polynucleotides. They also can be
used to identify agents that modify the function of the
polypeptides of this invention. These antibodies include polyclonal
antisera, monoclonal antibodies, and various reagents derived from
these preparations that are familiar to those practiced in the art
and described above.
[0439] Antibodies that neutralize the activities of proteins
encoded by identified genes can also be used in vivo and in vitro
to demonstrate function by adding such neutralizing antibodies into
in vivo and in vitro test systems. They also are useful as
pharmaceutical agents to modulate the activity of polypeptides of
the invention.
[0440] Various antibody preparations can also be used in analytical
methods such as ELISA assays or Western blots to demonstrate the
expression of proteins encoded by the identified genes by test
cells in vitro or in vivo. Fragments of such proteins generated by
protease degradation during metabolism can also be identified by
using appropriate polyclonal antisera with samples derived from
experimental samples.
[0441] The antibodies of the invention may be used for vaccination
or to boost vaccination, alone or in combination with peptides or
protein-based vaccines or dendritic-cell based vaccines.
Compositions
[0442] Compositions are further provided. The compositions comprise
a carrier and one or more of an isolated polypeptide of the
invention, an isolated polynucleotide of the invention, a vector of
the invention, an isolated host cell of the invention, a small
molecule or an antibody of the invention. The carriers can be one
or more of a solid support or a pharmaceutically acceptable
carrier. The compositions can further comprise an adjuvant or other
components suitable for administrations as vaccines. In one aspect,
the compositions are formulated with one or more pharmaceutically
acceptable excipients, diluents, carriers and/or adjuvants. In
addition, embodiments of the compositions of the present invention
include one or more of an isolated polypeptide of the invention, an
isolated polynucleotide of the invention, a vector of the
invention, a small molecule, an isolated host cell of the
invention, or an antibody of the invention, formulated with one or
more pharmaceutically acceptable auxiliary substances.
[0443] For oral preparations, any one or more of an isolated or
recombinant polypeptide as described herein, an isolated or
recombinant polynucleotide as described herein, a vector as
described herein, an isolated host cell as described herein, a
small molecule or an antibody as described herein can be used alone
or in pharmaceutical formulations of the invention comprising, or
consisting essentially of, the compound in combination with
appropriate additives to make tablets, powders, granules or
capsules, for example, with conventional additives, such as
lactose, mannitol, corn starch or potato starch; with binders, such
as crystalline cellulose, cellulose derivatives, acacia, corn
starch or gelatins; with disintegrators, such as corn starch,
potato starch or sodium carboxymethylcellulose; with lubricants,
such as talc or magnesium stearate; and if desired, with diluents,
buffering agents, moistening agents, preservatives and flavoring
agents. Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring.
[0444] Pharmaceutical formulations and unit dose forms suitable for
oral administration are particularly useful in the treatment of
chronic conditions, infections, and therapies in which the patient
self-administers the drug. In one aspect, the formulation is
specific for pediatric administration.
[0445] The invention provides pharmaceutical formulations in which
the one or more of an isolated polypeptide of the invention, an
isolated polynucleotide of the invention, a vector of the
invention, an isolated host cell of the invention, or an antibody
of the invention can be formulated into preparations for injection
in accordance with the invention by dissolving, suspending or
emulsifying them in an aqueous or nonaqueous solvent, such as
vegetable or other similar oils, synthetic aliphatic acid
glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers
and preservatives or other antimicrobial agents. A non-limiting
example of such is a antimicrobial agent such as other vaccine
components such as surface antigens, e.g. an OMP P5, rsPilA, OMP
26, OMP P2, or Type IV Pilin protein (see Jurcisek and Bakaletz
(2007) J. of Bacteriology 189(10):3868-3875 and Murphy, T F,
Bakaletz, L O and Smeesters, P R (2009) The Pediatric Infectious
Disease Journal, 28:S121-S126) and antibacterial agents. For
intravenous administration, suitable carriers include physiological
saline, bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany,
N.J.), or phosphate buffered saline (PBS). In all cases, a
composition for parenteral administration must be sterile and
should be fluid to the extent that easy syringability exists.
[0446] Aerosol formulations provided by the invention can be
administered via inhalation and can be propellant or non-propellant
based. For example, embodiments of the pharmaceutical formulations
of the invention comprise a compound of the invention formulated
into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like. For
administration by inhalation, the compounds can be delivered in the
form of an aerosol spray from a pressurized container or dispenser
which contains a suitable propellant, e.g., a gas such as carbon
dioxide, or a nebulizer. A non-limiting example of a non-propellant
is a pump spray that is ejected from a closed container by means of
mechanical force (i.e., pushing down a piston with one's finger or
by compression of the container, such as by a compressive force
applied to the container wall or an elastic force exerted by the
wall itself (e.g. by an elastic bladder)).
[0447] Suppositories of the invention can be prepared by mixing a
compound of the invention with any of a variety of bases such as
emulsifying bases or water-soluble bases. Embodiments of this
pharmaceutical formulation of a compound of the invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0448] Unit dosage forms for oral or rectal administration, such as
syrups, elixirs, and suspensions, may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more compounds of the invention. Similarly, unit
dosage forms for injection or intravenous administration may
comprise a compound of the invention in a composition as a solution
in sterile water, normal saline or another pharmaceutically
acceptable carrier.
[0449] Embodiments of the pharmaceutical formulations of the
invention include those in which one or more of an isolated
polypeptide of the invention, an isolated polynucleotide of the
invention, a vector of the invention, a small molecule for use in
the invention, an isolated host cell of the invention, or an
antibody of the invention is formulated in an injectable
composition. Injectable pharmaceutical formulations of the
invention are prepared as liquid solutions or suspensions; or as
solid forms suitable for solution in, or suspension in, liquid
vehicles prior to injection. The preparation may also be emulsified
or the active ingredient encapsulated in liposome vehicles in
accordance with other embodiments of the pharmaceutical
formulations of the invention.
[0450] In an embodiment, one or more of an isolated polypeptide of
the invention, an isolated polynucleotide of the invention, a
vector of the invention, an isolated host cell of the invention, or
an antibody of the invention is formulated for delivery by a
continuous delivery system. The term "continuous delivery system"
is used interchangeably herein with "controlled delivery system"
and encompasses continuous (e.g., controlled) delivery devices
(e.g., pumps) in combination with catheters, injection devices, and
the like, a wide variety of which are known in the art.
[0451] Mechanical or electromechanical infusion pumps can also be
suitable for use with the present disclosure. Examples of such
devices include those described in, for example, U.S. Pat. Nos.
4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589;
5,643,207; 6,198,966; and the like. In general, delivery of a
compound of the invention can be accomplished using any of a
variety of refillable, pump systems. Pumps provide consistent,
controlled release over time. In some embodiments, a compound of
the invention is in a liquid formulation in a drug-impermeable
reservoir, and is delivered in a continuous fashion to the
individual.
[0452] In one embodiment, the drug delivery system is an at least
partially implantable device. The implantable device can be
implanted at any suitable implantation site using methods and
devices well known in the art. An implantation site is a site
within the body of a subject at which a drug delivery device is
introduced and positioned. Implantation sites include, but are not
necessarily limited to, a subdermal, subcutaneous, intramuscular,
or other suitable site within a subject's body. Subcutaneous
implantation sites are used in some embodiments because of
convenience in implantation and removal of the drug delivery
device.
[0453] Drug release devices suitable for use in the disclosure may
be based on any of a variety of modes of operation. For example,
the drug release device can be based upon a diffusive system, a
convective system, or an erodible system (e.g., an erosion-based
system). For example, the drug release device can be an
electrochemical pump, osmotic pump, an electroosmotic pump, a vapor
pressure pump, or osmotic bursting matrix, e.g., where the drug is
incorporated into a polymer and the polymer provides for release of
drug formulation concomitant with degradation of a drug-impregnated
polymeric material (e.g., a biodegradable, drug-impregnated
polymeric material). In other embodiments, the drug release device
is based upon an electrodiffusion system, an electrolytic pump, an
effervescent pump, a piezoelectric pump, a hydrolytic system,
etc.
[0454] Drug release devices based upon a mechanical or
electromechanical infusion pump can also be suitable for use with
the present disclosure. Examples of such devices include those
described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019;
4,487,603; 4,360,019; 4,725,852, and the like. In general, a
subject treatment method can be accomplished using any of a variety
of refillable, non-exchangeable pump systems. Pumps and other
convective systems are generally preferred due to their generally
more consistent, controlled release over time. Osmotic pumps are
used in some embodiments due to their combined advantages of more
consistent controlled release and relatively small size (see, e.g.,
PCT published application no. WO 97/27840 and U.S. Pat. Nos.
5,985,305 and 5,728,396). Exemplary osmotically-driven devices
suitable for use in the disclosure include, but are not necessarily
limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770;
3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880;
4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139;
4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614;
5,137,727; 5,234,692; 5,234,693; 5.728,396; and the like. A further
exemplary device that can be adapted for the present disclosure is
the Synchromed infusion pump (Medtronic).
[0455] In some embodiments, the drug delivery device is an
implantable device. The drug delivery device can be implanted at
any suitable implantation site using methods and devices well known
in the art. As noted herein, an implantation site is a site within
the body of a subject at which a drug delivery device is introduced
and positioned. Implantation sites include, but are not necessarily
limited to a subdermal, subcutaneous, intramuscular, or other
suitable site within a subject's body.
[0456] Suitable excipient vehicles for a compound of the invention
are, for example, water, saline, dextrose, glycerol, ethanol, or
the like, and combinations thereof. In addition, if desired, the
vehicle may contain minor amounts of auxiliary substances such as
wetting or emulsifying agents or pH buffering agents. Methods of
preparing such dosage forms are known, or will be apparent upon
consideration of this disclosure, to those skilled in the art. See,
e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 17th edition, 1985. The composition or formulation to
be administered will, in any event, contain a quantity of the
compound adequate to achieve the desired state in the subject being
treated.
[0457] Compositions of the present invention include those that
comprise a sustained-release or controlled release matrix. In
addition, embodiments of the present invention can be used in
conjunction with other treatments that use sustained-release
formulations. As used herein, a sustained-release matrix is a
matrix made of materials, usually polymers, which are degradable by
enzymatic or acid-based hydrolysis or by dissolution. Once inserted
into the body, the matrix is acted upon by enzymes and body fluids.
A sustained-release matrix desirably is chosen from biocompatible
materials such as liposomes, polylactides (polylactic acid),
polyglycolide (polymer of glycolic acid), polylactide co-glycolide
(copolymers of lactic acid and glycolic acid), polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen,
chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids,
polysaccharides, nucleic acids, polyamino acids, amino acids such
as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene, polyvinylpyrrolidone and silicone. Illustrative
biodegradable matrices include a polylactide matrix, a
polyglycolide matrix, and a polylactide co-glycolide (co-polymers
of lactic acid and glycolic acid) matrix.
[0458] In another embodiment, the interfering agent (as well as
combination compositions) is delivered in a controlled release
system. For example, a compound of the invention may be
administered using intravenous infusion, an implantable osmotic
pump, a transdermal patch, liposomes, or other modes of
administration. In one embodiment, a pump may be used (Sefton
(1987) CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al. (1980)
Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321:574). In
another embodiment, polymeric materials are used. In yet another
embodiment a controlled release system is placed in proximity of
the therapeutic target, i.e., the liver, thus requiring only a
fraction of the systemic dose. In yet another embodiment, a
controlled release system is placed in proximity of the therapeutic
target, thus requiring only a fraction of the systemic. Other
controlled release systems are discussed in the review by Langer
(1990) Science 249:1527-1533.
[0459] In another embodiment, the compositions of the present
invention (as well as combination compositions separately or
together) include those formed by impregnation of an inhibiting
agent described herein into absorptive materials, such as sutures,
bandages, and gauze, or coated onto the surface of solid phase
materials, such as surgical staples, zippers and catheters to
deliver the compositions. Other delivery systems of this type will
be readily apparent to those skilled in the art in view of the
instant disclosure.
[0460] The present invention provides methods and compositions for
the administration of a one or more of an interfering agent to a
host (e.g., a human) for the treatment of a microbial infection. In
various embodiments, these methods of the invention span almost any
available method and route suitable for drug delivery, including in
vivo and ex vivo methods, as well as systemic and localized routes
of administration.
Screening Assays
[0461] The present invention provides methods for screening for
equivalent agents, such as equivalent monoclonal antibodies to a
polyclonal antibody as described herein and various agents that
modulate the activity of the active agents and pharmaceutical
compositions of the invention or the function of a polypeptide or
peptide product encoded by the polynucleotide of this invention.
For the purposes of this invention, an "agent" is intended to
include, but not be limited to a biological or chemical compound
such as a simple or complex organic or inorganic molecule, a
peptide, a protein (e.g. antibody), a polynucleotide (e.g.
anti-sense) or a ribozyme. A vast array of compounds can be
synthesized, for example polymers, such as polypeptides and
polynucleotides, and synthetic organic compounds based on various
core structures, and these are also included in the term "agent."
In addition, various natural sources can provide compounds for
screening, such as plant or animal extracts, and the like. It
should be understood, although not always explicitly stated that
the agent is used alone or in combination with another agent,
having the same or different biological activity as the agents
identified by the inventive screen.
[0462] One embodiment is a method for screening agents capable of
interacting with, binding to, or inhibiting the DNA-DNABII (e.g.,
IHF) interaction. The present invention provides in FIG. 6B the
three-dimensional structure of the microbial DNA and IHF.
Accordingly, the disclosure permits the use of virtual design
techniques, also known as computer-aided, in silico design or
modeling, to design, select, and synthesize agents capable of
interacting with, binding to, or inhibiting the DNA-DNABII (e.g.,
IHF) interaction. In turn, the candidate agents may be effective in
the treatment of biofilms and associated diseases or conditions
(medical, industrial or veterinary) as described herein. Thus, the
present disclosure also provides agents identified or designed by
the in silico methods.
[0463] Three-dimensional structure of a IHF-DNA complex is
illustrated in FIG. 6B and a representative structure, with X, Y
and Z coordinates, are provided in Protein Data Bank Accession
Number: 1IHF, with relevant details provided in Rice et al. Cell
87:1295-1306 (1996). The three-dimensional structure of the IHF
protein in the IHF-DNA complex can be used for the screening
method. A suitable agent is one that can be positioned relative to
the IHF protein structure in the IHF-DNA complex with interactions
at least one, or alternatively two, or three, or four, or five, or
six, or seven, or eight, or nine, or at least ten of the amino acid
residues that are identified to be involved in interacting with
DNA.
[0464] FIG. 6A illustrates the amino acid residues involved in
IHF-DNA interaction, using the E. coli IHF sequence (SEQ ID NO: 42)
as an example. Such amino acid residues, indicated by the lower
level of arrows in FIG. 6A, are further described below, indicated
with bold and underlined letters. Namely, with the E. coli IHF, the
amino acids involved in DNA binding are T4, K5, A6, E28, Q43, K45,
S47, G48, N51, R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or
Q85.
TABLE-US-00004 (SEQ ID NO: 42) MALTKAEMSE YLFDKLGLSK RDAKELVELF
FEEIRRALEN GEQVKLSGFG NFDLRDKNQR PGRNPKTGED IPITARRVVT FRPGQKLKSR
VENASPKDE
[0465] Thus, one embodiment of the present disclosure provides a
computer-implemented method for identifying an agent that inhibits,
competes or titrates the binding of a DNABII polypeptide or protein
to a microbial DNA, that inhibits, prevents or breaks down a
microbial biofilm, that inhibits, prevents or breaks down a biofilm
in a subject, or that inhibits, prevents or treats a microbial
infection that produces a biofilm in a subject, comprising
positioning a three-dimensional structure of a candidate agent
against a three-dimensional structure of an integration host factor
(IHF) protein, wherein the three-dimensional structure of the IHF
protein is based on X, Y and Z atomic structure coordinates
determined from a crystalline form of an IHF and DNA complex,
wherein interaction of the agent with the IHF at two or more IHF
amino acids selected from T4, K5, A6, E28, Q43, K45, S47, G48, N51,
R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or Q85 as
represented in SEQ ID NO: 42, or the equivalent of each, identifies
that the agent inhibits, competes or titrates the binding of a
DNABII polypeptide or protein to a microbial DNA, inhibits,
prevents or breaks down a microbial biofilm, inhibits, prevents or
breaks down a biofilm, or inhibits, prevents or treats a microbial
infection that produces a biofilm.
[0466] In one aspect, a candidate agent interacts with the IHF
protein at least one, or two, or three of amino acids 63, 64, 65,
or 66. In one aspect, a candidate agent interacts with the IHF
protein at least one, or two, or three of R63, N64, P65, K66. In
another aspect, a candidate agent interacts with the IHF protein at
least at one of 63 or 66. In another aspect, a candidate agent
interacts with the IHF protein at least at one of R63 or K66.
[0467] It would be appreciated in the art that the exact locations
and amino acid residues vary depending on the IHF sequence. One of
the skill in the art, however, can readily identify such locations
and amino acid residues based on the sequences. For instance, an
IHF sequence can be aligned with the IHF sequence of E. coli (SEQ
ID NO: 42), as illustrated in Table 9, to reveal those that
correspond to the amino acids in E. coli IHF that interact with
DNA. Likewise, the three-dimensional structure of such an IHF
sequence in an IHF-DNA complex can be used for the screening.
[0468] In addition to the computer-implemented methods as provided
herein, the present disclosure also provides custom computer system
that includes, e.g., processor, memory and/or program, for
performing the methods, as well as a computer readable medium, such
as a non-transitory computer readable medium that stores suitable
computer program or code for carrying out the methods.
[0469] Accordingly, another embodiment provides a custom computing
apparatus comprising:
[0470] at least one processor;
[0471] a memory coupled to the at least one processor;
[0472] a storage medium in communication with the memory and the at
least one processor, the storage medium containing a set of
processor executable instructions that, when executed by the
processor configure the custom computing apparatus to identify an
agent that inhibits, competes or titrates the binding of a DNABII
polypeptide or protein to a microbial DNA, that inhibits, prevents
or breaks down a microbial biofilm, that inhibits, prevents or
breaks down a biofilm in a subject, or that inhibits, prevents or
treats a microbial infection that produces a biofilm in a subject,
wherein the configuration comprises:
[0473] positioning a three-dimensional structure of a candidate
agent against a three-dimensional structure of an integration host
factor (IHF) protein, wherein the three-dimensional structure of
the IHF protein is based on X, Y and Z atomic structure coordinates
determined from a crystalline form of an IHF and DNA complex,
wherein interaction of the agent with the IHF at two or more IHF
amino acids selected from T4, K5, A6. E28, Q43, K45, S47. G48. N51,
R55, K57, R60, R63, N64, P65, K66, R76, T80, R82 or Q85 as
represented in SEQ ID NO: 42, or the equivalent of each, identifies
that the agent inhibits, competes or titrates the binding of a
DNABII polypeptide or protein to a microbial DNA, inhibits,
prevents or breaks down a microbial biofilm, inhibits, prevents or
breaks down a biofilm, or inhibits, prevents or treats a microbial
infection that produces a biofilm.
[0474] Yet another embodiment provides a non-transitory computer
medium comprising a set of processor executable instructions that,
when executed by a processor, identifying an agent that inhibits,
competes or titrates the binding of a DNABII polypeptide or protein
to a microbial DNA, that inhibits, prevents or breaks down a
microbial biofilm, that inhibits, prevents or breaks down a biofilm
in a subject, or that inhibits, prevents or treats a microbial
infection that produces a biofilm in a subject, comprising
positioning a three-dimensional structure of a candidate agent
against a three-dimensional structure of an integration host factor
(IHF) protein, wherein the three-dimensional structure of the IHF
protein is based on X, Y and Z atomic structure coordinates
determined from a crystalline form of an IHF and DNA complex,
wherein interaction of the agent with the IHF at two or more IHF
amino acids selected from T4, K5, A6, E28, Q43, K45, S47, G48, N51,
R55, K57, R60, R63, N64, P65, K66, R76, T80. R82 or Q85 as
represented in SEQ ID NO: 42, or the equivalent of each, identifies
that the agent inhibits, competes or titrates the binding of a
DNABII polypeptide or protein to a microbial DNA, inhibits,
prevents or breaks down a microbial biofilm, inhibits, prevents or
breaks down a biofilm, or inhibits, prevents or treats a microbial
infection that produces a biofilm.
[0475] Methods of in silico molecule or drug designs are well known
in the art, see generally Kapetanovic (2008) Chem Biol. Interact.,
171(2):165-76. Briefly, the atomic coordinates of the
three-dimensional structure are input into a computer so that
images of the structure and various parameters are shown on the
display. The design typically involves positioning a
three-dimensional structure to the three-dimensional structure of
the target molecule. The positioning can be controlled by the user
with assistance from a computer's graphic interface, and can be
further guided by a computer algorithm looking for potential good
matches. Positioning also involves moving either or both of the
three-dimensional structures around at any dimension.
[0476] Then, the resultant data are input into a virtual compound
and/or agent library. Since a virtual library is contained in a
virtual screening software such as DOCK-4 (Kuntz, UCSF), the
above-described data may be input into such a software. Candidate
agents may be searched for, using a three-dimensional structure
database of virtual or non-virtual drug candidate compounds, such
as MDDR (Prous Science, Spain).
[0477] A candidate agent is found to be able to bind to DNA and/or
DNABII protein if a desired interaction between the candidate agent
and either or both is found. The interaction can be quantitative,
e.g, strength of interaction and/or number of interaction sites, or
qualitative, e.g., interaction or lack of interaction. The output
of the method, accordingly, can be quantitative or qualitative. In
one aspect, therefore, the present disclosure also provides a
method for identifying an agent that does not inhibit the
interaction or alternatively, strengthens the interation between
the DNA and protein.
[0478] The potential inhibitory or binding effect (i.e.,
interaction or association) of an agent such as a small molecule
compound may be analyzed prior to its actual synthesis and testing
by the use of computer modeling techniques. If the theoretical
structure of the given compound suggests insufficient interaction
and association between it and microbial DNA in the biofilm and/or
DNABII protein, synthesis and testing of the agent can be obviated.
However, if computer modeling indicates a strong interaction, the
agent can then be synthesized and tested for its ability to bind to
or inhibit the interaction using various methods such as in vitro
or in vivo experiments. Methods of testing an agent's ability to
inhibit or titrate a biofilm, alone or in connection with another
agent, are disclosed herein. In this manner, synthesis of
inoperative agents and compounds can be avoided.
[0479] One skilled in the art may use any of several methods to
screen chemical or biological entities or fragments for their
ability to associate with DNABII or microbial DNA and more
particularly with the specific binding sites. Selected fragments or
chemical entities may then be positioned in a variety of
orientations, or docked, within an individual binding site of DNA
or DNABII polypeptide. Docking may be accomplished using software
such as QUANTA, SYBYL, followed by energy minimization and
molecular dynamics with standard molecular mechanics forcefields,
such as CHARMM and AMBER.
[0480] Commercial computer programs are also available for in
silico design. Examples include, without limitation, GRID (Oxford
University, Oxford, UK). MCSS (Molecular Simulations, Burlington,
Mass.), AUTODOCK (Scripps Research Institute, La Jolla, Calif.),
DOCK (University of California, San Francisco, Calif.), GLIDE
(Schrodinger Inc.), FlexX (Tripos Inc.) and GOLD (Cambridge
Crystallographic Data Centre).
[0481] Once an agent or compound has been designed or selected by
the above methods, the efficiency with which that agent or compound
may bind to each other can be tested and optimized by computational
evaluation. For example, an effective DNABII fragment or may
preferably demonstrate a relatively small difference in energy
between its bound and free states (i.e., a small deformation energy
of binding).
[0482] A compound designed or selected can be further
computationally optimized so that in its bound state it would
preferably lack repulsive electrostatic interaction with the target
protein. Such non-complementary (e.g., electrostatic) interactions
include repulsive charge-charge, dipole-dipole, and charge-dipole
interactions. Specifically, the sum of all electrostatic
interactions between the agent and DNABII and/or microbial DNA in
the biofilm when the agent or compound is bound to either agent,
preferably make a neutral or favorable contribution to the enthalpy
of binding.
[0483] Computer softwares are also available in the art to evaluate
compound deformation energy and electrostatic interaction. Examples
include, without limitation, Gaussian 92 [Gaussian, Inc.,
Pittsburgh, Pa.]; AMBER [University of California at San
Francisco]; QUANTA/CHARMM [Molecular Simulations, Inc., Burlington,
Mass.]; and Insight I/Discover [Biosysm Technologies Inc., San
Diego, Calif.].
[0484] Once an binding agent has been optimally selected or
designed, as described above, substitutions may then be made in
some of its atoms or side groups in order to improve or modify its
binding properties. Generally, initial substitutions are
conservative, i.e., the replacement group will have approximately
the same size, shape, hydrophobicity and charge as the original
group. It should, of course, be understood that components known in
the art to alter conformation should be avoided. Such substituted
chemical compounds may then be analyzed for efficiency of fit to
the DNABII protein and/or microbial DNA in the biofilm by the same
computer methods described in detail, above.
[0485] One preferred embodiment is a method for screening small
molecules capable of interacting with the protein or polynucleotide
of the invention. For the purpose of this invention, "small
molecules" are molecules having low molecular weights (MW) that
are, in one embodiment, capable of binding to a protein of interest
thereby altering the function of the protein. Preferably, the MW of
a small molecule is no more than 1,000. Methods for screening small
molecules capable of altering protein function are known in the
art. For example, a miniaturized arrayed assay for detecting small
molecule-protein interactions in cells is discussed by You et al.
(1997) Chem. Biol. 4:961-968.
[0486] To practice the screening method in vitro, suitable cell
culture or tissue infected with the microbial to be treated are
first provided. The cells are cultured under conditions
(temperature, growth or culture medium and gas (CO.sub.2)) and for
an appropriate amount of time to attain exponential proliferation
without density dependent constraints. It also is desirable to
maintain an additional separate cell culture that is not infected
as a control.
[0487] As is apparent to one of skill in the art, suitable cells
can be cultured in micro-titer plates and several agents can be
assayed at the same time by noting genotypic changes, phenotypic
changes or a reduction in microbial titer.
[0488] When the agent is a composition other than a DNA or RNA,
such as a small molecule as described above, the agent can be
directly added to the cell culture or added to culture medium for
addition. As is apparent to those skilled in the art, an
"effective" a mount must be added which can be empirically
determined.
[0489] When the agent is an antibody or antigen binding fragment,
the agent can be contacted or incubated with the target antigen and
polyclonal antibody as described herein under conditions to perform
a competitive ELISA. Such methods are known to the skilled
artisan.
[0490] The assays also can be performed in a subject. When the
subject is an animal such as a rat, chinchilla, mouse or simian,
the method provides a convenient animal model system that can be
used prior to clinical testing of an agent in a human patient. In
this system, a candidate agent is a potential drug if symptoms of
the disease or microbial infection is reduced or eliminated, each
as compared to untreated, animal having the same infection. It also
can be useful to have a separate negative control group of cells or
animals that are healthy and not treated, which provides a basis
for comparison.
[0491] The agents and compositions can be used in the manufacture
of medicaments and for the treatment of humans and other animals by
administration in accordance with conventional procedures, such as
an active ingredient in pharmaceutical compositions.
Combination Therapy
[0492] The compositions and related methods of the present
invention may be used in combination with the administration of
other therapies. These include, but are not limited to, the
administration of DNase enzymes, antibiotics, antimicrobials, or
other antibodies.
[0493] In some embodiments, the methods and compositions include a
deoxyribonuclease (DNase) enzyme that acts synergistically with the
anti-DNABII antibody. A DNase is any enzyme that catalyzes the
cleavage of phosphodiester linkages in the DNA backbone. Three
non-limiting examples of DNase enzymes that are known to target not
only cruciform structures, but also a variety of secondary
structure of DNA include DNAse I, T4 EndoVII and T7 Endo I. In
certain embodiments, the effective amount of anti-DNABII antibody
needed to destabilize the biofilm is reduced when combined with a
DNase. When administered in vitro, the DNase can be added directly
to the assay or in a suitable buffer known to stabilize the enzyme.
The effective Unit dose of DNase and the assay conditions may vary,
and can be optimized according to procedures known in the art.
[0494] In other embodiments, the methods and compositions can be
combined with antibiotics and/or antimicrobials. Antimicrobials are
substances that kill or inhibit the growth of microorganisms such
as bacteria, fungi, or protozoans. Although biofilms are generally
resistant to the actions of antibiotics, compositions and methods
described herein can be used to sensitize the infection involving a
biofilm to traditional therapeutic methods for treating infections.
In other embodiments, the use of antibiotics or antimicrobials in
combination with methods and compositions described herein allow
for the reduction of the effective amount of the antimicrobial
and/or biofilm reducing agent. Some non-limiting examples of
antimicrobials and antibiotics useful in combination with methods
of the current invention include amoxicillin,
amoxicillin-clavulanate, cefdinir, azithromycin, and
sulfamethoxazole-trimethoprim. The therapeutically effective dose
of the antimicrobial and/or antibiotic in combination with the
biofilm reducing agent can be readily determined by traditional
methods. In some embodiments the dose of the antimicrobial agent in
combination with the biofilm reducing agent is the average
effective dose which has been shown to be effective in other
bacterial infections, for example, bacterial infections wherein the
etiology of the infection does not include a biofilm. In other
embodiments, the dose is 0.1, 0.15, 0.2, 0.25, 0.30, 0.35, 0.40,
0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.8, 0.85, 0.9, 0.95,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0 or 5
times the average effective dose. The antibiotic or antimicrobial
can be added prior to, concurrent with, or subsequent to the
addition of the anti-DNABII antibody.
[0495] In other embodiments, the methods and compositions can be
combined with antibodies that treat the bacterial infection. One
example of an antibody useful in combination with the methods and
compositions described herein is an antibody directed against an
unrelated outer membrane protein (i.e. OMP P5). Treatment with this
antibody alone does not debulk a biofilm in vitro. Combined therapy
with this antibody and a biofilm reducing agent results in a
greater effect than that which could be achieved by either reagent
used alone at the same concentration. Other antibodies that may
produce a synergistic effect when combined with a biofilm reducing
agent or methods to reduce a biofilm include anti-rsPilA,
anti-OMP26, anti-OMP P2, and anti-whole OMP preparations.
[0496] The compositions and methods described herein can be used to
sensitize the bacterial infection involving a biofilm to common
therapeutic modalities effective in treating bacterial infections
without a biofilm but are otherwise ineffective in treating
bacterial infections involving a biofilm. In other embodiments, the
compositions and methods described herein can be used in
combination with therapeutic modalities that are effective in
treating bacterial infections involving a biofilm, but the
combination of such additional therapy and biofilm reducing agent
or method produces a synergistic effect such that the effective
dose of either the biofilm reducing agent or the additional
therapeutic agent can be reduced. In other instances the
combination of such additional therapy and biofilm reducing agent
or method produces a synergistic effect such that the treatment is
enhanced. An enhancement of treatment can be evidenced by a shorter
amount of time required to treat the infection.
[0497] The additional therapeutic treatment can be added prior to,
concurrent with, or subsequent to methods or compositions used to
reduce the biofilm, and can be contained within the same
formulation or as a separate formulation.
Kits
[0498] Kits containing the agents and instructions necessary to
perform the in vitro and in vivo methods as described herein also
are claimed. Accordingly, the invention provides kits for
performing these methods which may include an interfering of this
invention as well as instructions for carrying out the methods of
this invention such as collecting tissue and/or performing the
screen, and/or analyzing the results, and/or administration of an
effective amount of an interfering agent as defined herein. These
can be used alone or in combination with other suitable
antimicrobial agents.
[0499] For example, a kit can comprise, or alternatively consist
essentially of, or yet further consist of any one or more agent of
the group of an isolated or recombinant integration host factor
(IHF) polypeptide or a fragment or an equivalent of each thereof;
an isolated or recombinant protein polypeptide identified in Table
8, Table 9, Table 10 a DNA binding peptide identified in FIG. 6, or
a fragment or an equivalent of each thereof; an isolated or
recombinant polypeptide of SEQ ID NO. 1 through 33, or a fragment
or an equivalent of each thereof; an isolated or recombinant
C-terminal polypeptide of SEQ ID NO. 5 through 11, 28, 29 or those
identified in Table 8, Table 10 or a fragment or an equivalent of
each thereof; a polypeptide that competes with an integration host
factor on binding to a microbial DNA; a four-way junction
polynucleotide resembling a Holliday junction, a 3 way junction
polynucleotide resembling a replication fork, a polynucleotide that
has inherent flexibility or bent polynucleotide; an isolated or
recombinant polynucleotide encoding any one of the above noted
polypeptides; an antibody that specifically recognizes or binds any
one of the above noted polypeptides, or an equivalent or fragment
thereof; or a small molecule that competes with the binding of a
DNABII protein or polypeptide to a microbial DNA, and instructions
for use. The kit can further comprising one or more of an adjuvant,
an antigenic peptide or an antimicrobial. Examples of carriers
include a liquid carrier, a pharmaceutically acceptable carrier, a
solid phase carrier, a pharmaceutically acceptable carrier, a
pharmaceutically acceptable polymer, a liposome, a micelle, an
implant, a stent, a paste, a gel, a dental implant, or a medical
implant.
[0500] The following examples are intended to illustrate, and not
limit, the inventions disclosed herein.
EXPERIMENTAL
Experiment No. 1
[0501] IHF antibodies, proteins and polypeptides were a generous
gift from Nash. The methods to produce them are well known to the
skilled artisan, e.g., as described in Granston and Nash, (1993) J.
Mol. Biol., 234:45-59; Nash et al., (1987) Journal of Bacteriology,
169(9):4124-4127; and Rice et al., (1996) Cell, 87:1295-1306.
Briefly, to overproduce IHF-.alpha., the himA gene was inserted
downstream from the P.sub.L promoter in the bacterial plasmid
pAD284. Transformants of strain K5607, a lambda lysogen of strain
C600himA42 that had received the desired plasmid, were identified
by screening ampicillin-resistant transformants for the ability to
grow bacteriophage Mu (13). DNA was prepared from himA.sup.+
transformants according to standard DNA isolation techniques, and
the orientation of the himA gene was determined by restriction
enzyme cleavage. Plasmid pP.sub.LhimA-1, which has the himA gene in
the proper orientation for expression by the P.sup.L promoter, was
transformed into strain N5271, which contains a cryptic lambda
prophage expressing the cI857 thermoinducible repressor, to yield
strain K5770.
[0502] To overproduce IHF-.beta., plasmid pKT23-hip323, which
contains a fusion of the IHF-coding sequence to the bacteriophage
lambda P.sub.L promoter was used. pKT23-hip323 was introduced into
N5271 to give strain E443. To facilitate the selection of
pKT23-hip323 in the presence of another plasmid, changed its
selectable marker was changed from ampicillin resistance
(bla.sup.+) to chloramphenicol resistance (cat.sup.+). A
cat-containing fragment was isolated from plasmid pBR325 as
described by Flamm and Weisberg and was inserted into the unique
ScaI site in bla. The ligated DNA was introduced into strain E403,
which carries a hip mutation and which synthesizes
temperature-sensitive X repressor, and chloramphenicol-resistant
transformants were selected at low temperature. One such
transformant (E735) was hip.sup.+ and ampicillin sensitive; it
therefore appears to carry a bla cat.sup.+ derivative of
pKT23-hip323 (pE735).
[0503] To generate a strain that overproduces both subunits of IHF,
E735 was transformed with plasmid pP.sub.LhimA-1, selecting a
transformant (E738) that had become ampicillin resistant and had
retained chloramphenicol resistance. The generation of a second
strain that overproduces both subunits of IHF depended on the
construction of plasmid pP.sub.Lhip himA-5. which was made by
ligating blunted (SstII restriction enzyme site) containing the
pheT and himA genes into the pKT23-hip323 plasmid. This is
described in further detail in (Nash et al., (1987) J. of
Bacteriology 169(9): 4124-4127. himA.sup.+ transformants of strain
K5607 were identified by screening for HimA.sup.+, and the plasmid
DNA was analyzed by restriction digestion. In all cases where the
plasmid structure was obvious, two copies of himA had been ligated
as a tandem direct repeat into the vector. It is not known if the
presence of two copies of the himA gene on this plasmid is demanded
by the selection, but it should be recalled that a single copy of
the himA gene in plasmid pP.sub.LhimA-1 is sufficient to complement
a himA mutant. Plasmid pP.sub.LhiphimA-5 was used to transform
strain N5271 to yield strain K5746.
[0504] Cells were grown in shaking water bath at 31.degree. C. in
TBY medium (10 g of tryptone, 5 g of yeast extract, and 5 g of
sodium chloride per liter). At mid-log phase (optical density at
650 nm, ca. 0.6), the cells were shifted to a 42.degree. C. water
bath and shaking was continued. Typically, 300 ml of culture was
centrifuged and suspended in 0.6 to 0.9 ml of TEG (20 mM Tris
hydrochloride (pH 7.4), 1 mM sodium EDTA, 10% glycerol) containing
20 mM NaCl. The cells were disrupted with six 20-s bursts of
sonication, with 40 s between each burst. A portion of the sonic
extract was centrifuged in a Sorvall SS34 rotor for 20 min at
15,000 rpm. Samples of the sonic extract were analyzed by sodium
dodecyl sulfate (SDS) gel electrophoresis according to standard
molecular biology techniques.
[0505] Purification of IHF was done according to the following: A
3.6-liter batch of cells was induced for 3 h. All subsequent steps
were carried out at 0 to 4.degree. C. The cell pellet from
3.3-liters was suspended in 10 ml of TEG containing 20 mM NaCl to
give a total volume of 29 ml; this suspension was disrupted in two
batches, each receiving six bursts of 3 min of sonication separated
by 90-s intervals. The sonic extract was centrifuged for 20 min at
15,000 rpm, yielding 16.9 ml of clarified extract. A 10% (vol/vol)
solution (1.1 ml) of polymin P (BDH Chemicals Ltd.) was added
slowly to the clarified extract; after being stirred for 20 min,
the mixture was centrifuged for 30 min at 10,000 rpm. The resulting
pellet was suspended in 10 ml of TEG containing 500 mM NaCl; after
being stirred for 15 min, the mixture was centrifuged for 20 min at
12,000 rpm. The supernatant (10.3 ml) was adjusted to 50%
saturation by the addition of 3.2 g of ammonium sulfate, stirred
for 20 min, and centrifuged for 15 min at 15.000 rpm. The resulting
supernatant was adjusted to 70% saturation by the addition of 1.64
g of ammonium sulfate, stirred for 20 min, and centrifuged for 15
min at 15,000 rpm. The resulting pellet was suspended in 1 ml of TG
(50 mM Tris hydrochloride (pH 7.4) containing 10% glycerol) and
dialyzed against two changes of TG. The dialyzed material (2.0 ml)
was loaded onto a 1-ml column (0.5 by 5.8 cm) of phosphocellulose
(P11; Whatman, Inc.) that had been equilibrated with TG. The column
was washed with 3 ml of TG and developed with 20 ml of a linear
gradient (0 to 1.2 M) of KCl in TG. Fractions of 0.5 ml were
collected, stored at -20.degree. C., and assayed for IHF
activity.
[0506] Polyclonal anti-IHF was prepared as follows. Rabbits were
injected with 250 .mu.g of purified IHF with Freund's complete
adjuvant. Booster immunizations of 250.mu. of IHF with Freund's
incomplete adjuvant were given 1, 7, and 12 weeks later. As
determined by immunoblotting of IHF, sera collected 13 weeks after
the initial injection had a high titer of IHF-reactive material.
The animals were maintained for several years and, when necessary,
given further booster immunization in order to maintain a high
titer of anti-IHF in their sera. The antibody was not purified
further. Crude sera was stored at -70.degree. C.
[0507] Antibodies specific to each subunit were generated by using
synthetic polypeptides corresponding to the most C-terminal 20
amino acid residues of each subunit (SEQ ID Nos. 34 and 35), to
immunize rabbits according to Ditto et al., (1994) J. of
Bacteriology, 176(12):3738-3748.
Experiment 2
[0508] This experiment describes an in vitro model for reversal of
an established biofilm in 8-well chamber slide. The materials used
in this experiment were: Chocolate Agar, sBHI (BHI with 2 mg
heme/mL and 2 mg b-NAD/mL); 8-well Chamber slides (Nunc* Lab-Tek*
Fisher catalog #12-565-18); Sterile 0.9% saline; LIVE/DEAD BacLight
Bacterial Viability Kit (Fisher catalog #NC9439023) and
Formalin.
[0509] On day 1, NTHI was struck for isolation on chocolate agar.
It was then incubated for 20 hrs at 37.degree. C. and 5% CO.sub.2.
The next day, bacteria were suspended in 5 mL equilibrated
(37.degree. C., 5% CO.sub.2) sBHI and optical density was adjusted
to OD.sub.490=0.65 in sBHI. Bacteria was diluted 1:6 in
equilibrated sBHI (1 mL bacterial suspension+5 mL sBHI). Bacteria
was then incubated for 3 hours at 37.degree. C. in 5% CO.sub.2,
static (OD.sub.490 should be approx 0.65). Next, the bacteria was
again diluted 1:2500 in equilibrated sBHI and 200 mL of the
bacterial suspension was added to each well of the chamber slide.
For dilution, 10 .mu.L bacteria was added to 990 .mu.L sBHI in an
eppendorf tube and 8 .mu.L dilution was added to 192 .mu.L sBHI in
each chamber and incubated at 37.degree. C., 5% CO.sub.2,
static.
[0510] On the third day after 16 hours of incubation medium was
aspirated from chamber by aspirating medium from the corner of the
well so as not to disturb biofilm. Then 200 mL of equilibrated sBHI
was added to each chamber and incubated for 37.degree. C., 5%
CO.sub.2, static for 8 hours. After 8 hours, the medium was
aspirated and 200 mL equilibrated sBHI was added to each untreated
chamber; and 200 mL of interfering agent such as Rabbit
anti-rsPilA; diluted 1:50 in sBHI and 200 mL Naive rabbit serum (or
other appropriate serum control) diluted 1:50 in sBHI was added.
They were then incubate at 37.degree. C. and 5% CO.sub.2,
static.
[0511] On day 4, after approximately 16 hours of incubation,
aspirate sBHI was aspirated and the biofilm was washed twice with
200 mL sterile saline. The saline was aspirated and 200 mL
Live/Dead stain was added. Next, 3 mL component A plus 3 mL
component B in 1 mL sterile 10 mM phosphate buffered saline was
added. It was then incubated for 15 minutes at room temperature,
static, protected from light. Stain was aspirated and biofilm was
washed twice with 200 mL sterile saline. Saline was aspirated and
200 mL formalin was added to fix biofilm. It was then incubated 15
minutes at room temperature, static, protected from light. Formalin
was aspirated and biofilm was washed twice with 200 mL sterile
saline. Gasket was removed and coverslip were placed on slide:
coverslip were sealed with nail polish and dried prior to viewing
by confocal microscopy.
[0512] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Haemophilus influenzae which is prevalent in
sinusitis, bronchitis, otitis media and exacerbations of chronic
obstructive pulmonary disease (COPD). Untreated biofilm mass was
measured to be 4.24 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness
of 11.68 .mu.m. After treatment, the biofilm mass was reduced to
0.53 .mu.m/.mu.m.sup.2 with a mean thickness of 1.31 .mu.m. Thus,
this shows a 88.8% reduction in mean thickness and an 87.5%
reduction in biomass.
[0513] Polyclonal antisera directed against the E. coli IHF was
prepared in rabbits according to standard techniques using purified
Integration Host Factor (IHF). Experiment 1 describes the
expression and purification of IHF from E. coli.
[0514] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Streptococcus mutans which is prevalent in
initiation and progression of dental caries. Untreated biofilm mass
was measured to be 1.17 .mu.m.sup.3/.mu.m.sup.2 with a mean
thickness of 5.43 .mu.m. After treatment, the biofilm mass was
reduced to 0.1 .mu.m/.mu.m.sup.2 with a mean thickness of 0.47
.mu.m. Thus, this shows a 91.3% reduction in mean thickness and an
91.6% % reduction in biomass. In vitro biofilm assays were repeated
3 times, on separate days. The percent reduction in the max height,
average thickness, and biomass is depicted in Table 1 below.
TABLE-US-00005 TABLE 1 Assay 1 Assay 2 Assay 3 S. mutans Max height
(.mu.m) 56 24 41 Ave thickness (.mu.m) 65 65 67 biomass
(.mu.m.sup.3/.mu.m.sup.2) 37 58 66
[0515] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Staphylococcus aureus which is prevalent in
localized and diffuse skin infections, chronic rhinosinusitis and
nosocomial infections. Untreated biofilm mass was measured to be
0.3 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness of 2.2 .mu.m and
a biofilm height of 17.5 .mu.m. After treatment, the biofilm mass
was reduced to 0.3 .mu.m.sup.3/.mu.m with a mean thickness of 1.1
.mu.m and a biofilm height of 8 .mu.m. Thus, this shows a 48.8%
reduction in mean thickness and an 2.7% reduction in biomass (FIG.
7).
[0516] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Moraxella catarrhalis which is prevalent in
exacerbation of COPD and otitis media. Untreated biofilm mass was
measured to be 0.72 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness
of 1.48 .mu.m. After treatment, the biofilm mass was reduced to
0.13 .mu.m/.mu.m.sup.2 with a mean thickness of 0.65 .mu.m. Thus,
this shows a 55.8% reduction in mean thickness and an 82.1%
reduction in biomass. In vitro biofilm assays were repeated 3
times, on separate days. The percent reduction in the max height,
average thickness, and biomass is depicted in Table 2 below.
TABLE-US-00006 TABLE 2 Assay 1 Assay 2 Assay 3 M. catarrhalis Max
height (.mu.m) 61 33 36 Ave thickness (.mu.m) 92 34 84 biomass
(.mu.m.sup.3/.mu.m.sup.2) 92 35 92
[0517] Using this method and anti-IHF antibody. Applicants reduced
a biofilm produced by Streptococcus pneumoniae which is prevalent
in sinusitis, pneumonia and otitis media. Untreated biofilm mass
was measured to be 0.64 .mu.m.sup.3/.mu.m.sup.2 with a mean
thickness of 3.99 .mu.m. After treatment, the biofilm mass was
reduced to 0.14 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness of
0.82 .mu.m. Thus, this shows a 79.5% reduction in mean thickness
and an 78.6% reduction in biomass. In vitro biofilm assays were
repeated 3 times, on separate days. The percent reduction in the
max height, average thickness, and biomass is depicted in Table 3
below.
TABLE-US-00007 TABLE 3 Assay 1 Assay 2 Assay 3 S. pneumoniae Max
height (.mu.m) 49 51 44 Ave thickness (.mu.m) 25 64 79 biomass
(.mu.m.sup.3/.mu.m.sup.2) 51 61 79
[0518] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Pseudomonas aeruginosa which is prevalent in
cystic fibrosis, pneumonia, skin and soft tissue infections and on
medical devices. Untreated biofilm mass was measured to be 7.0
.mu.m.sup.3/.mu.m.sup.2 with a mean thickness of 25.70 .mu.m and a
biofilm height of 40 .mu.m. After treatment, the biofilm mass was
reduced to 3.4 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness of
10.3 .mu.m and a biofilm height of 27.5 .mu.m. Thus, this shows a
60.1% reduction in mean thickness and an 50.8% reduction in biomass
(FIG. 7).
[0519] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Neisseria gonorrhoeae which is in gonorrhea.
Untreated biofilm mass was measured to be 9.5
.mu.m.sup.3/.mu.m.sup.2 with a mean thickness of 22.02 .mu.m and a
biofilm height of 40 .mu.m. After treatment, the biofilm mass was
reduced to 0.8 .mu.m.sup.3/.mu.m.sup.2 with a mean thickness of 3.4
.mu.m and a biofilm height of 27.5 .mu.m. Thus, this shows a 84.5%
reduction in mean thickness and an 92.1% reduction in biomass (FIG.
7).
[0520] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Uropathogenic E. coli which is prevalent in
urinary tract infections. Untreated biofilm mass was measured to be
1.75 .mu.m/.mu.m.sup.2 with a mean thickness of 31.73 .mu.m. After
treatment, the biofilm mass was reduced to 0.75
.mu.m.sup.3/.mu.m.sup.2 with a mean thickness of 1.62 .mu.m. Thus,
this shows a 94.9% reduction in mean thickness and an 56.9%
reduction in biomass. In vitro biofilm assays were repeated 3
times, on separate days. The percent reduction in the max height,
average thickness, and biomass is depicted in Table 4 below.
TABLE-US-00008 TABLE 4 Assay 1 Assay 2 Assay 3 UPEC Max height
(.mu.m) 69 65 76 Ave thickness 97 33 95 (.mu.m) biomass 98 96 57
(.mu.m.sup.3/.mu.m.sup.2)
[0521] Using this method and anti-IHF antibody, Applicants reduced
a biofilm produced by Staphylococcus epidermidis which is prevalent
in infections of the skin. In vitro biofilm assays were repeated 3
times, on separate days. The percent reduction in the max height,
average thickness, and biomass is depicted in Table 5 below.
TABLE-US-00009 TABLE 5 Assay 1 Assay 2 Assay 3 S. epidermidis Max
height (.mu.m) 42 38 56 Ave thickness (.mu.m) 62 71 92 biomass
(.mu.m.sup.3/.mu.m.sup.2) 62 76 88
Experiment No. 3
[0522] Middle ear infection (or otitis media, OM) is a highly
prevalent disease worldwide, afflicting 50-330 million children
globally each year. The socioeconomic burden of OM is also great,
with cost estimates between $5-6 billion in the United States alone
annually. All three of the predominant bacterial pathogens of OM
are known to form biofilms both in vitro and in vivo and recently,
clinicians have come to appreciate that the chronicity and
recurrence of OM is due, at least in part, to the formation of
bacterial biofilms within the middle ear cavity. In one chinchilla
model of OM, juvenile chinchillas are first given a viral `cold`,
followed a week later by their being challenged intranasally with
an inoculum of viable bacteria. Similar to the human condition
wherein "my child has a cold and a week later gets an ear
infection" chinchillas will also develop a bacterial OM
approximately one week after a challenge, and while experiencing
the viral upper respiratory tract infection. Once bacteria gain
access to the middle ear (either via ascension of the Eustachian
tube or following direct challenge to the middle ear space), they
will form a robust biofilm. Applicants thus contemplate and indeed
have already used chinchilla models as reported herein to
demonstrate the protective efficacy of IHF immunization which
results in rapid resolution of existing biofilms. This model is
also useful for therapeutic approaches via either passive delivery
of anti-DNABII antibody or via delivery of a small molecule or
other agent known to bind to IHF or other DNABII family
members.
[0523] Because the chinchilla model is used for development and
pre-clinical testing of human vaccines, it is important to
establish meaningful immunological parallels with the human host,
particularly the child. Applicants have shown that effusions
recovered from children with AOM due to NTHI, and middle ear fluids
from chinchillas with experimental NTHI-induced OM, recognized
immunodominant regions of OMP P5 in a similar hierarchical manner
(see for e.g. Novotny L A, et al., (2000) Infect Immun.
68(4):2119-28; Novotny L A, et al., (2007) 9.sup.th International
Symposium on Recent Advances in Otitis Media; St. Pete Beach, Fla.;
Novotny L A, et al., (2002) Vaccine 20(29-30):3590-97). Applicants
have also shown that chinchillas with experimental OM, children
with natural OM, and adults with exacerbations of COPD, all
recognized peptides representing PilA in a highly analogous manner
(see for e.g. Adams L D, et al., (2007) 107th General Meeting,
American Society for Microbiology; 2007; Toronto, ON; Adams L D, et
al., (2007) 9th International Symposium on Recent Advances in
Otitis Media; St. Pete Beach, Fla.). Thus, chinchillas with
experimental OM and children with natural disease respond similarly
immunologically to at least two unrelated NTHI protein adhesins.
This parallel was put to the ultimate test recently, when the
chinchilla AV-NTHI superinfection model was used to conduct
pre-clinical efficacy testing of a novel 11-valent Protein
D-pneumococcal polysaccharide conjugate vaccine. Data obtained in
the chinchilla predicted an efficacy of 34% whereas, when tested in
children, the efficacy obtained against H. influenzae-induced OM
was 35.6% (see for e.g. Novotny L A, et al., (2006) Vaccine
24(22):4804-11 and Prymula R, et al., (2006) Lancet.
367(9512):740-8), thus lending strong support to the relevancy of
this model for the development and testing of OM vaccine
candidates.
[0524] Applicants have shown a dramatic reduction in the pre-formed
biofilm remaining within the middle ears of chinchillas after
receipt of 2 weekly TC immunizations with IHF delivered with a
mucosal/systemic adjuvant.
[0525] 48 adults were ordered from Rauscher's Chinchilla Ranch
(LaRue, Ohio) and were acclimated to the vivarium for 7-10 days
prior to the beginning of the study. Prior to TB [transbullar or
direct challenge into the middle ear cavity] challenge, baseline
otoscopy and tympanometry were performed as well as a limited
volume prebleed to collect serum. Chinchillas were anesthetized and
300 .mu.l of NTHI (strain #86-028NP) suspension containing
approximately 1000 cfu were introduced into the middle ear space
via transbullar challenge. Animals were allowed to recover, then
monitored daily for adverse reactions as per IACUC accepted animal
use protocols. Routine otoscopy and tympanometry were performed
every 2-3 days throughout the study.
[0526] Four days after challenge (day+4), chinchillas were
anesthetized and a CT scan was performed to visualize biofilms
present within the middle ear and to obtain pre-immunization
images. The surface of the pinnae were cleaned and hydrated by
wiping with a sterile gauze pad moistened with sterile pyrogen free
saline. After .about.2 minutes (to allow the pinnae to dry), 50
.mu.l of either the dmLT, IHF (purified E. coli IHF according to
Experiment 1)+dmLT, or IHF+rsPilA+dmLT solution will be added to
the pinna and gently massaged in. Three chinchillas per cohort were
sacrificed and tissues/samples collected (on days+4, and +11) to
begin to determine mechanism of action.
[0527] Eleven days after challenge (day+11), the secondary
immunization occurred as described above wherein the pinnae are
cleaned/hydrated and immunogens were topically administered. Three
chinchillas per cohort were sacrificed and tissues/samples
collected to begin to determine mechanism of action.
[0528] Eighteen days after challenge (day+18), chinchillas were
anesthetized and a CT scan were performed to identify any biofilms
present, as well as to compare to the pre-immunization CT scans.
The animals were then bled to collect serum and euthanized. The
bullae are dissected and any fluids present were aseptically
collected, the bullae were then be opened to visualize the inferior
bulla and any biofilm present. Images were collected and the bulla
were washed with 1 ml of sterile saline and re-imaged. The mucosa,
along with any biofilm present, were collected from the right bulla
and placed in a preweighed tube. These tissues were homogenized,
serially diluted, and plated to determine cfu NTHI/mg wet weight
tissue. The left bullae were filled with OCT compound and snap
frozen for histological analysis.
[0529] Images of the left and right middle ear cavities, with
resident biofilms, were scrambled and two images per animal were
compiled into a single file for ranking by blinded evaluators. The
relative amount of biomass remaining within the middle ear of each
animal was ranked on a 0 to 4+ scale by blinded reviewers using the
scale shown in Table 6 below. Titer ELISAs, cytokine arrays, and
Biacore were run on the serum as well as on the collected middle
ear effusions. The OCT embedded middle ears were sectioned and
stained either for basic morphology and architecture (H&E) or
for the presence of IHF using inmmunohistochemistry and/or
immunofluoresence.
TABLE-US-00010 TABLE 6 Score Criteria 0 No evidence of biomass. 1+
Biomass fills .ltoreq.25% of middle ear space. Junction of the bony
septa to inferior bulla is visible. 2+ Biomass fills >25% to
.ltoreq.50% of middle ear space. Unable to visualize where the bony
septa meet the inferior bulla. 3+ Biomass fills >50% to
.ltoreq.75% of middle ear space. Biomass covers >50% of the
length of bony septa. 4+ Biomass fills >75% to .ltoreq.100% of
middle ear space. Bony septa not visible; obscured by biomass.
[0530] Immunofluoresence imaging of frozen sections of biofilms
formed in vivo was preformed according to the following. After
dissection, the middle ear of each chinchilla was filled with OCT
embedding compound (Fisher Scientific, Pittsburgh, Pa.) and flash
frozen over liquid nitrogen. The bone which forms the inferior
bulla was removed to leave the middle ear mucosa and existing
biofilm intact. The resulting block was then bisected to reveal a
cross section of the biofilm and re-embedded in OCT. Ten micron
serial sections were cut using a Microm rotary cryotome, adhered to
glass slides (Mercedes Medical, Sarasota, Fla.) and stored at
-80.degree. C. Sections were later stained to determine the
relative incorporation of IHF within biofilms that had formed in
vivo. Briefly, slides were air-dried, fixed in cold acetone, then
equilibrated in buffer (0.05M Tris-HCl, 0.15M NaCl and 0.05% Tween
20, pH 7.4). Sections were blocked with image-iT FX signal enhancer
(Molecular Probes, Eugene, Oreg.) and with Background Sniper
(BioCare Medical, Concord, Calif.) per manufacturer's instructions.
Sections were then incubated with a 1:200 dilution of polyclonal
rabbit anti-IHF overnight at 4.degree. C., in a humidified chamber.
Slides were further rinsed and incubated with goat anti-rabbit IgG
conjugated to AlexaFluor 594 (Invitrogen) for 30 minutes at room
temp. As a counterstain, sections were incubated with DAPI and
cover-slipped using ProLong Gold antifade reagent (Molecular
Probes, Eugene, Oreg.). Naive rabbit serum served as the negative
control. Sections were viewed with a Zeiss LSM 510 Meta confocal
system attached to a Zeiss Axiovert 200 inverted microscope (Carl
Zeiss Inc., Thornwood, N.Y.).
[0531] Significant differences in mean CFU/mg tissue and mean CFU
NTHI/ml supernatant were determined by paired t-test. A
p-value.ltoreq.0.05 was considered significant. Significance in
relative biomass among cohorts was assessed by unpaired t-test. A
p-value.ltoreq.0.05 was considered significant.
[0532] As shown in FIG. 9, Panel A, the mean score for remaining
biomass within the middle ears of chinchillas immunized with
adjuvant only was 2.8 which indicated significant remaining disease
and a lack of resolution of pre-formed biomass in most animals by
day 18 after bacterial challenge of the middle ear. In contrast,
the mean score for an E. coli IHF+adjuvant immunized animal was
1.5. Representative images of a residual biomass that received a
mean score of +2.8 and one that received a mean score of +1.5 are
shown in FIG. 9, Panel B. Also, as shown in FIG. 10, the disease
resolution was additionally measured by both histological evidence
of altered biomass architecture within the middle ear (see Panel A)
as well as a statistically significant reduction of bacterial load
present within remaining biomass as measured by homogenization of
the biomass and culture on chocolate agar (see Panel B).
Furthermore, all animals immunized with isolated E. coli IHF
mounted a strong local and systemic immune response and no animal
presented with obvious secondary sequelae as the result of
immunization as noted upon necropsy (data not shown).
[0533] The notable observed efficacy when anti-IHF used in vitro to
debulk biofilms and also of purified E. coli IHF, when used as an
immunogen in vivo to induce the formation of polyclonal antibodies
that could resolve an ongoing biofilm disease, created a conundrum
as to why mammalian hosts do not naturally mount an effective
immune response to DNABII proteins that are associated with eDNA
within the bacterial biofilms of recurrent and/or chronic disease
states. In review of the solved structure of IHF when it is bound
to DNA (Rice et al., (1996) Cell 87(7): 1295-1306), it is clear
that a significant portion of the protein structure is occluded by
bound DNA, which suggested the potential for occlusion of
protective epitopes or domains of IF and/or HU when bound to eDNA
within a bacterial biofilm. It was hypothesized that use of native
IHF, to which no DNA was bound, as the immunogen might provide a
mechanism to overcome such occlusion and thereby foster production
of protective antibodies.
[0534] To determine if eDNA could indeed prevent the development of
protective antibodies directed against a DNABII family member upon
immunization, whereas use of native protein was effective, a second
larger cohort study was performed wherein the chinchilla study as
detailed previously was essentially repeated.
[0535] For the second animal immunization study, twenty adult
chinchillas (body mass between 500-700 g), shown to have no
evidence of middle ear disease by tympanometry and video otoscopy,
were enrolled and divided into four cohorts of 5 animals each. All
animals were again challenged transbullarly with approximately 1000
CFU NTHI strain 86-028NP per bulla. Four days later, after a
biofilm formed in the middle ear cavities of these animals, they
were immunized by a transcutaneous route (TCI) as described above.
Formulations consisted of either: 10 .mu.g IHF admixed with 10
.mu.g dmLT, 10 .mu.g IHF that had been pre-bound to DNA+dmLT,
DNA+dmLT, or 10 .mu.g dmLT alone.
[0536] To determine if TCI with IHF pre-bound to DNA resulted in a
similar immune response to that induced when the same nucleoprotein
complex was delivered subcutaneously (SQ), and compared to that
induced by IHF alone, two adult chinchillas were immunized to
generate antisera. Each animal received a priming dose followed by
two identical boosts delivered at 30-day intervals. Immunogens were
admixed with the adjuvant monophosphoryl lipid A (MPL) (10
.mu.g/dose) (Sigma-Aldrich. St. Louis, Mo.) due to its demonstrated
strong adjuvant properties in the chinchilla host (See for example
Bakaletz et al., (1999) Infect Immun. 67(6):2746-2762 and Kennedy
et al., (2000) Infect. Immun. 68(5):2756-2765. One chinchilla was
immunized with 10 .mu.g of IHF+MPL and the other received 10 .mu.g
IHF bound to DNA+MPL. All doses were delivered SQ in a total volume
of 200 .mu.l. Fifteen days after receiving the final boost, animals
were bled to procure serum and sera were assayed via Western blot
and ELISA to determine both reciprocal titer and specificity of
antibody reactivity to IHF.
[0537] Middle ears were again subsequently scored from 0 to 4+ for
disease severity upon completion of the study. To better assure
that the IHF and DNA remained in complex for immunization,
synthetic oligonucleotides identical to those used in the published
co-crystallization study (see for e.g. Rice et al., (1996) Cell
87(7): 1295-1306) of a high affinity IF binding site from the
bacteriophage lambda recombination site attP at a molar ratio of
2:1 [DNA (10 .mu.M) to IHF (5 .mu.M)] was used. This amount was at
least 3 orders of magnitude over the K.sub.d of IHF bound to this
DNA target. As shown in FIG. 11, IHF indeed bound dsDNA as
demonstrated by its reduced mobility in an electrophoretic mobility
shift assay.
[0538] As shown in FIG. 12, animals immunized with isolated E. coli
IHF showed a dramatic reduction in disease state with a mean
residual middle ear biomass score of 0.9 as compared to the
controls that had been immunized with adjuvant alone (mean biomass
score=2.2) or to those that had been immunized with DNA that had
been admixed with adjuvant (mean biomass score=2.8). Interestingly,
those animals that had been immunized with the THF-DNA complex also
demonstrated middle ears with significant remaining bacterial
biomass, yielding a mean biomass score of 2.5 which was not
statistically significantly different from cohorts that received
either the adjuvant alone or DNA that had been admixed with
adjuvant. This outcome strongly suggested that if sufficient eDNA
fragments were present, as one could hypothesize would be the case
during natural disease, this situation could result in occlusion of
critical IHF epitopes necessary for the generation of neutralizing
antibodies.
[0539] To be assured that the observed DNA occlusion result was not
specific to the use of a transcutaneous immunization route, these
immunizations using a subcutaneous (SQ) immunization route to
ensure delivery of the antigens to antigen presenting cells within
the chinchilla host were repeated. As shown in FIG. 13, whereas SQ
immunization with isolated E. coli IHF yielded the generation of a
strong immune response to isolated IHF, immunization with E. coli
IHF that had been pre-bound to an excess of DNA failed to induce
detectable antibodies that recognized IHF when assayed by Western
blot. When assayed by ELISA, reciprocal titer versus isolated IHF
was 1000 for the animal immunized with IHF that had been pre-bound
to oligonucleotides, whereas that for the animal immunized with
native THF was 8000 (both animal's pre-immune reciprocal titers
against IHF were 100). Collectively, these results are consistent
with our hypothesis that the binding of IHF to eDNA, as would occur
during a natural disease state, has the potential to block epitopes
or domains of IHF necessary for generation of a protective acquired
immune response. Further, immunization with native IHF (to which no
DNA is bound) appeared to allow for the effective direction of the
immune response toward the generation of protective or neutralizing
antibodies, as demonstrated in both pre-clinical chinchilla studies
described here.
Experiment No. 4
[0540] A number of oral bacteria (e.g., Aggregatibacter
aclinomycelemcomitans, Porphyromonas gingivalis) have been
implicated in the pathogenesis of inflammatory diseases such as
periodontitis and peri-implantitis, which destroy alveolar bone and
gingiva. Investigations of the pathogenesis of these bacteria are
hampered by lack of effective animal models. One of the challenges
of investigating the pathogenicity of specific bacteria is the
difficulty of establishing a biofilm when exogenous bacteria are
introduced into the oral cavity of animals. Though animal models of
periodontitis have been developed, cultivable bacteria are rarely
recovered from the oral cavity of inoculated animals. Developing an
effective animal model which can assess the pathogenicity of
specific bacteria will greatly aid in elucidating their pathogenic
mechanisms.
[0541] The surface of machined titanium dental implants
(1.2.times.4.5 mm) was modified by grit blasting with AlO3 (100
.mu.m) and HCl etching (pH 7.8 for 20 min at 80.degree. C.).
Machined and nano-textured implants were incubated in TSB medium
inoculated with D7S clinical strain of Aggregatibacter
actinomycetemcomitans (Aa) for 1 to 3 days at 37.degree. C. The
bacterial biofilm on the implants were analyzed by SEM, as well as
by confocal laser scanning microscopy following staining with
LIVE/DEAD.RTM. BacLight.TM.. Implants with and without established
Aa biofilm were transmucosally placed into the alveolar bone of
female rats between premolar and incisor region of the maxillae. To
detect the presence of Aa biofilm on the implants placed in vivo,
bacterial samples were collected from saliva and the oral surfaces
of implants after 2 days. Aa was detected by culture, as well as by
PCR analysis. Micro-CT and histological analysis of peri-implant
bone and mucosal tissues was performed six weeks after
implantation.
[0542] After one day of cultivation, agglomerates of coccoid-shaped
Aa cells were found scattered throughout the implant. After two
days, the number and size of the agglomerates decreased and more
cells of varying lengths were observed ranging between bacteria
with coccoid morphology. After three days of incubation, the
agglomerates had almost disappeared, while large areas of the
implant surface were covered with bacteria with rod-shaped
morphology, forming a densely packed biofilm. LIVE/DEAD.RTM.
staining of such three days Aa biofilm on the implants showed green
signal for 75-80% of all biofilm bacteria, indicating living cells
with uncompromised membrane integrity. Microbiological and PCR
detection of Aa biofilm on implants placed in vivo were positive
for samples from the implant surfaces and negative for the saliva
samples as well as control implants. Clinical examination
demonstrated significant peri-implant mucosal inflammation around
implants with Aa biofilm, compared with control untreated implants.
Micro-CT and histological analysis of peri-implant bone and mucosal
tissues is pending. Nano-textured implant surfaces favor the
establishment of Aa biofilm and increase risk of
peri-implantitis.
Experiment No. 5
[0543] This experiment provides a mouse model for pre-clinical
testing of interfering agents to treat lyme disease. See Dresser et
al. Pathogens 5(12)e1000680. Epub 2009 Dec. 4. Lyme disease is the
most common tick-borne disease in the United States. Reported cases
have more than doubled between 1992 and 2006, with approximately
29,000 new cases confirmed in 2008. Estimates are that the actual
number of cases of Lyme disease may exceed that reported by a
factor of 6-12 in endemic areas. By definition, these endemic areas
are expanding as populations continue to move from cities to
suburban and rural areas and whitetail deer (which carry the tick
species Ixodes) increasingly roam these areas. Lyme disease is
caused by the microorganism Borrelia burgdorferi, a spirochete. B.
burgdorferi is transmitted via the bite of the Ixodes tick and
subsequently disseminates, via the bloodstream, to other tissues
and organs.
[0544] In this animal model, C3H/HeN mice are injected with
spirochetes via dorsal subcutaneous and intraperitoneal injection,
or via intravenous injection. Blood and biopsy specimens are
recovered at approximately 7 days post infection for evaluation of
microbial burden and assessment of pathology in tissues and organs.
The methods and compositions of this invention are contemplated to
develop both therapeutic as well as preventative strategies for
reduction and/or elimination of the resulting B. burgdorferi
biofilms which form subsequent to challenge and are believed to
contribute to both the pathogenesis and chronic nature of the
disease.
Experiment No. 6
[0545] This experiment provides a porcine model for pre-clinical
testing of interfering agents to treat cystic fibrosis. See Stoltz
et al. (2010) Science Translational Medicine 2(29): 29ra31. Cystic
fibrosis is an autosomal recessive disease due to mutations in a
gene that encodes the CF transmembrane conductance regulator
(called CFTR) anion channel. In this model, pigs which have been
specifically bred to carry a defect in the genes called "CFTR" and
called CF pigs spontaneously develop hallmark features of CF lung
disease that includes infection of the lower airway by multiple
bacterial species. The pigs can be immunized with the interfering
agents to either 1) immunize these CF pigs with a polypeptide or
other immunogenic agent thereby inducing the formation of
antibodies which will eradicate bacterial biofilms in the lungs
(similarly to how antibodies to IHF eradicated biofilms resident
within the middle ears of chinchillas following active immunization
as shown in Experiment No. 1, to deliver anti-IHF (or other
interfering agent) to the lungs of these animals by nebulization to
assess the amelioration of the signs of disease and associated
pathologies.
Experiment No. 7
[0546] Applicants also provide a pre-clinical model for
tuberculosis (TB). See Ordway et al. (2010) Anti. Agents and
Chemotherapy 54:1820. The microorganism Mycobacterium tuberculosis
is responsible for a growing global epidemic. Current figures
suggest that there are approximately 8 million new cases of TB and
about 2.7 million deaths due to TB annually. In addition to the
role of this microbe as a co-infection of individuals with HIV (of
the .about.45 million infected with HIV, estimates are that
.about.1/3 are also co-infected with M. tuberculosis), its
particularly troublesome that isolates have become highly resistant
to multiple drugs and no new drug for TB has been introduced in
over a quarter of a century. In this animal model, SPF guinea pigs
are maintained in a barrier colony and infected via aerosolized
spray to deliver .about.20 cfu of M. tuberculosis strain Erdman K01
bacilli into their lungs. Animals are sacrificed with determination
of bacterial load and recovery of tissues for histopathological
assessment on days 25, 50, 75, 100, 125 and 150 days
post-challenge. Unlike mice which do not develop classic signs of
TB, guinea pigs challenged in this manner develop well-organized
granulomas with central necrosis, a hallmark of human disease.
Further, like humans, guinea pigs develop severe pyogranulomatous
and necrotizing lymphadenitis of the draining lymph nodes as part
of the primary lesion complex. Use of this model will provide a
pre-clinical screen to confirm and identify therapeutic as well as
preventative strategies for reduction and/or elimination of the
resulting M. tuberculosis biofilms which have been observed to form
in the lungs of these animals subsequent to challenge and are
believed to contribute to both the pathogenesis and chronicity of
the disease.
Experiment No. 8
[0547] Multiple animal models of catheter/indwelling device biofilm
infections are known. See Otto (2009) Nature Reviews Microbiology,
7:555. While typically considered normal skin flora, the microbe
Staphylococcus epidermidis has become what many regard as a key
opportunistic pathogen, ranking first among causative agents of
nosocomial infections. Primarily, this bacterium is responsible for
the majority of infections that develop on indwelling medical
devices which are contaminated by this common skin colonizer during
device insertion. While not typically life-threatening, the
difficulty associated with treatment of these biofilm infections,
combined with their frequency, makes them a serious public health
burden. Current costs associated with treatment of vascular
catheter associated bloodstream infections alone that are due to S.
epidermidis amount to $2 billion annually in the United States. In
addition to S. epidermidis, E. faecalis and S. aureus are also
contaminations found on indwelling medical devices. There are
several animal models of catheter-associated S. epidermidis
infections including rabbits, mice, guinea pigs and rats all of
which are used to study the molecular mechanisms of pathogenesis
and which lend themselves to studies of prevention and/or
therapeutics. Rat jugular vein catheters have been used to evaluate
therapies that interfere with E. faecalis, S. aureus and S.
epidermidis biofilm formation. Biofilm reduction is often measured
three ways--(i) sonicate catheter and calculate CFUs, (ii) cut
slices of catheter or simply lay on a plate and score, or (iii) the
biofilm can be stained with crystal violet or another dye, eluted,
and OD measured as a proxy for CFUs.
Experiment No. 9
[0548] Methods described herein may be used to elicit immune
responses in humans and animals. Immunogenic compositions may be
administered to a human and animal subjects in the presence of
adjuvants such as but not limited to aluminum salts and liposomes.
Those skilled in the art will understand that any number of
pharmaceutically acceptable adjuvants can also be used. Immunogenic
compositions may be administered to a human or animal subjects
intramuscularly, subdermally, intranasally, or through any other
suitable route. Immunogenic compositions may be prepared in a
manner consistent with the selected mode of administration.
Immunogenic compositions may take the form of polypeptides, nucleic
acids, or a combination thereof, and may comprise full-length or
partial antigens. Additionally or alternatively, immunogenic
compositions may take the form of APCs pulsed with a particular
antigen, or APCs transfected with one or more polynucleotides
encoding a particular antigen. Administration may comprise a single
dose of an immunogenic composition, or an initial administration,
followed by one or more booster doses. Booster doses may be
provided a day, two days, three days, a week, two weeks, three
weeks, one, two, three, six or twelve months, or at any other time
point after an initial dose. A booster dose may be administered
after an evaluation of the subject's antibody titer.
Experiment No. 10
[0549] Methods described herein may be used to confer passive
immunity on a non-immune subject. Passive immunity against a given
antigen may be conferred through the transfer of antibodies or
antigen binding fragments that specifically recognize or bind to a
particular antigen. Antibody donors and recipients may be human or
non-human subjects. Additionally or alternatively, the antibody
composition may comprise an isolated or recombinant polynucleotide
encoding an antibody or antigen binding fragment that specifically
recognizes or binds to a particular antigen.
[0550] Passive immunity may be conferred in cases where the
administration of immunogenic compositions poses a risk for the
recipient subject, the recipient subject is immuno-compromised, or
the recipient subject requires immediate immunity. Immunogenic
compositions may be prepared in a manner consistent with the
selected mode of administration. Compositions may comprise whole
antibodies, antigen binding fragments, polyclonal antibodies,
monoclonal antibodies, antibodies generated in vivo, antibodies
generated in vitro, purified or partially purified antibodies, or
whole serum. Administration may comprise a single dose of an
antibody composition, or an initial administration followed by one
or more booster doses. Booster doses may be provided a day, two
days, three days, a week, two weeks, three weeks, one, two, three,
six or twelve months, or at any other time point after an initial
dose. A booster dose may be administered after an evaluation of the
subject's antibody titer.
Experiment 11
[0551] Members of the DNABII family are highly pleiotropic for
multiple nucleoprotein systems including gene transcription, and
moreover, are thus often essential. In contrast, both HU and IHF
mutants have been generated in laboratory strains of E. coli and
studied extensively. To determine what role IHF and HU have in eDNA
formation, biofilms formed by E. coli strain MG1655 or its HU and
IHF deficient derivatives were incubated with anti-IHF (prepared as
described in Granston and Nash (1993) J. Mol. Biol., 234:45-59. As
shown in FIG. 8, whereas the parental isolate, strain MG1655
produced a robust biofilm in vitro (Panel A), both HU and IHF
mutant strains were less robust (Panels C and E, respectively). An
HU deficient mutant yielded a biofilm that was approximately 1/2
the height of the wild type biofilm, whereas IHF deficient strains
produced a biofilm that was approximately 2/3rds the height of the
parental isolate. This result suggested that both proteins were
involved in either the production and/or integrity of the E. coli
biofilm EPS. After treatment with anti-IHF, the biofilm formed by
the wild type isolate showed notable debulking and a mean percent
reduction of 58.1% in height, 73.1% percent reduction in biomass
and 87% reduction in mean thickness (Panel B), whereas that formed
by the HU mutant showed no reduction in height and only a 30.9%
reduction on biomass and 37.2% reduction on mean thickness (Panel
D). In contrast, no effect on the biofilm formed by the IHF mutant
was observed in terms of loss of height (-3.4% reduction), biomass
(-20.5% reduction) or mean thickness (-19.8% reduction) (Panel F).
Collectively, these data indicated that for this strain of E. coli,
IHF was likely the only structural element that could be targeted
by use of the anti-IHF antibody. Since to date only the highly
structured interwoven eDNA in biofilms that have been formed in
vivo have been seen, confirmation of the expression of remaining
DNABII family member by each respective mutant and any change in
the resultant DNA structure awaits an in vivo immunofluorescent
analysis.
Experiment 12
[0552] Whereas it was demonstrated in this application in vitro and
in vivo that both anti-THF and the use of IHF as an immunogen show
utility in debulking biofilms and/or resolving biofilm disease
respectively, it was unknown if debulking of NTHI biofilms with
anti-IHF might also allow synergism when used in conjunction with
other therapeutic modalities. To this end the ability to induce
augmented structural destabilization of pre-formed NTHI biofilms
was assessed when a sub-optimal concentration of anti-IHF was used
along with one of each of three unique reagents. These three
reagents include 1) a DNA degrading enzyme (DNaseI) known to be
able to degrade an NTHI biofilm in vitro (but used here at a
suboptimal concentration), 2) antisera to an outer membrane protein
of NTHI that did not destabilize a pre-formed NTHI biofilm when
used alone (anti-OMP P5) (data not shown), and 3) an antibiotic
typically used as a first line choice in children with chronic
and/or recurrent OM (amoxicillin) but which has limited efficacy
against bacteria resident within a biofilm community.
[0553] FIG. 14A shows that treatment of an NTHI biofilm with a
concentration of DNase shown to be sub-optimal has a marginal
effect (see 14A, panel II). Likewise, a 1:200 dilution of anti-IHF
had little effect on the pre-formed NTHI biofilm (see 14A, panel
III). In contrast however, when these two reagents were used in
concert, the biofilm was notably diminished (see 14A, panel IV).
Upon repeat of this experiment three times, we found that the most
marked synergistic effect of debulking of the biofilm was measured
as a diminution in height. Table 7 below depicts these results.
TABLE-US-00011 TABLE 7 DNase + DNase Anti-IHF anti-IHF Max height
(.mu.m) 3.9 37.3 51.0 Assay 1 Max height (.mu.m) -11.5 16.4 37.7
Assay 2 Max height (.mu.m) -1.6 19.4 38.7 Assay 3
[0554] One simple explanation for this outcome was that as the
DNABII protein was being titrated away from the periphery of the
biofilm, the eDNA became more accessible to the action of the
DNase.
[0555] FIG. 14B shows the results of treatment with anti-OMP P5 on
pre-formed NTHI biofilms. Although this antisera is strongly
reactive with isolated NTHI OMP P5 (data not shown) and further,
active immunization with isolated OMP P5 is effective in mediating
significant protection against experimental OM in chinchilla models
(see for e.g. 25. Bakaletz et al., (1997) Vaccine 15(9): 955-961;
Bakaletz et al., (1999) Infect Immun 67(6): 2746-2762; Kennedy et
al., (2000) Infect Immun 68(5): 2756-2765; Kyd J M, et al., (2003)
Infect Immun 71(8): 4691-4699; Novotny L A, et al., (2000) Infect
Immun 68(4): 2119-2128; Novotny L A, et al., (2002) Vaccine
20(29-30): 3590-3597 and Novotny L A, et al., (2003) J Immunol
171(4): 1978-1983) this antiserum does not induce a change in the
structural integrity of an NTHI biofilm that has been formed in
vitro when used at a dilution of 1:50 (see 14B, panel II). Likewise
a marginal effect when these biofilms were incubated with a
sub-optimal dilution of anti-IHF (used at a 1:100 dilution here)
was observed (see 14B, panel I). When combined, however, the use of
anti-PS plus anti-IHF resulted in a reduction in the height of the
biofilm that exceeded the sum of the two antisera when used singly
(see 14B, panel III), thus indicating a synergistic outcome. Hence,
it was concluded that use of anti-IHF to destabilize the NTHI EPS
matrix resulted in the exposure of the targeted bacterial cell
surface protein (e.g. OMP P5) that would otherwise be obscured by
eDNA as well as perhaps other components of the EPS, thus allowing
immune mediated bacterial clearance by as yet unknown
mechanisms.
[0556] Lastly, FIG. 14C shows the results of treating pre-formed
NTHI biofilms with amoxicillin. When used at a concentration of 64
.mu.g/ml, amoxicillin had no measurable effect on the architecture
of pre-formed NTHI biofilms (see 14C, panel II) despite evidence of
limited bacterial cell death. Treatment with IHF antisera at a 1:50
dilution substantially reduced the height of the biofilm as shown
previously (see 14C, panel III). Interestingly however, when the
two reagents were used simultaneously, not only was there a
dramatic reduction in the height of the biofilm (see 14C, panel
IV), but use of a vital dye indicated that the majority of the
bacteria remaining in the biofilm were now dead as noted by the
predominant fluorescence in the red channel within the imaged
biofilm. This result showed that debulking of the biofilm with
anti-IHF likely exposed the bacteria sufficiently so as to create
conditions more akin to susceptibility to amoxicillin
concentrations known to be effective when assayed against
planktonic NTHI. This outcome may have been mediated by either
increased physical exposure of bacteria within the remaining
biofilm matrix to the action of amoxicillin and/or via increased
release of bacteria into the planktonic phase as we showed earlier
can occur during biofilm debulking by exposure to anti-IHF
antibodies (see FIG. 4).
Experiment 13
[0557] Applicants have shown that the induction of anti-IHF
antibodies by transcutaneous immunization is effective in resolving
NTHI induced biofilms within the chinchilla middle ear. It was also
shown that use of native IHF is essential to induction of a
protective immune response, as when it is bound to eDNA, protective
epitopes appear to have been masked as shown following both TCI and
SQ immunization. Nonetheless, the rational design of vaccine
candidates that will likely have broad protective efficacy requires
a detailed understanding of the immunodominant and/or protective
epitopes of that protein, which may in fact be neither obvious nor
necessarily continuous. Current vaccine candidate design strategies
are more reductionist in their approach, and often aimed at
identifying and using only that portion of the targeted protein
that is absolutely necessary for induction of high avidity and
protective antibodies. To this end, epitope mapping is done to
determine the most effective immunogen.
[0558] Epitope mapping can be performed as described in Novotny, et
al., (2009) Vaccine 28:(1):279-289 and according to the following
procedure. To generate immune serum Chinchillas can be parenterally
immunized by subcutaneous (SQ) injection of 10 .mu.g of the native
IHF protein, a polypeptide of the C-terminal 20 amino acids of
IHF.alpha., a polypeptide of the C-terminal 20 amino acids of
IHF.beta., or a polypeptide of amino acids 60-76 of TBP-DNABII
administered co-mixed with the adjuvant monophosphoryl lipid A
(MPL; Corixa) or with MPL alone (adjuvant-only control cohort). Two
identical boosts can be delivered at 21-day intervals. Blood for
serum can be collected 10 days after receipt of the final
immunizing dose. Enzyme-linked immunosorbent assay (ELISA) and
Western blotting of chinchilla antisera can be performed. For
ELISA, sera can be assayed in a 96-well plate format against the
immunogen delivered (0.2 .mu.g protein/well). Titer can be defined
as the reciprocal of the serum dilution that yielded an OD450 value
of 0.1 greater than control wells which contain all components
except serum. ELISA assays can be performed a minimum of three
times and results are reported as the geometric mean. Western
blotting can be performed against 1 .mu.g of the respective
immunogen using immune serum diluted 1:100 and detected with
HRP-protein A (Zymed, South San Francisco, Calif.). Color was
developed with CN/DAB substrate (Pierce, Rockford, Ill.).
[0559] A series of 20-mer synthetic peptides with a 5-residue
overlap can be synthesized in order to epitope map the IHF protein
of NTHI. Synthesis, purification and sequence confirmation of all
synthetic peptides can be performed according to standard
techniques (see for e.g. Bakaletz L O. et al., (1997) Infect.
Immun. 71(8):4691-9 and Bakaletz L O., et al., (2001) Vaccine
68(4):2119-28). Amino acid analysis and mass spectral analysis can
be used to confirm the purity, composition and amino acid sequence
of each peptide.
[0560] Analysis of the interaction between synthetic peptides and
antibodies present in sera, can be examined using a Biacore 3000
instrument as described and all reagents can be purchased from
Biacore (Uppsala, Sweden). Briefly, peptides can be suspended in
sodium acetate buffer, prior to immobilization to the surface of an
activated reagent grade CM5 sensor chip using amine coupling
chemistry. For interaction analysis, 15 .mu.l of serum (diluted 1:2
in HBS-EP buffer plus 1 mg carboxymethyldextran/ml) can be injected
across the sensor chip surface at a flow rate of 5 .mu.l/min. The
relative amount of antigen- or immunogen-specific antibody in each
sample can be calculated as the difference in resonance units (RU)
obtained 5 seconds before and 45 seconds after each sample can be
injected. The sensor chip surface can be regenerated between
samples with 25 mM NaOH. The synthetic peptides with the strongest
interaction with the antibodies present in the sera can be the most
promising candidates of protective epitopes capable of inducing
high avidity protective antibodies in vivo.
[0561] Rabbit polyclonal antiserum can be generated against the
most immunogenic peptides from the epitope mapping. This antiserum
can be used to affinity purify neutralizing antibodies from
non-neutralizing antibodies by column chromatography. To do so,
both rabbit and chinchilla polyclonal antisera (the latter has been
shown to be protective) can be used. Sera can be assayed both prior
to as well as after running each over a column to which either
native IHF was bound or to which IHF pre-admixed with DNA has been
bound. Only the antibodies that do not bind to the DNA-IHF complex
should be neutralizing. All flow-through and eluted antisera will
then be assayed against the original antiserum pools via our in
vitro biofilm assay for relative ability to debulk and NTHI biofilm
as well as via ELISA, Western blot and biosensor assays to
determine specificity and titer. As an additional test,
epitope-targeted vaccine candidates can be bound to the column to
determine if they can remove antibodies now proven to be
neutralizing.
[0562] It is to be understood that while the invention has been
described in conjunction with the above embodiments, that the
foregoing description and examples are intended to illustrate and
not limit the scope of the invention. Other aspects, advantages and
modifications within the scope of the invention will be apparent to
those skilled in the art to which the invention pertains.
[0563] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
nucleotide sequences provided herein are presented in the 5' to 3'
direction.
[0564] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising". "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed.
[0565] Thus, it should be understood that although the present
invention has been specifically disclosed by preferred embodiments
and optional features, modification, improvement and variation of
the inventions embodied therein herein disclosed may be resorted to
by those skilled in the art, and that such modifications,
improvements and variations are considered to be within the scope
of this invention. The materials, methods, and examples provided
here are representative of preferred embodiments, are exemplary,
and are not intended as limitations on the scope of the
invention.
[0566] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0567] In addition, where features or aspects of the invention are
described in terms of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0568] All publications, patent applications, patents, and other
references mentioned herein are expressly incorporated by reference
in their entirety, to the same extent as if each were incorporated
by reference individually. In case of conflict, the present
specification, including definitions, will control.
SEQUENCE LISTING
[0569] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Mar. 25, 2011, is named 64189381.txt and is 113,218 bytes in
size.
TABLE-US-00012 TABLE 8 Gram (+) - only HU, Gram (-) - all have HU
some also IHF Bacteria strain Abbreviation Protein name(s) S.
sobrinus 6715 Ss 1310 (HU) (not fully sequenced) S. pyogenes
MGAS10270 Spyog Spy1239 (HU) S. gordonii Challis NCTC7868 Sg
SGO_0701 (HlpA) S. agalactiae (Group B Strep)2603V/R GBS SAG_0505
(Hup) S. mutans UA159 Sm Smu_589 (HU) S. pneumoniae R6 Spneu
spr1020 (HU) S. gallolyticus UCN34 (S. bovis) Sgall YP_003430069
(HlpA) S. aureus MW2 Sa MW1362 (HU) S. epidermidis RP62A Se
SERP1041 (Hup) E. coli K12-MG1655 Ec b1712 (HimA) b0912 (HimD)
(HupA) (HupB) H. influenza KW20 Rd Hi HI1221 (HimA) HI1313 (HimD)
HI0430 (HupA) Salmonella enteric serovar typhi CT18 Salm Sty1771
(HimA) Sty0982 (HimD) Aggregatibacter actinomycetemcomitans D11S-1
Aa YP_003255965 (IHFalpha) YP_003256209 (IhfB) YP_003255304 (HU) P.
gingivalis W83 Pg PG_0121 (Hup-1) PG_1258 (Hup-2) N. gonorrhoeae
FA1090 (Oklahoma) Ng NGO603 (IHF.beta.) NGO030 (IHF.alpha.) N.
meningitides MC58 Nm NMB_0729 (HimA) NMB_1302 (HimA) P. aeruginosa
Pa PA3161 (HimD) PA1804 (HupB) PA2758 (HimA) H. pylori 26695 Hp
Hp0835 (Hup) B. burgdorferi B31 Bb BB_0232 (Hbb) Moraxella
catarrhalis RH4 Mc YP_003626307 (HimA) YP_003627027 (HimD)
YP_003626775 (HupB) V. cholera El Tor N16961 Vc VC_0273 (HupA)
VC_1914 (HipB) VC_1919 (HupB) VC_1222 (HimA) Burkholderia
cenocepacia HI2424 Bc Bcen2424_1048 (IHFB) Bcen2424_1481 (IHFA)
Burkholderia pseudomallei 668 Bp BURPS668_2881 (IHFB) BURPS668_1718
(IHFA) Mycobacterium tuberculosis CDC1551 Mtb MT_3064 (HU)
Mycobacterium smegmatis MC2 Ms MSMEG_2389 (Hup) Treponema denticola
ATCC 35405 Td TDE_1709 (HU) Treponema palladum Nichols Tp TP_0251
(DNA binding protein II) Prevotella melaninogenica ATCC 25845 Pm
PREME0022_2103 (HupB) PREME0022_0268 (HupA) PREME0022_0341 (Hup)
PREME0022_0340 (HimA) Prevotella intermedia 17 Pi PIN_A0704 (Hup)
PIN_A1504 (Hup-2) PIN_0345 (HimA) PIN_0343 (Hypothetical protein)
Bordetella pertusis Tohama 1 Bpert BP2572 (IhfA) BP3530 (HupB)
BP0951 (IhfB) Enterococcus faecalis V583 Ef Ef1550 (hup)
TABLE-US-00013 TABLE 9A1 SEQ ID NOS 42-72, respectively, in order
of appearance ##STR00001## ##STR00002##
TABLE-US-00014 TABLE 9A2 SEQ ID NOS 73-76, respectively, in order
of appearance ##STR00003##
TABLE-US-00015 TABLE 9A3 SEQ ID NOS 77-100, respectively, in order
of appearance ##STR00004## ##STR00005##
TABLE-US-00016 TABLE 9B SEQ ID NOS 101-128, respectively, in order
of appearance Comparison to Liu et al 16 as peptide motif Strep
inter HU EVRERAARK-GRNPQTG Ec_HimA ##STR00006## Salm_HimA
##STR00007## Vc_HimA ##STR00008## Pa_HimA ##STR00009## Hi_HimA
##STR00010## Aa_IHFalpha ##STR00011## Mc HimA ##STR00012##
Ng_IHFalpha ##STR00013## Nm HimA ##STR00014## Bc IHFA ##STR00015##
Bp IHFA ##STR00016## Bpert IhfA ##STR00017## Pm HimA ##STR00018##
Pi HimA ##STR00019## Tp Dbp II ##STR00020## Pm Hup ##STR00021## Pi
hypo ##STR00022## Sa_HU ##STR00023## Ec hupA ##STR00024## Se_Hup
##STR00025## Ss Hu ##STR00026## Spyog_HU ##STR00027## Sgall_HlpA
##STR00028## GBS_Hup ##STR00029## Spneu_HU ##STR00030## Sg_HlpA
##STR00031## Sm_HU ##STR00032## Ef Hup ##STR00033## Hi_HupA
##STR00034## Vc_HupA ##STR00035## Bpert HupB ##STR00036## Pa_HupB
##STR00037## Aa HU ##STR00038## Pm HupB ##STR00039## Pi Hup
##STR00040## Td HU ##STR00041## Pg_Hup-1 ##STR00042## Hp_Hup
##STR00043## Pm HupA ##STR00044## Pi Hup-2 ##STR00045## Pg_Hup-2
##STR00046## Mt HU ##STR00047## Ms Hup ##STR00048## Ec_HimD
##STR00049## Salm_HimD ##STR00050## Vc_HipB ##STR00051## Ec hupB
##STR00052## Mc HupB ##STR00053## Pa_HimD ##STR00054## Hi_HimD
##STR00055## Aa_IHFB ##STR00056## Ng_IHF.beta. ##STR00057## Nm HimD
##STR00058## Bc IHFB ##STR00059## Bp IHFB ##STR00060## Bpert IhfB
##STR00061## Mc HimD ##STR00062## Bb_ Hbb ##STR00063##
TABLE-US-00017 TABLE 10 (SEQ ID NOS 160-336, respectively, in order
of appearance) Bacteria strain, protein name .beta.3 sequence
.alpha.3 sequence C-terminal 20 aa S. pyogenes MGAS10270, HU AFKAGK
ALKDAVK IAASKVPAFKAGKALKDAVK S. gallolyticus UCN34 (S. bovis), HlpA
AFKAGK ALKDAVK IAASKVPAFKAGKALKDAVK S. sobrinus 6715 HU AFKAGK
ALKDAVK IAASKVPAFKAGKALKDAVK S. agalactiae (Group B Strep) 2603V/R
Hup AFKAGK ALKDAVK IAASKVPAFKAGKALKDAVK S. pneumoniae R6 HU AFKAGK
ALKDAVK IAASKVPAFKAGKALKDAVK S. gordonii Challis NCTC7868,HlpA
AFKAGK ALKDAVK IAASKVPAFKAGKALKDAVK S. mutans UA159,HU AFKAGK
ALKDAVK IKASKVPAFKAGKALKDAVK Enterococcus faecalis V583, Hup AFKPGK
ALKDAVK IAASKVPAFKPGKALKDAVK S. aureus MW2, HU AFKAGK ALKDAVK
IPASKVPAFKAGKALKDAVK S. epidermidis RP62A Hup AFKAGK ALKDAVK
IPASKVPAFKAGKALKDAVK H. influenza KW20 Rd HupA AFVSGK ALKDAIK
IAASKVPAFVSGKALKDAIK Aggregatibacter actinomycetemcomitans AFVSGK
ALKDAVK IAASKVPAFVSGKALKDAVK D11S-1 HU V. cholera El Tor N16961,
HupA AFVAGK ALKDAIK IAAANVPAFVAGKALKDAIK E. coli K12-MG1655 hupA
AFVSGK ALKDAVK IAAANVPAFVSGKALKDAVK P. aeruginosa HupB GFKAGK
ALKDAVN IAAAKIPGFKAGKALKDAVN E. coli K12-MG1655 hupB SFRAGK ALKDAVN
IAAAKVPSFRAGKALKDAVN V. cholera El Tor N16961 HupB SFRAGK ALKDACN
IAEAKVPSFKAGKALKDACN Bordetella pertusis Tohama 1 HupB KFRPGK
ALKDAVN IKKAKVPKFRPGKALKDAVN Prevotella melaninogenica ATCC 25845
HupB KFKAGA ELADAVNK AAKKVAKFKAGAELADAVNK Prevotella intermedia 17
Hup KFKPGA ELADAVNA AAKKVAKFKPGAELADAVNA Moraxella catarrhalis RH4
HupB SFKAGK VLKESVN IAASKVPSFKAGKVLKESVN P. gingivalis W83 Hup-1
RFKPGS TLELK ISIPARKVVRFKPGSTLELK H. pylori 2669 Hup KFKPGK
TLKQKVEEGK KRVPKFKPGKTLKQKVEEGK Prevotella melaninogenica ATCC
25845 HupA SFKPAK TFIEDMKK PAHDFPSFKPAKTFIEDMKK Prevotella
intermedia 17 Hup-2 SFKPAK TFIEDMKK PAHDFPSFKPAKTFIEDMKK P.
gingivalis W83 Hup-2 AFKPSK IFMSQMKQD KRNIPAFKPSKIFMSQMKQD
Mycobacterium tuberculosis CDC1551 HU AFRPGA QFKAVVSGAQRLPAEGPAVKRG
AKRPATKAPAKKATAPRGRK Mycobacterium smegmatis MC2 Hup AFRPGA
QFKAVISGAQKLPADGPAVKRG TKAPAKKAAAKKARAKKGRR Prevotella
melaninogenica ATCC 25845 HimA NFKPAA TIKGHVRKGGQDNG
NFKPAATIKGHVRKGGQDNG Prevotella intermedia 17 HimA NFRATA
SVKEKLKKGGAE VLNFRATASVKEKLKKGGAE E. coli K12-MG1655 HimA TFRPGQ
KLKSRVENASPKDE TFRPGQKLKSRVENASPKDE Salmonella enteric serovar
typhi CT18 HimA TFRPGQ KLKSRVENASPKEE TFRPGQKLKSRVENASPKEE V.
cholera El Tor N1696 HimA TFRPGQ KLKARVENIKVEK VTFRPGQKLKARVENIKVEK
P. aeruginosa HimA TFRPGQ KLKARVEAYAGTKS TFRPGQKLKARVEAYAGTKS
Burkholderia cenocepacia HI2424 IHFA TFHASQ KLKALVENGAE
RVVTFHASQKLKALVENGAE Burkholderia pseudomallei 668 IHFA TFHASQ
KLKALVENGAEPDLAR HASQKLKALVENGAEPDLAR Bordetella pertusis Tohama 1
IhfA TFHASQ KLKSVVEQPNSPPDPASAE, QKLKSVVEQPNSPPDPASAE N.
gonorrhoeae FA1090 (Oklahoma) IHF.alpha. TFHASQ KLKGMVEHYYDKQR
TFHASQKLKGMVEHYYDKQR N. meningitides MC58 HimA TFHASQ
KLKSMVEHYYDKQR TFHASQKLKSMVEHYYDKQR H. influenza KW20 Rd HimA
TFKPGQ KLRRARVEKTK RRVVTFKPGQKLRARVEKTK Aggregatibacter
actinomycetemcomitans VFKPGQ KLRNRVEKVKPKA VVFKPGQKLRNRVEKVKPKA
D11S-1 HimA Moraxella catarrhalis RH4 HimA TFKPGQ KLRGWIDSQNEG
VVTFKAGQKLRGWIDSQNEG Treponema palladium Nichols VFRPSK
RLKSAVRGYRSGEVGAD PSKRLKSAVRGYRSGEVGAD DNA_binding_protein_II
Prevotella melaninogenica ATCC 25845 Hup SFTPDT
VMKELVNKPFSQFETVVINDGV MQAGDTMKVPKVELRPEYRK Prevotella intermedia
17 hypothetical SFTPDA TMKELVNKPFAQFETVVLNDGV SAGDTMKVPKVELRPQYRTK
E. coli K12-MG1655 HimD HFKPGK ELRDRANIYG KYVPHFKPGKELRDRANIYG
Salmonella enteric serovar typhi HFKPGK ELRDRANIYG
KYVPHFKPGKELRDRANIYG CT18 bHimD V. cholera El Tor N1696 HipB HFKPGK
ELRERVNL EGKYVPHFKPGKELRERVNL P. aeruginosa HimD HFKPGK ELRDRVNEPE
KFVPHFKPGKELRDRVNEPE H. influenza KW20 Rd HimD YFKAGK ELKARVDVQA
KSVPYFKAGKELKARVDVQA Aggregatibacter actinomycetemcomitans YFKAGK
ELRERVDVYAA CVPYFKAGKELRERVDVYAA D11S-1 IHFB N. gonorrhoeae FA1090
(Oklahoma) IHF.beta. HFKPGK ELRERVDLALKENAN FKPGKELRERVDLALKENAN N.
meningitides MC58 HimD HFKPGK ELRERVDLALKENAN FKPGKELRERVDLALKENAN
Burkholderia cenocepacia HI2424 IHFB HFKPGK ELRERVDGRAGEPLKADDPDDDR
ERVDGRAGEPLKADDPDDDR Burkholderia pseudomallei 668 IHFB HFKPGK
ELRERVDGRAGEPLKNDEPEDAQ ERVDGRAGEPLKKDEPEDAQ Bordetella pertusis
Tohama 1 Ihfb HFKPGK ELREWVDLVGNDQGDDSSNGSS DSSNGSSDPLQSVMDMHAMH
Moraxella catarrhalis RH4 HimD YFKPGK ALRESVNLVND
ATPYFKPGKALRESVNLVND B. burgdorferi B31 Hbb YFRPGK DLKERVWGIKG
HVAYFRPGKDLKERVWGIKR Treponema denticola ATCC 35405 HU RFKPGK
ELKEALHKIDTQELIES PGKELKEALHKIDTQELIES
Sequence CWU 1
1
34019PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Xaa Xaa Xaa Xaa Xaa Phe Gly Xaa Phe 1 5
29PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Val Lys Lys Ser Gly Phe Gly Asn Phe 1 5
37PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 45PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Asn
Pro Xaa Thr Gly 1 5 57PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Gly Arg Asn Pro Xaa Thr Gly
1 5 696PRTHaemophilus influenzae 6Met Ala Thr Ile Thr Lys Leu Asp
Ile Ile Glu Tyr Leu Ser Asp Lys 1 5 10 15 Tyr His Leu Ser Lys Gln
Asp Thr Lys Asn Val Val Glu Asn Phe Leu 20 25 30 Glu Glu Ile Arg
Leu Ser Leu Glu Ser Gly Gln Asp Val Lys Leu Ser 35 40 45 Gly Phe
Gly Asn Phe Glu Leu Arg Asp Lys Ser Ser Arg Pro Gly Arg 50 55 60
Asn Pro Lys Thr Gly Asp Val Val Pro Val Ser Ala Arg Arg Val Val 65
70 75 80 Thr Phe Lys Pro Gly Gln Lys Leu Arg Ala Arg Val Glu Lys
Thr Lys 85 90 95 7136PRTHaemophilus influenzae 7Met Arg Phe Val Thr
Ile Phe Ile Asn His Ala Phe Asn Ser Ser Gln 1 5 10 15 Val Arg Leu
Ser Phe Ala Gln Phe Leu Arg Gln Ile Arg Lys Asp Thr 20 25 30 Phe
Lys Glu Ser Asn Phe Leu Phe Asn Arg Arg Tyr Lys Phe Met Asn 35 40
45 Lys Thr Asp Leu Ile Asp Ala Ile Ala Asn Ala Ala Glu Leu Asn Lys
50 55 60 Lys Gln Ala Lys Ala Ala Leu Glu Ala Thr Leu Asp Ala Ile
Thr Ala 65 70 75 80 Ser Leu Lys Glu Gly Glu Pro Val Gln Leu Ile Gly
Phe Gly Thr Phe 85 90 95 Lys Val Asn Glu Arg Ala Ala Arg Thr Gly
Arg Asn Pro Gln Thr Gly 100 105 110 Ala Glu Ile Gln Ile Ala Ala Ser
Lys Val Pro Ala Phe Val Ser Gly 115 120 125 Lys Ala Leu Lys Asp Ala
Ile Lys 130 135 896PRTHaemophilus influenzae 8Met Ala Thr Ile Thr
Lys Leu Asp Ile Ile Glu Tyr Leu Ser Asp Lys 1 5 10 15 Tyr His Leu
Ser Lys Gln Asp Thr Lys Asn Val Val Glu Asn Phe Leu 20 25 30 Glu
Glu Ile Arg Leu Ser Leu Glu Ser Gly Gln Asp Val Lys Leu Ser 35 40
45 Gly Phe Gly Asn Phe Glu Leu Arg Asp Lys Ser Ser Arg Pro Gly Arg
50 55 60 Asn Pro Lys Thr Gly Asp Val Val Pro Val Ser Ala Arg Arg
Val Val 65 70 75 80 Thr Phe Lys Pro Gly Gln Lys Leu Arg Ala Arg Val
Glu Lys Thr Lys 85 90 95 996PRTHaemophilus influenzae 9Met Ala Thr
Ile Thr Lys Leu Asp Ile Ile Glu Tyr Leu Ser Asp Lys 1 5 10 15 Tyr
His Leu Ser Lys Gln Asp Thr Lys Asn Val Val Glu Asn Phe Leu 20 25
30 Glu Glu Ile Arg Leu Ser Leu Glu Ser Gly Gln Asp Val Lys Leu Ser
35 40 45 Gly Phe Gly Asn Phe Glu Leu Arg Asp Lys Ser Ser Arg Pro
Gly Arg 50 55 60 Asn Pro Lys Thr Gly Asp Val Val Pro Val Ser Ala
Arg Arg Val Val 65 70 75 80 Thr Phe Lys Pro Gly Gln Lys Leu Arg Ala
Arg Val Glu Lys Thr Lys 85 90 95 1099PRTEscherichia coli 10Met Ala
Leu Thr Lys Ala Glu Met Ser Glu Tyr Leu Phe Asp Lys Leu 1 5 10 15
Gly Leu Ser Lys Arg Asp Ala Lys Glu Leu Val Glu Leu Phe Phe Glu 20
25 30 Glu Ile Arg Arg Ala Leu Glu Asn Gly Glu Gln Val Lys Leu Ser
Gly 35 40 45 Phe Gly Asn Phe Asp Leu Arg Asp Lys Asn Gln Arg Pro
Gly Arg Asn 50 55 60 Pro Lys Thr Gly Glu Asp Ile Pro Ile Thr Ala
Arg Arg Val Val Thr 65 70 75 80 Phe Arg Pro Gly Gln Lys Leu Lys Ser
Arg Val Glu Asn Ala Ser Pro 85 90 95 Lys Asp Glu
11100PRTPseudomonas aeruginosa 11Met Gly Ala Leu Thr Lys Ala Glu
Ile Ala Glu Arg Leu Tyr Glu Glu 1 5 10 15 Leu Gly Leu Asn Lys Arg
Glu Ala Lys Glu Leu Val Glu Leu Phe Phe 20 25 30 Glu Glu Ile Arg
Gln Ala Leu Glu His Asn Glu Gln Val Lys Leu Ser 35 40 45 Gly Phe
Gly Asn Phe Asp Leu Arg Asp Lys Arg Gln Arg Pro Gly Arg 50 55 60
Asn Pro Lys Thr Gly Glu Glu Ile Pro Ile Thr Ala Arg Arg Val Val 65
70 75 80 Thr Phe Arg Pro Gly Gln Lys Leu Lys Ala Arg Val Glu Ala
Tyr Ala 85 90 95 Gly Thr Lys Ser 100 126PRTEscherichia coli 12Thr
Phe Arg Pro Gly Gln 1 5 1314PRTEscherichia coli 13Lys Leu Lys Ser
Arg Val Glu Asn Ala Ser Pro Lys Asp Glu 1 5 10 146PRTEscherichia
coli 14His Phe Lys Pro Gly Lys 1 5 1510PRTEscherichia coli 15Glu
Leu Arg Asp Arg Ala Asn Ile Tyr Gly 1 5 10 166PRTHaemophilus
influenzae 16Thr Phe Lys Pro Gly Gln 1 5 1710PRTHaemophilus
influenzae 17Lys Leu Arg Ala Arg Val Glu Lys Thr Lys 1 5 10
186PRTHaemophilus influenzae 18Thr Phe Lys Pro Gly Gln 1 5
1910PRTHaemophilus influenzae 19Lys Leu Arg Ala Arg Val Glu Asn Thr
Lys 1 5 10 206PRTHaemophilus influenzae 20Thr Phe Lys Pro Gly Gln 1
5 2110PRTHaemophilus influenzae 21Lys Leu Arg Ala Arg Val Glu Lys
Thr Lys 1 5 10 226PRTHaemophilus influenzae 22Thr Phe Lys Pro Gly
Gln 1 5 2310PRTHaemophilus influenzae 23Lys Leu Arg Ala Arg Val Glu
Lys Thr Lys 1 5 10 246PRTEscherichia coli 24Thr Phe Arg Pro Gly Gln
1 5 2514PRTEscherichia coli 25Lys Leu Lys Ser Arg Val Glu Asn Ala
Ser Pro Lys Asp Glu 1 5 10 266PRTPseudomonas aeruginosa 26Thr Phe
Arg Pro Gly Gln 1 5 2714PRTPseudomonas aeruginosa 27Lys Leu Lys Ala
Arg Val Glu Ala Tyr Ala Gly Thr Lys Ser 1 5 10 2890PRTEscherichia
coli 28Met Asn Lys Thr Gln Leu Ile Asp Val Ile Ala Glu Lys Ala Glu
Leu 1 5 10 15 Ser Lys Thr Gln Ala Lys Ala Ala Leu Glu Ser Thr Leu
Ala Ala Ile 20 25 30 Thr Glu Ser Leu Lys Glu Gly Asp Ala Val Gln
Leu Val Gly Phe Gly 35 40 45 Thr Phe Lys Val Asn His Arg Ala Glu
Arg Thr Gly Arg Asn Pro Gln 50 55 60 Thr Gly Lys Glu Ile Lys Ile
Ala Ala Ala Asn Val Pro Ala Phe Val 65 70 75 80 Ser Gly Lys Ala Leu
Lys Asp Ala Val Lys 85 90 2990PRTEscherichia coli 29Met Asn Lys Ser
Gln Leu Ile Asp Lys Ile Ala Ala Gly Ala Asp Ile 1 5 10 15 Ser Lys
Ala Ala Ala Gly Arg Ala Leu Asp Ala Ile Ile Ala Ser Val 20 25 30
Thr Glu Ser Leu Lys Glu Gly Asp Asp Val Ala Leu Val Gly Phe Gly 35
40 45 Thr Phe Ala Val Lys Glu Arg Ala Ala Arg Thr Gly Arg Asn Pro
Gln 50 55 60 Thr Gly Lys Glu Ile Thr Ile Ala Ala Ala Lys Val Pro
Ser Phe Arg 65 70 75 80 Ala Gly Lys Ala Leu Lys Asp Ala Val Asn 85
90 306PRTEscherichia coli 30Ala Phe Val Ser Gly Lys 1 5
317PRTEscherichia coli 31Ala Leu Lys Asp Ala Val Lys 1 5
326PRTEscherichia coli 32Ser Phe Arg Ala Gly Lys 1 5
337PRTEscherichia coli 33Ala Leu Lys Asp Ala Val Asn 1 5
3420PRTEscherichia coli 34Thr Phe Arg Pro Gly Gln Lys Leu Lys Ser
Arg Val Glu Asn Ala Ser 1 5 10 15 Pro Lys Asp Glu 20
3520PRTEscherichia coli 35Lys Tyr Val Pro His Phe Lys Pro Gly Lys
Glu Leu Arg Asp Arg Ala 1 5 10 15 Asn Ile Tyr Gly 20
3613DNAArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 36watcaannnn ttr 13376PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 37Gly
Pro Ser Leu Lys Leu 1 5 384PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 38Gly Pro Ser Leu 1
394PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 39Pro Ser Leu Lys 1 405PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 40Gly
Pro Ser Leu Lys 1 5 414PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 41Ser Leu Lys Leu 1
4299PRTEscherichia coli 42Met Ala Leu Thr Lys Ala Glu Met Ser Glu
Tyr Leu Phe Asp Lys Leu 1 5 10 15 Gly Leu Ser Lys Arg Asp Ala Lys
Glu Leu Val Glu Leu Phe Phe Glu 20 25 30 Glu Ile Arg Arg Ala Leu
Glu Asn Gly Glu Gln Val Lys Leu Ser Gly 35 40 45 Phe Gly Asn Phe
Asp Leu Arg Asp Lys Asn Gln Arg Pro Gly Arg Asn 50 55 60 Pro Lys
Thr Gly Glu Asp Ile Pro Ile Thr Ala Arg Arg Val Val Thr 65 70 75 80
Phe Arg Pro Gly Gln Lys Leu Lys Ser Arg Val Glu Asn Ala Ser Pro 85
90 95 Lys Asp Glu 4399PRTSalmonella enterica 43Met Ala Leu Thr Lys
Ala Glu Met Ser Glu Tyr Leu Phe Asp Lys Leu 1 5 10 15 Gly Leu Ser
Lys Arg Asp Ala Lys Glu Leu Val Glu Leu Phe Phe Glu 20 25 30 Glu
Ile Arg Arg Ala Leu Glu Asn Gly Glu Gln Val Lys Leu Ser Gly 35 40
45 Phe Gly Asn Phe Asp Leu Arg Asp Lys Asn Gln Arg Pro Gly Arg Asn
50 55 60 Pro Lys Thr Gly Glu Asp Ile Pro Ile Thr Ala Arg Arg Val
Val Thr 65 70 75 80 Phe Arg Pro Gly Gln Lys Leu Lys Ser Arg Val Glu
Asn Ala Ser Pro 85 90 95 Lys Glu Glu 4498PRTVibrio cholerae 44Met
Ala Leu Thr Lys Ala Glu Leu Ala Glu Ala Leu Phe Glu Gln Leu 1 5 10
15 Gly Met Ser Lys Arg Asp Ala Lys Asp Thr Val Glu Val Phe Phe Glu
20 25 30 Glu Ile Arg Lys Ala Leu Glu Ser Gly Glu Gln Val Lys Leu
Ser Gly 35 40 45 Phe Gly Asn Phe Asp Leu Arg Asp Lys Asn Glu Arg
Pro Gly Arg Asn 50 55 60 Pro Lys Thr Gly Glu Asp Ile Pro Ile Thr
Ala Arg Arg Val Val Thr 65 70 75 80 Phe Arg Pro Gly Gln Lys Leu Lys
Ala Arg Val Glu Asn Ile Lys Val 85 90 95 Glu Lys
45100PRTPseudomonas aeruginosa 45Met Gly Ala Leu Thr Lys Ala Glu
Ile Ala Glu Arg Leu Tyr Glu Glu 1 5 10 15 Leu Gly Leu Asn Lys Arg
Glu Ala Lys Glu Leu Val Glu Leu Phe Phe 20 25 30 Glu Glu Ile Arg
Gln Ala Leu Glu His Asn Glu Gln Val Lys Leu Ser 35 40 45 Gly Phe
Gly Asn Phe Asp Leu Arg Asp Lys Arg Gln Arg Pro Gly Arg 50 55 60
Asn Pro Lys Thr Gly Glu Glu Ile Pro Ile Thr Ala Arg Arg Val Val 65
70 75 80 Thr Phe Arg Pro Gly Gln Lys Leu Lys Ala Arg Val Glu Ala
Tyr Ala 85 90 95 Gly Thr Lys Ser 100 4696PRTHaemophilus influenzae
46Met Ala Thr Ile Thr Lys Leu Asp Ile Ile Glu Tyr Leu Ser Asp Lys 1
5 10 15 Tyr His Leu Ser Lys Gln Asp Thr Lys Asn Val Val Glu Asn Phe
Leu 20 25 30 Glu Glu Ile Arg Leu Ser Leu Glu Ser Gly Gln Asp Val
Lys Leu Ser 35 40 45 Gly Phe Gly Asn Phe Glu Leu Arg Asp Lys Ser
Ser Arg Pro Gly Arg 50 55 60 Asn Pro Lys Thr Gly Asp Val Val Pro
Val Ser Ala Arg Arg Val Val 65 70 75 80 Thr Phe Lys Pro Gly Gln Lys
Leu Arg Ala Arg Val Glu Lys Thr Lys 85 90 95 4798PRTAggregatibacter
actinomycetemcomitans 47Met Thr Leu Thr Lys Val Glu Leu Ala Glu Asn
Leu Ile Glu Lys Phe 1 5 10 15 His Leu Ser Lys Arg Glu Ala Lys Asp
Leu Val Glu Ser Phe Phe Glu 20 25 30 Glu Ile Arg Val Ala Leu Glu
Thr Gly Asn Asp Val Lys Leu Ser Gly 35 40 45 Phe Gly Asn Phe Glu
Leu Arg Asp Lys Ala Ser Arg Pro Gly Arg Asn 50 55 60 Pro Lys Thr
Gly Glu Ser Val Pro Val Ser Ala Arg Arg Val Val Val 65 70 75 80 Phe
Lys Pro Gly Gln Lys Leu Arg Asn Arg Val Glu Lys Val Lys Pro 85 90
95 Lys Ala 4898PRTMoraxella catarrhalis 48Met Gly Ala Leu Thr Lys
Ala Asp Met Val Asp Glu Leu Thr Ile Arg 1 5 10 15 Leu Arg Leu Thr
Arg Gln Gln Ala Arg Lys Leu Val Asp Gly Phe Phe 20 25 30 Glu Glu
Ile Ser Gln Ser Leu Ala Gln Gly His Glu Val Lys Leu Ser 35 40 45
Gly Phe Gly Asn Phe Glu Leu Lys Asp Lys Lys Pro Arg Pro Gly Arg 50
55 60 Asn Pro Lys Thr Gly Glu Ser Val Pro Ile Gln Ala Arg Arg Val
Val 65 70 75 80 Thr Phe Lys Ala Gly Gln Lys Leu Arg Gly Trp Ile Asp
Ser Gln Asn 85 90 95 Glu Gly 49100PRTNeisseria gonorrhoeae 49Met
Thr Leu Thr Lys Ala Glu Leu Ala Asp Ile Leu Val Asp Lys Val 1 5 10
15 Ser Asn Val Thr Lys Asn Asp Ala Lys Glu Ile Val Glu Leu Phe Phe
20 25 30 Glu Glu Ile Arg Ser Thr Leu Ala Ser Gly Glu Glu Ile Lys
Ile Ser 35 40 45 Gly Phe Gly Asn Phe Gln Leu Arg Asp Lys Pro Gln
Arg Pro Gly Arg 50 55 60 Asn Pro Lys Thr Gly Glu Glu Val Pro Ile
Thr Ala Arg Arg Val Val 65 70 75 80 Thr Phe His Ala Ser Gln Lys Leu
Lys Gly Met Val Glu His Tyr Tyr 85 90 95 Asp Lys Gln Arg 100
50100PRTNeisseria meningitidis 50Met Thr Leu Thr Lys Ala Glu Leu
Ala Asp Ile Leu Val Asp Lys Val 1 5 10 15 Ser Asn Val Thr Lys Asn
Asp Ala Lys Glu Ile Val Glu Leu Phe Phe 20 25 30 Glu Glu Ile Arg
Ser Thr Leu Ala Ser Gly Glu Glu Ile Lys Ile Ser 35 40 45 Gly Phe
Gly Asn Phe Gln Leu Arg Asp Lys Pro Gln Arg Pro Gly Arg 50 55 60
Asn Pro Lys Thr Gly Glu Glu Val Pro Ile Thr Ala Arg Arg Val Val 65
70 75 80 Thr Phe His Ala Ser Gln Lys Leu Lys Ser Met Val Glu His
Tyr Tyr 85 90 95 Asp Lys Gln Arg 100 51101PRTBurkholderia
cenocepacia 51Ala Ser Thr Glu Thr Pro Thr Leu Thr Lys Ala Glu Leu
Ala Glu Leu 1 5 10 15 Leu Phe Asp Ser Val Gly Leu Asn Lys Arg Glu
Ala Lys Asp Met Val 20 25 30 Glu Ala Phe Phe Glu Val Ile Arg Asp
Ala Leu Glu Asn Gly Glu Ser 35 40 45 Val Lys Leu Ser Gly Phe Gly
Asn Phe Gln Leu Arg Asp Lys Pro Gln 50 55
60 Arg Pro Gly Arg Asn Pro Lys Thr Gly Glu Ala Ile Pro Ile Ala Ala
65 70 75 80 Arg Arg Val Val Thr Phe His Ala Ser Gln Lys Leu Lys Ala
Leu Val 85 90 95 Glu Asn Gly Ala Glu 100 52107PRTBurkholderia
pseudomallei 52Thr Ser Ala Gly Asp Thr Pro Thr Leu Thr Lys Ala Glu
Leu Ala Glu 1 5 10 15 Leu Leu Phe Asp Ser Val Gly Leu Asn Lys Arg
Glu Ala Lys Asp Met 20 25 30 Val Glu Ala Phe Phe Glu Val Ile Arg
Asp Ala Leu Glu Asn Gly Glu 35 40 45 Ser Val Lys Leu Ser Gly Phe
Gly Asn Phe Gln Leu Arg Asp Lys Pro 50 55 60 Gln Arg Pro Gly Arg
Asn Pro Asn Thr Gly Glu Ala Ile Pro Ile Ala 65 70 75 80 Ala Arg Arg
Val Val Thr Phe His Ala Ser Gln Lys Leu Lys Ala Leu 85 90 95 Val
Glu Asn Gly Ala Glu Pro Asp Leu Ala Arg 100 105 53113PRTBordetella
pertussis 53Met Gly Thr Thr Met Leu Ala Glu Pro Arg Thr Leu Thr Lys
Ala Glu 1 5 10 15 Leu Ala Glu Leu Leu Phe Glu Arg Val Gly Leu Asn
Lys Arg Glu Ala 20 25 30 Lys Asp Ile Val Asp Thr Phe Phe Glu Glu
Ile Arg Asp Ala Leu Ala 35 40 45 Arg Gly Asp Ser Val Lys Leu Ser
Gly Phe Gly Asn Phe Gln Val Arg 50 55 60 Asp Lys Pro Pro Arg Pro
Gly Arg Asn Pro Lys Thr Gly Glu Thr Ile 65 70 75 80 Pro Ile Ala Ala
Arg Arg Val Val Thr Phe His Ala Ser Gln Lys Leu 85 90 95 Lys Ser
Val Val Glu Gln Pro Asn Ser Pro Pro Asp Pro Ala Ser Ala 100 105 110
Glu 5497PRTPrevotella melaninogenica 54Met Asn Asn Lys Glu Phe Ile
Ala Ala Leu Ala Ala Arg Thr Gly Tyr 1 5 10 15 Thr Gln Asp Glu Ser
Gln Lys Met Val Lys Thr Val Val Asp Met Met 20 25 30 Gly Lys Ser
Phe Glu Thr Gly Asp Pro Val Pro Val Ile Gly Phe Gly 35 40 45 Thr
Phe Glu Val Lys Lys Arg Leu Glu Arg Val Met Val Asn Pro Ser 50 55
60 Thr Gly Leu Arg Met Leu Val Pro Pro Lys Leu Val Leu Asn Phe Lys
65 70 75 80 Pro Ala Ala Thr Ile Lys Gly His Val Arg Lys Gly Gly Gln
Asp Asn 85 90 95 Gly 5595PRTPrevotella intermedia 55Met Asn Asn Lys
Glu Phe Ile Thr Ala Leu Ala Asn Arg Val Gly Arg 1 5 10 15 Ser Gln
Asp Glu Thr Gln Lys Leu Val Lys Thr Ala Leu Gln Ala Met 20 25 30
Gly Asp Asn Phe Glu Ser Gly Glu Pro Val Leu Val Ser Gly Phe Gly 35
40 45 Ser Phe Glu Val Lys Lys Arg Leu Glu Arg Ile Met Thr Asn Pro
Ala 50 55 60 Thr Gly Leu Arg Met Leu Val Pro Pro Lys Leu Val Leu
Asn Phe Arg 65 70 75 80 Ala Thr Ala Ser Val Lys Glu Lys Leu Lys Lys
Gly Gly Ala Glu 85 90 95 56105PRTTreponema palladium 56Met Lys Arg
Val Arg Arg Thr Arg Ser Phe Val Val Asp Ala Leu Cys 1 5 10 15 Asp
Glu Val Asp Leu Ser Arg Arg His Val Ala Arg Val Val Asp Ser 20 25
30 Phe Val Ser Val Val Thr Ala Ala Leu Glu Arg Gly Glu Thr Val Glu
35 40 45 Leu Arg Asp Phe Gly Val Phe Glu Ser Arg Val Arg Lys Ala
Ser Val 50 55 60 Gly Lys Ser Ile Asn Thr Gly Glu Val Val Ser Ile
Pro Ser His Cys 65 70 75 80 Val Val Val Phe Arg Pro Ser Lys Arg Leu
Lys Ser Ala Val Arg Gly 85 90 95 Tyr Arg Ser Gly Glu Val Gly Ala
Asp 100 105 57101PRTPrevotella melaninogenica 57Met Ala Lys Ser Ala
Ile Gln Leu Ile Thr Ser Ala Leu Ala Lys Gln 1 5 10 15 His Asn Leu
Ser Ala Asp Asp Ala Ala Ala Phe Val Asp Ala Phe Phe 20 25 30 Asp
Ile Ile Ser Ser Glu Leu Lys Asn Gly Asn Gln Val Lys Ile Lys 35 40
45 Gly Leu Gly Thr Phe Lys Val Gln Ala Val Lys Pro Arg Glu Ser Val
50 55 60 Asn Val Asn Thr Gly Glu Arg Val Leu Ile Glu Gly His Asp
Lys Ile 65 70 75 80 Ser Phe Thr Pro Asp Thr Val Met Lys Glu Leu Val
Asn Lys Pro Phe 85 90 95 Ser Gln Phe Glu Thr 100 58101PRTPrevotella
intermedia 58Met Ala Lys Thr Ala Leu Gln Leu Ile Ala Asp Ala Val
Ala Lys Lys 1 5 10 15 His Lys Ile Thr Val Lys Glu Ala Glu Lys Phe
Val Ser Ala Ile Phe 20 25 30 Asp Val Val Asn Glu Gly Leu Lys Thr
Asp Lys Leu Val Lys Val Lys 35 40 45 Gly Leu Gly Thr Phe Lys Val
Gln Ala Val Lys Pro Arg Glu Ser Val 50 55 60 Asn Val Asn Thr Gly
Glu Arg Val Leu Ile Glu Gly His Glu Lys Val 65 70 75 80 Ser Phe Thr
Pro Asp Ala Thr Met Lys Glu Leu Val Asn Lys Pro Phe 85 90 95 Ala
Gln Phe Glu Thr 100 5990PRTStaphylococcus aureus 59Met Asn Lys Thr
Asp Leu Ile Asn Ala Val Ala Glu Gln Ala Asp Leu 1 5 10 15 Thr Lys
Lys Glu Ala Gly Ser Ala Val Asp Ala Val Phe Glu Ser Ile 20 25 30
Gln Asn Ser Leu Ala Lys Gly Glu Lys Val Gln Leu Ile Gly Phe Gly 35
40 45 Asn Phe Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro
Gln 50 55 60 Thr Gly Lys Glu Ile Asp Ile Pro Ala Ser Lys Val Pro
Ala Phe Lys 65 70 75 80 Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85
90 6090PRTEscherichia coli 60Met Asn Lys Thr Gln Leu Ile Asp Val
Ile Ala Glu Lys Ala Glu Leu 1 5 10 15 Ser Lys Thr Gln Ala Lys Ala
Ala Leu Glu Ser Thr Leu Ala Ala Ile 20 25 30 Thr Glu Ser Leu Lys
Glu Gly Asp Ala Val Gln Leu Val Gly Phe Gly 35 40 45 Thr Phe Lys
Val Asn His Arg Ala Glu Arg Thr Gly Arg Asn Pro Gln 50 55 60 Thr
Gly Lys Glu Ile Lys Ile Ala Ala Ala Asn Val Pro Ala Phe Val 65 70
75 80 Ser Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6190PRTStaphylococcus epidermidis 61Met Asn Lys Thr Asp Leu Ile Asn
Ala Val Ala Glu Gln Ala Asp Leu 1 5 10 15 Thr Lys Lys Glu Ala Gly
Ser Ala Val Asp Ala Val Phe Glu Ser Ile 20 25 30 Gln Asn Ser Leu
Ala Lys Gly Glu Lys Val Gln Leu Ile Gly Phe Gly 35 40 45 Asn Phe
Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln 50 55 60
Thr Gly Lys Glu Ile Asp Ile Pro Ala Ser Lys Val Pro Ala Phe Lys 65
70 75 80 Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6291PRTStreptococcus sobrinus 62Met Ala Asn Lys Gln Asp Leu Ile Ala
Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser Ala
Ala Ala Val Asp Thr Val Phe Ser Ser 20 25 30 Ile Glu Gly Phe Leu
Ser Lys Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn Phe
Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60 Gln
Thr Gly Ala Glu Ile Lys Ile Ala Ala Ser Lys Val Pro Ala Phe 65 70
75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6391PRTStreptococcus pyogeneses 63Met Ala Asn Lys Gln Asp Leu Ile
Ala Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser
Ala Ala Ala Val Asp Ala Val Phe Ser Thr 20 25 30 Ile Glu Ala Phe
Leu Ala Glu Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn
Phe Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60
Gln Thr Gly Ala Glu Ile Glu Ile Ala Ala Ser Lys Val Pro Ala Phe 65
70 75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6491PRTStreptococcus gallolyticus 64Met Ala Asn Lys Gln Asp Leu Ile
Ala Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser
Ala Ala Ala Val Asp Ala Val Phe Ser Ala 20 25 30 Ile Glu Ser Phe
Leu Ser Glu Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn
Phe Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60
Gln Thr Gly Glu Glu Ile Glu Ile Ala Ala Ser Lys Val Pro Ala Phe 65
70 75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6591PRTStreptococcus agalactiae 65Met Ala Asn Lys Gln Asp Leu Ile
Ala Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser
Ala Ala Ala Val Asp Ala Val Phe Ala Ala 20 25 30 Val Ala Asp Tyr
Leu Ala Glu Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn
Phe Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60
Gln Thr Gly Ala Glu Ile Glu Ile Ala Ala Ser Lys Val Pro Ala Phe 65
70 75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6691PRTStreptococcus pneumoniae 66Met Ala Asn Lys Gln Asp Leu Ile
Ala Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser
Ala Ala Ala Val Glu Ala Val Phe Ala Ala 20 25 30 Val Ala Asp Tyr
Leu Ala Ala Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn
Phe Glu Val Arg Glu Arg Ala Glu Arg Lys Gly Arg Asn Pro 50 55 60
Gln Thr Gly Lys Glu Met Thr Ile Ala Ala Ser Lys Val Pro Ala Phe 65
70 75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6791PRTStreptococcus gordonii 67Met Ala Asn Lys Gln Asp Leu Ile Ala
Lys Val Ala Ala Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser Ala
Ala Ala Val Asp Ala Val Phe Ala Ala 20 25 30 Val Thr Glu Tyr Leu
Ser Lys Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn Phe
Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60 Gln
Thr Gly Lys Glu Ile Lys Ile Ala Ala Ser Lys Val Pro Ala Phe 65 70
75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6891PRTStreptococcus mutans 68Met Ala Asn Lys Gln Asp Leu Ile Ala
Lys Val Ala Glu Ala Thr Glu 1 5 10 15 Leu Thr Lys Lys Asp Ser Ala
Ala Ala Val Asp Ala Val Phe Ser Ala 20 25 30 Val Ser Ser Tyr Leu
Ala Lys Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn Phe
Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60 Gln
Thr Gly Glu Glu Ile Lys Ile Lys Ala Ser Lys Val Pro Ala Phe 65 70
75 80 Lys Ala Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
6991PRTEnterococcus faecalis 69Met Ala Asn Lys Ala Glu Leu Ile Glu
Asn Val Ala Ser Ser Thr Gly 1 5 10 15 Leu Thr Lys Lys Asp Ala Thr
Ala Ala Val Asp Ala Val Phe Ser Thr 20 25 30 Ile Gln Glu Thr Leu
Ala Lys Gly Glu Lys Val Gln Leu Ile Gly Phe 35 40 45 Gly Asn Phe
Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro 50 55 60 Gln
Thr Gly Gln Glu Ile Gln Ile Ala Ala Ser Lys Val Pro Ala Phe 65 70
75 80 Lys Pro Gly Lys Ala Leu Lys Asp Ala Val Lys 85 90
7090PRTHaemophilus influenzae 70Met Asn Lys Thr Asp Leu Ile Asp Ala
Ile Ala Asn Ala Ala Glu Leu 1 5 10 15 Asn Lys Lys Gln Ala Lys Ala
Ala Leu Glu Ala Thr Leu Asp Ala Ile 20 25 30 Thr Ala Ser Leu Lys
Glu Gly Glu Pro Val Gln Leu Ile Gly Phe Gly 35 40 45 Thr Phe Lys
Val Asn Glu Arg Ala Ala Arg Thr Gly Arg Asn Pro Gln 50 55 60 Thr
Gly Ala Glu Ile Gln Ile Ala Ala Ser Lys Val Pro Ala Phe Val 65 70
75 80 Ser Gly Lys Ala Leu Lys Asp Ala Ile Lys 85 90 7190PRTVibrio
cholerae 71Met Asn Lys Thr Gln Leu Ile Asp Phe Ile Ala Glu Lys Ala
Asp Leu 1 5 10 15 Thr Lys Val Gln Ala Lys Ala Ala Leu Glu Ala Thr
Leu Gly Ala Val 20 25 30 Glu Gly Ala Leu Lys Asp Gly Asp Gln Val
Gln Leu Ile Gly Phe Gly 35 40 45 Thr Phe Lys Val Asn His Arg Ser
Ala Arg Thr Gly Arg Asn Pro Gln 50 55 60 Thr Gly Glu Glu Ile Lys
Ile Ala Ala Ala Asn Val Pro Ala Phe Val 65 70 75 80 Ala Gly Lys Ala
Leu Lys Asp Ala Ile Lys 85 90 7290PRTPseudomonas aeruginosa 72Met
Asn Lys Ser Glu Leu Ile Asp Ala Ile Ala Ala Ser Ala Asp Ile 1 5 10
15 Pro Lys Ala Val Ala Gly Arg Ala Leu Asp Ala Val Ile Glu Ser Val
20 25 30 Thr Gly Ala Leu Lys Ala Gly Asp Ser Val Val Leu Val Gly
Phe Gly 35 40 45 Thr Phe Ala Val Lys Glu Arg Ala Ala Arg Thr Gly
Arg Asn Pro Gln 50 55 60 Thr Gly Lys Pro Ile Lys Ile Ala Ala Ala
Lys Ile Pro Gly Phe Lys 65 70 75 80 Ala Gly Lys Ala Leu Lys Asp Ala
Val Asn 85 90 7390PRTAggregatibacter actinomycetemcomitans 73Met
Asn Lys Thr Asp Leu Ile Asp Ala Ile Ala Ser Ser Ala Glu Leu 1 5 10
15 Asn Lys Lys Gln Ala Lys Ala Ala Leu Glu Ala Thr Leu Asp Ala Ile
20 25 30 Thr Gly Ser Leu Lys Lys Gly Glu Ala Val Gln Leu Ile Gly
Phe Gly 35 40 45 Thr Phe Lys Val Asn Ala Arg Lys Ala Arg Thr Gly
Arg Asn Pro Gln 50 55 60 Thr Gly Ala Glu Ile Lys Ile Ala Ala Ser
Lys Val Pro Ala Phe Val 65 70 75 80 Ser Gly Lys Ala Leu Lys Asp Ala
Val Lys 85 90 7490PRTVibrio cholerae 74Met Asn Lys Thr Gln Leu Val
Glu Gln Ile Ala Ala Asn Ala Asp Ile 1 5 10 15 Ser Lys Ala Ser Ala
Gly Arg Ala Leu Asp Ala Phe Ile Glu Ala Val 20 25 30 Ser Gly Thr
Leu Gln Ser Gly Asp Gln Val Ala Leu Val Gly Phe Gly 35 40 45 Thr
Phe Ser Val Arg Thr Arg Ala Ala Arg Thr Gly Arg Asn Pro Lys 50 55
60 Thr Gly Glu Glu Ile Lys Ile Ala Glu Ala Lys Val Pro Ser Phe Lys
65 70 75 80 Ala Gly Lys Ala Leu Lys Asp Ala Cys Asn 85 90
7590PRTEscherichia coli 75Met Asn Lys Ser Gln Leu Ile Asp Lys Ile
Ala Ala Gly Ala Asp Ile 1 5
10 15 Ser Lys Ala Ala Ala Gly Arg Ala Leu Asp Ala Ile Ile Ala Ser
Val 20 25 30 Thr Glu Ser Leu Lys Glu Gly Asp Asp Val Ala Leu Val
Gly Phe Gly 35 40 45 Thr Phe Ala Val Lys Glu Arg Ala Ala Arg Thr
Gly Arg Asn Pro Gln 50 55 60 Thr Gly Lys Glu Ile Thr Ile Ala Ala
Ala Lys Val Pro Ser Phe Arg 65 70 75 80 Ala Gly Lys Ala Leu Lys Asp
Ala Val Asn 85 90 7690PRTMoraxella catarrhalis 76Met Asn Lys Ser
Glu Leu Val Asp Ser Ile Ala Gln Ser Ala Gly Leu 1 5 10 15 Thr Lys
Glu Gln Ala Ala Lys Ala Val Asn Ala Phe Thr Glu Ser Val 20 25 30
Gln Gly Ala Leu Gln Arg Gly Asp Asp Val Val Leu Val Gly Phe Gly 35
40 45 Thr Phe Ser Val Lys Glu Arg Ala Ala Arg Met Gly Arg Asn Pro
Lys 50 55 60 Thr Gly Glu Ala Ile Gln Ile Ala Ala Ser Lys Val Pro
Ser Phe Lys 65 70 75 80 Ala Gly Lys Val Leu Lys Glu Ser Val Asn 85
90 7790PRTBordetella pertussis 77Met Asn Lys Thr Glu Leu Ile Asp
His Ile Ala Ser Lys Ala Asp Ile 1 5 10 15 Ser Lys Ala Ala Ala Gly
Arg Ser Leu Asp Ala Leu Ile Gly Ala Val 20 25 30 Lys Thr Thr Leu
Lys Lys Gly Gly Thr Val Thr Leu Val Gly Phe Gly 35 40 45 Thr Phe
Ala Val Ser Ala Arg Ala Ala Arg Thr Gly Arg Asn Pro Arg 50 55 60
Thr Gly Glu Thr Ile Lys Ile Lys Lys Ala Lys Val Pro Lys Phe Arg 65
70 75 80 Pro Gly Lys Ala Leu Lys Asp Ala Val Asn 85 90
7899PRTMoraxella catarrhalis 78Met Gln Ala Val Ile Asn Lys Ser Asn
Leu Ile Ala Asn Leu Ala Ser 1 5 10 15 Val Cys Glu Glu Leu Glu Glu
Asp Val Val Asp Glu Ala Val Arg Leu 20 25 30 Met Ile Ala Met Met
Val Asn Glu Leu Val Tyr Asp Gly Arg Ile Glu 35 40 45 Val Arg Gly
Phe Gly Ser Phe Cys Leu His His Arg Ser Ala Arg Ile 50 55 60 Ala
Arg Asn Pro Arg Thr Gly Glu Ser Val Ser Val Lys Ala Lys Ala 65 70
75 80 Thr Pro Tyr Phe Lys Pro Gly Lys Ala Leu Arg Glu Ser Val Asn
Leu 85 90 95 Val Asn Asp 7991PRTPrevotella melaninogenica 79Met Asn
Lys Thr Glu Leu Ile Glu Lys Ile Ala Ala Asn Ala Glu Val 1 5 10 15
Ser Lys Ala Ala Ala Lys Lys Ala Leu Asp Ala Thr Thr Glu Ala Ile 20
25 30 Lys Glu Ala Leu Ala Ala Gly Asp Lys Val Gln Leu Val Gly Phe
Gly 35 40 45 Thr Phe Ala Thr Thr Glu Arg Pro Ala His Glu Gly Ile
Asn Pro Arg 50 55 60 Ser Lys Glu Lys Ile Lys Ile Ala Ala Lys Lys
Val Ala Lys Phe Lys 65 70 75 80 Ala Gly Ala Glu Leu Ala Asp Ala Val
Asn Lys 85 90 8091PRTPrevotella intermedia 80Met Asn Lys Thr Glu
Leu Ile Glu Lys Ile Ala Ala Gly Ala Gly Leu 1 5 10 15 Ser Lys Ala
Asp Ser Lys Lys Ala Leu Asp Ala Met Thr Ala Ala Ile 20 25 30 Lys
Glu Ala Leu Val Ala Gly Asp Lys Val Gln Leu Val Gly Phe Gly 35 40
45 Thr Tyr Ser Val Thr Glu Arg Pro Ala His Glu Gly Ile Asn Pro Ala
50 55 60 Thr Lys Gln Lys Ile Gln Ile Ala Ala Lys Lys Val Ala Lys
Phe Lys 65 70 75 80 Pro Gly Ala Glu Leu Ala Asp Ala Val Asn Ala 85
90 81106PRTTreponema denticola 81Met Lys Gln Lys Arg Ser Lys Ile
Asp Ile Ile Asp Ser Val Tyr Arg 1 5 10 15 Asn Asn Pro Gln Tyr Gln
Leu Lys Gln Ile Asn Ala Ile Ala Asn Leu 20 25 30 Phe Leu Asp Glu
Leu Ser Val Leu Leu Gln Gln Gly Ile Pro Val Glu 35 40 45 Ile Arg
Gly Leu Gly Ser Phe Asp Phe Ala Val Leu His Gly Arg Lys 50 55 60
Asn Ala Arg Asn Pro Lys Thr Gly Glu Ala Val Leu Thr Ala Asp Arg 65
70 75 80 Cys Lys Val Arg Phe Lys Pro Gly Lys Glu Leu Lys Glu Ala
Leu His 85 90 95 Lys Ile Asp Thr Gln Glu Leu Ile Glu Ser 100 105
8288PRTPorphyromonas gingivalis 82Met Asn Lys Thr Asp Phe Ile Ala
Ala Val Ala Glu Lys Ala Asn Leu 1 5 10 15 Thr Lys Ala Asp Ala Gln
Arg Ala Val Asn Ala Phe Ala Glu Val Val 20 25 30 Thr Glu Gln Met
Asn Ala Gly Glu Lys Ile Ala Leu Ile Gly Phe Gly 35 40 45 Thr Phe
Ser Val Ser Glu Arg Ala Ala Arg Lys Gly Ile Asn Pro Lys 50 55 60
Thr Lys Lys Ser Ile Ser Ile Pro Ala Arg Lys Val Val Arg Phe Lys 65
70 75 80 Pro Gly Ser Thr Leu Glu Leu Lys 85 8394PRTHelicobacter
pylori 83Met Asn Lys Ala Glu Phe Ile Asp Leu Val Lys Glu Ala Gly
Lys Tyr 1 5 10 15 Asn Ser Lys Arg Glu Ala Glu Glu Ala Ile Ser Ala
Phe Thr Leu Ala 20 25 30 Val Glu Thr Ala Leu Ser Lys Gly Glu Ser
Val Glu Leu Ile Gly Phe 35 40 45 Gly Lys Phe Glu Thr Ala Glu Gln
Lys Gly Lys Glu Gly Lys Val Pro 50 55 60 Gly Ser Asp Lys Thr Tyr
Lys Thr Glu Asp Lys Arg Val Pro Lys Phe 65 70 75 80 Lys Phe Gly Lys
Thr Leu Lys Gln Lys Val Glu Glu Gly Lys 85 90 8492PRTPrevotella
melaninogenica 84Met Thr Lys Ala Asp Ile Ile Asn Glu Ile Ala Thr
Ser Thr Gly Ile 1 5 10 15 Ala Lys Lys Asp Val Ser Ala Val Val Glu
Ser Phe Met Glu Thr Ile 20 25 30 Lys Asp Ser Leu Leu Glu Lys Lys
Glu Asn Val Tyr Leu Arg Gly Phe 35 40 45 Gly Ser Phe Ile Val Lys
His Arg Ala Glu Lys Thr Ala Arg Asn Ile 50 55 60 Ser Lys Asn Thr
Thr Ile Thr Ile Pro Ala His Asp Phe Pro Ser Phe 65 70 75 80 Lys Pro
Ala Lys Thr Phe Ile Glu Asp Met Lys Lys 85 90 8592PRTPrevotella
intermedia 85Met Thr Lys Ala Asp Ile Ile Asn Glu Ile Ala Ser Ser
Thr Gly Ile 1 5 10 15 Ser Lys Lys Asp Val Ser Ala Val Val Glu Ser
Phe Met Asp Ala Ile 20 25 30 Lys Asp Ser Leu Leu Glu Asn Lys Glu
Asn Val Tyr Leu Arg Gly Phe 35 40 45 Gly Ser Phe Ile Val Lys His
Arg Ala Glu Lys Thr Ala Arg Asn Ile 50 55 60 Ser Lys Asn Thr Thr
Ile Thr Ile Pro Ala His Asp Phe Pro Ser Phe 65 70 75 80 Lys Pro Ala
Lys Thr Phe Ile Glu Asp Met Lys Lys 85 90 8692PRTPorphyromonas
gingivalis 86Met Thr Lys Ala Asp Val Val Asn Ala Ile Ala Lys Ser
Thr Gly Ile 1 5 10 15 Asp Lys Glu Thr Thr Leu Lys Val Val Glu Ser
Phe Met Asp Thr Ile 20 25 30 Lys Asp Ser Leu Ser Glu Gly Asp Asn
Val Tyr Leu Arg Gly Phe Gly 35 40 45 Ser Phe Ile Val Lys Glu Arg
Ala Glu Lys Thr Ala Arg Asn Ile Ser 50 55 60 Lys Gln Thr Thr Ile
Ile Ile Pro Lys Arg Asn Ile Pro Ala Phe Lys 65 70 75 80 Pro Ser Lys
Ile Phe Met Ser Gln Met Lys Gln Asp 85 90 87100PRTMycobacterium
tuberculosis 87Met Asn Lys Ala Glu Leu Ile Asp Val Leu Thr Gln Lys
Leu Gly Ser 1 5 10 15 Asp Arg Arg Gln Ala Thr Ala Ala Val Glu Asn
Val Val Asp Thr Ile 20 25 30 Val Arg Ala Val His Lys Gly Asp Ser
Val Thr Ile Thr Gly Phe Gly 35 40 45 Val Phe Glu Gln Arg Arg Arg
Ala Ala Arg Val Ala Arg Asn Pro Arg 50 55 60 Thr Gly Glu Thr Val
Lys Val Lys Pro Thr Ser Val Pro Ala Phe Arg 65 70 75 80 Phe Gly Ala
Gln Phe Lys Ala Val Val Ser Gly Ala Gln Arg Leu Pro 85 90 95 Ala
Glu Gly Pro 100 88100PRTMycobacterium smegmatis 88Met Asn Lys Ala
Glu Leu Ile Asp Val Leu Thr Thr Lys Met Gly Thr 1 5 10 15 Asp Arg
Arg Gln Ala Thr Ala Ala Val Glu Asn Val Val Asp Thr Ile 20 25 30
Val Arg Ala Val His Lys Gly Asp Ser Val Thr Ile Thr Gly Phe Gly 35
40 45 Val Phe Glu Gln Arg Arg Arg Ala Ala Arg Val Ala Arg Asn Pro
Arg 50 55 60 Thr Gly Glu Thr Val Lys Val Lys Pro Thr Ser Val Pro
Ala Phe Arg 65 70 75 80 Phe Gly Ala Gln Phe Lys Ala Val Ile Ser Gly
Ala Gln Lys Leu Pro 85 90 95 Ala Asp Gly Pro 100 8994PRTEscherichia
coli 89Met Thr Lys Ser Glu Leu Ile Glu Arg Leu Ala Thr Gln Gln Ser
His 1 5 10 15 Ile Pro Ala Lys Thr Val Glu Asp Ala Val Lys Glu Met
Leu Glu His 20 25 30 Met Ala Ser Thr Leu Ala Gln Gly Glu Arg Ile
Glu Ile Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu His Tyr Arg Ala
Pro Arg Thr Gly Arg Asn Pro 50 55 60 Lys Thr Gly Asp Lys Val Glu
Leu Glu Gly Lys Tyr Val Pro His Phe 65 70 75 80 Lys Pro Gly Lys Glu
Leu Arg Asp Arg Ala Asn Ile Tyr Gly 85 90 9094PRTSalmonella
enterica 90Met Thr Lys Ser Glu Leu Ile Glu Arg Leu Ala Thr Gln Gln
Ser His 1 5 10 15 Ile Pro Ala Lys Ala Val Glu Asp Ala Val Lys Glu
Met Leu Glu His 20 25 30 Met Ala Ser Thr Leu Ala Gln Gly Glu Arg
Ile Glu Ile Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu His Tyr Arg
Ala Pro Arg Thr Gly Arg Asn Pro 50 55 60 Lys Thr Gly Asp Lys Val
Glu Leu Glu Gly Lys Tyr Val Pro His Phe 65 70 75 80 Lys Pro Gly Lys
Glu Leu Arg Asp Arg Ala Asn Ile Tyr Gly 85 90 9192PRTVibrio
cholerae 91Met Thr Lys Ser Glu Leu Ile Glu Arg Leu Cys Ala Glu Gln
Thr His 1 5 10 15 Leu Ser Ala Lys Glu Ile Glu Asp Ala Val Lys Asn
Ile Leu Glu His 20 25 30 Met Ala Ser Thr Leu Glu Ala Gly Glu Arg
Ile Glu Ile Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu His Tyr Arg
Glu Pro Arg Val Gly Arg Asn Pro 50 55 60 Lys Thr Gly Asp Lys Val
Glu Leu Glu Gly Lys Tyr Val Pro His Phe 65 70 75 80 Lys Pro Gly Lys
Glu Leu Arg Glu Arg Val Asn Leu 85 90 9294PRTPseudomonas aeruginosa
92Met Thr Lys Ser Glu Leu Ile Glu Arg Ile Val Thr His Gln Gly Gln 1
5 10 15 Leu Ser Ala Lys Asp Val Glu Leu Ala Ile Lys Thr Met Leu Glu
Gln 20 25 30 Met Ser Gln Ala Leu Ala Thr Gly Asp Arg Ile Glu Ile
Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu His Tyr Arg Ala Pro Arg
Val Gly Arg Asn Pro 50 55 60 Lys Thr Gly Glu Ser Val Arg Leu Asp
Gly Lys Phe Val Pro His Phe 65 70 75 80 Lys Pro Gly Lys Glu Leu Arg
Asp Arg Val Asn Glu Pro Glu 85 90 9394PRTHaemophilus influenzae
93Met Thr Lys Ser Glu Leu Met Glu Lys Leu Ser Ala Lys Gln Pro Thr 1
5 10 15 Leu Pro Ala Lys Glu Ile Glu Asn Met Val Lys Gly Ile Leu Glu
Phe 20 25 30 Ile Ser Gln Ser Leu Glu Asn Gly Asp Arg Val Glu Val
Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu His His Arg Gln Pro Arg
Leu Gly Arg Asn Pro 50 55 60 Lys Thr Gly Asp Ser Val Asn Leu Ser
Ala Lys Ser Val Pro Tyr Phe 65 70 75 80 Lys Ala Gly Lys Glu Leu Lys
Ala Arg Val Asp Val Gln Ala 85 90 9495PRTAggregatibacter
actinomycetemcomitans 94Met Thr Lys Ser Glu Leu Ile Glu Leu Leu Val
Gln Lys Asn Ser Asn 1 5 10 15 Ile Pro Val Lys His Val Glu Glu Ala
Val Lys Ala Ile Leu Glu Gln 20 25 30 Met Ser Tyr Val Leu Glu His
Gly Glu Arg Ile Glu Val Arg Gly Phe 35 40 45 Gly Ser Phe Ser Leu
His Cys Arg Gln Pro Arg Ile Gly Arg Asn Pro 50 55 60 Lys Thr Gly
Glu Gln Val Lys Leu Asp Ala Lys Cys Val Pro Tyr Phe 65 70 75 80 Lys
Ala Gly Lys Glu Leu Arg Glu Arg Val Asp Val Tyr Ala Ala 85 90 95
9598PRTNeisseria gonorrhoeae 95Met Val Arg Leu Ala Glu Val Phe Ala
Ala Lys Asn Gly Thr His Leu 1 5 10 15 Leu Ala Lys Asp Val Glu Tyr
Ser Val Lys Val Leu Val Asp Thr Met 20 25 30 Thr Arg Ser Leu Ala
Arg Gly Gln Arg Ile Glu Ile Arg Gly Phe Gly 35 40 45 Ser Phe Asp
Leu Asn His Arg Pro Ala Arg Ile Gly Arg Asn Pro Lys 50 55 60 Thr
Gly Glu Arg Val Glu Val Pro Glu Lys His Val Pro His Phe Lys 65 70
75 80 Pro Gly Lys Glu Leu Arg Glu Arg Val Asp Leu Ala Leu Lys Glu
Asn 85 90 95 Ala Asn 96104PRTNeisseria meningitidis 96Met Thr Lys
Ser Glu Leu Met Val Arg Leu Ala Glu Val Phe Ala Ala 1 5 10 15 Lys
Asn Gly Thr His Leu Leu Ala Lys Asp Val Glu Tyr Ser Val Lys 20 25
30 Val Leu Val Asp Thr Met Thr Arg Ser Leu Ala Arg Gly Gln Arg Ile
35 40 45 Glu Ile Arg Gly Phe Gly Ser Phe Asp Leu Asn His Arg Pro
Ala Arg 50 55 60 Ile Gly Arg Asn Pro Lys Thr Gly Glu Arg Val Glu
Val Pro Glu Lys 65 70 75 80 His Val Pro His Phe Lys Pro Gly Lys Glu
Leu Arg Glu Arg Val Asp 85 90 95 Leu Ala Leu Lys Glu Asn Ala Asn
100 97107PRTBurkholderia cenocepacia 97Met Thr Lys Ser Glu Leu Val
Ala Gln Leu Ala Ser Arg Phe Pro Gln 1 5 10 15 Leu Val Leu Lys Asp
Ala Asp Phe Ala Val Lys Thr Met Leu Asp Ala 20 25 30 Met Ser Asp
Ala Leu Ala Lys Gly His Arg Ile Glu Ile Arg Gly Phe 35 40 45 Gly
Ser Phe Gly Leu Asn Arg Arg Pro Ala Arg Val Gly Arg Asn Pro 50 55
60 Lys Ser Gly Glu Lys Val Gln Val Pro Glu Lys Phe Val Pro His Phe
65 70 75 80 Lys Pro Gly Lys Glu Leu Arg Glu Arg Val Asp Gly Arg Ala
Gly Glu 85 90 95 Pro Leu Lys Ala Asp Asp Pro Asp Asp Asp Arg 100
105 98107PRTBurkholderia pseudomallei 98Met Thr Lys Ser Glu Leu Val
Ala Gln Leu Ala Ser Arg Phe Pro Gln 1 5 10 15 Leu Val Leu Lys Asp
Ala Asp Phe Ala Val Lys Thr Met Leu Asp Ala 20 25 30 Met Ser Asp
Ala Leu Ser Lys Gly His Arg Ile Glu Ile Arg Gly Phe 35 40 45 Gly
Ser Phe Gly Leu Asn Arg Arg Pro Ala Arg Val Gly Arg Asn Pro 50 55
60 Lys Ser Gly Glu Lys Val Gln Val Pro Glu Lys His Val Pro His Phe
65 70 75
80 Lys Pro Gly Lys Glu Leu Arg Glu Arg Val Asp Gly Arg Ala Gly Glu
85 90 95 Pro Leu Lys Asn Asp Glu Pro Glu Asp Ala Gln 100 105
99101PRTBordetella pertussis 99Met Thr Lys Ser Glu Leu Ile Ala Ala
Leu Ala Ala Arg Tyr Pro Gln 1 5 10 15 Leu Ala Ala Arg Asp Thr Asp
Tyr Ala Val Lys Thr Met Leu Asp Ala 20 25 30 Met Thr Gln Ala Leu
Ala Ser Gly Gln Arg Ile Glu Ile Arg Gly Phe 35 40 45 Gly Ser Phe
Ser Leu Ser Gln Arg Ser Pro Arg Ile Gly Arg Asn Pro 50 55 60 Lys
Ser Gly Glu Gln Val Leu Val Pro Gly Lys Gln Val Pro His Phe 65 70
75 80 Lys Pro Gly Lys Glu Leu Arg Glu Trp Val Asp Leu Val Gly Asn
Asp 85 90 95 Gln Gly Asp Asp Ser 100 100108PRTBorrelia burgdorferi
100Met Ser Phe Ser Arg Arg Pro Lys Val Thr Lys Ser Asp Ile Val Asp
1 5 10 15 Gln Ile Ser Leu Asn Ile Lys Asn Asn Asn Leu Lys Leu Glu
Lys Lys 20 25 30 Tyr Ile Arg Leu Val Ile Asp Ala Phe Phe Glu Glu
Leu Lys Ser Asn 35 40 45 Leu Cys Ser Asn Asn Val Ile Glu Phe Arg
Ser Phe Gly Thr Phe Glu 50 55 60 Val Arg Lys Arg Lys Gly Arg Leu
Asn Ala Arg Asn Pro Gln Thr Gly 65 70 75 80 Glu Tyr Val Lys Val Leu
Asp His His Val Ala Tyr Phe Arg Pro Gly 85 90 95 Lys Asp Leu Lys
Glu Arg Val Trp Gly Ile Lys Gly 100 105 10116PRTStreptococcus sp.
101Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln Thr Gly
1 5 10 15 10216PRTEscherichia coli 102Asp Leu Arg Asp Lys Asn Gln
Arg Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 10316PRTSalmonella
enterica 103Asp Leu Arg Asp Lys Asn Gln Arg Pro Gly Arg Asn Pro Lys
Thr Gly 1 5 10 15 10416PRTVibrio cholerae 104Asp Leu Arg Asp Lys
Asn Glu Arg Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
10516PRTPseudomonas aeruginosa 105Asp Leu Arg Asp Lys Arg Gln Arg
Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 10616PRTHaemophilus
influenzae 106Glu Leu Arg Asp Lys Ser Ser Arg Pro Gly Arg Asn Pro
Lys Thr Gly 1 5 10 15 10716PRTAggregatibacter actinomycetemcomitans
107Glu Leu Arg Asp Lys Ala Ser Arg Pro Gly Arg Asn Pro Lys Thr Gly
1 5 10 15 10816PRTMoraxella catarrhalis 108Glu Leu Lys Asp Lys Lys
Pro Arg Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 10916PRTNeisseria
gonorrhoeae 109Gln Leu Arg Asp Lys Pro Gln Arg Pro Gly Arg Asn Pro
Lys Thr Gly 1 5 10 15 11016PRTNeisseria meningitidis 110Gln Leu Arg
Asp Lys Pro Gln Arg Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
11116PRTBurkholderia cenocepacia 111Gln Leu Arg Asp Lys Pro Gln Arg
Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 11216PRTBurkholderia
pseudomallei 112Gln Leu Arg Asp Lys Pro Gln Arg Pro Gly Arg Asn Pro
Asn Thr Gly 1 5 10 15 11316PRTBordetella pertussis 113Gln Val Arg
Asp Lys Pro Pro Arg Pro Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
11416PRTPrevotella melaninogenica 114Glu Val Lys Lys Arg Leu Glu
Arg Val Met Val Asn Pro Ser Thr Gly 1 5 10 15 11516PRTPrevotella
intermedia 115Glu Val Lys Lys Arg Leu Glu Arg Ile Met Thr Asn Pro
Ala Thr Gly 1 5 10 15 11616PRTTreponema palladium 116Glu Ser Arg
Val Arg Lys Ala Ser Val Gly Lys Ser Ile Asn Thr Gly 1 5 10 15
11716PRTPrevotella melaninogenica 117Lys Val Gln Ala Val Lys Pro
Arg Glu Ser Val Asn Val Asn Thr Gly 1 5 10 15 11816PRTPrevotella
intermedia 118Lys Val Gln Ala Val Lys Pro Arg Glu Ser Val Asn Val
Asn Thr Gly 1 5 10 15 11916PRTStaphylococcus aureus 119Glu Val Arg
Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15
12016PRTEscherichia coli 120Lys Val Asn His Arg Ala Glu Arg Thr Gly
Arg Asn Pro Gln Thr Gly 1 5 10 15 12116PRTStaphylococcus
epidermidis 121Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro
Gln Thr Gly 1 5 10 15 12216PRTStreptococcus sobrinus 122Glu Val Arg
Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15
12316PRTStreptococcus pyogeneses 123Glu Val Arg Glu Arg Ala Ala Arg
Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15 12416PRTStreptococcus
gallolyticus 124Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro
Gln Thr Gly 1 5 10 15 12516PRTStreptococcus agalactiae 125Glu Val
Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15
12616PRTStreptococcus pneumoniae 126Glu Val Arg Glu Arg Ala Glu Arg
Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15 12716PRTStreptococcus
gordonii 127Glu Val Arg Glu Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln
Thr Gly 1 5 10 15 12816PRTStreptococcus mutans 128Glu Val Arg Glu
Arg Ala Ala Arg Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15
12916PRTEnterococcus faecalis 129Glu Val Arg Glu Arg Ala Ala Arg
Lys Gly Arg Asn Pro Gln Thr Gly 1 5 10 15 13016PRTHaemophilus
influenzae 130Lys Val Asn Glu Arg Ala Ala Arg Thr Gly Arg Asn Pro
Gln Thr Gly 1 5 10 15 13116PRTVibrio cholerae 131Lys Val Asn His
Arg Ser Ala Arg Thr Gly Arg Asn Pro Gln Thr Gly 1 5 10 15
13216PRTBordetella pertussis 132Ala Val Ser Ala Arg Ala Ala Arg Thr
Gly Arg Asn Pro Arg Thr Gly 1 5 10 15 13316PRTPseudomonas
aeruginosa 133Ala Val Lys Glu Arg Ala Ala Arg Thr Gly Arg Asn Pro
Gln Thr Gly 1 5 10 15 13416PRTAggregatibacter actinomycetemcomitans
134Ser Val Arg Thr Arg Ala Ala Arg Thr Gly Arg Asn Pro Lys Thr Gly
1 5 10 15 13516PRTPrevotella melaninogenica 135Ala Thr Thr Glu Arg
Pro Ala His Glu Gly Ile Asn Pro Arg Ser Lys 1 5 10 15
13616PRTPrevotella intermedia 136Ser Val Thr Glu Arg Pro Ala His
Glu Gly Ile Asn Pro Ala Thr Lys 1 5 10 15 13716PRTTreponema
denticola 137Phe Ala Val Leu His Gly Arg Lys Asn Ala Arg Asn Pro
Lys Thr Gly 1 5 10 15 13816PRTPorphyromonas gingivalis 138Ser Val
Ser Glu Arg Ala Ala Arg Lys Gly Ile Asn Pro Lys Thr Lys 1 5 10 15
13916PRTHelicobacter pylori 139Glu Thr Ala Glu Gln Lys Gly Lys Glu
Gly Lys Val Pro Gly Ser Asp 1 5 10 15 14017PRTPrevotella
melaninogenica 140Phe Ile Val Lys His Arg Ala Glu Lys Thr Ala Arg
Asn Ile Ser Lys 1 5 10 15 Asn 14117PRTPrevotella intermedia 141Phe
Ile Val Lys His Arg Ala Glu Lys Thr Ala Arg Asn Ile Ser Lys 1 5 10
15 Asn 14216PRTPorphyromonas gingivalis 142Ile Val Lys Glu Arg Ala
Glu Lys Thr Ala Arg Asn Ile Ser Lys Gln 1 5 10 15
14316PRTMycobacterium tuberculosis 143Glu Gln Arg Arg Arg Ala Ala
Arg Val Ala Arg Asn Pro Arg Thr Gly 1 5 10 15 14416PRTMycobacterium
smegmatis 144Glu Gln Arg Arg Arg Ala Ala Arg Val Ala Arg Asn Pro
Arg Thr Gly 1 5 10 15 14516PRTEscherichia coli 145Ser Leu His Tyr
Arg Ala Pro Arg Thr Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
14616PRTSalmonella enterica 146Ser Leu His Tyr Arg Ala Pro Arg Thr
Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 14716PRTVibrio cholerae
147Ser Leu His Tyr Arg Glu Pro Arg Val Gly Arg Asn Pro Lys Thr Gly
1 5 10 15 14816PRTEscherichia coli 148Ala Val Lys Glu Arg Ala Ala
Arg Thr Gly Arg Asn Pro Gln Thr Gly 1 5 10 15 14916PRTMoraxella
catarrhalis 149Ser Val Lys Glu Arg Ala Ala Arg Met Gly Arg Asn Pro
Lys Thr Gly 1 5 10 15 15016PRTPseudomonas aeruginosa 150Ser Leu His
Tyr Arg Ala Pro Arg Val Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
15116PRTHaemophilus influenzae 151Ser Leu His His Arg Gln Pro Arg
Leu Gly Arg Asn Pro Lys Thr Gly 1 5 10 15 15216PRTAggregatibacter
actinomycetemcomitans 152Ser Leu His Cys Arg Gln Pro Arg Ile Gly
Arg Asn Pro Lys Thr Gly 1 5 10 15 15316PRTNeisseria gonorrhoeae
153Asp Leu Asn His Arg Pro Ala Arg Ile Gly Arg Asn Pro Lys Thr Gly
1 5 10 15 15416PRTNeisseria meningitidis 154Asp Leu Asn His Arg Pro
Ala Arg Ile Gly Arg Asn Pro Lys Thr Gly 1 5 10 15
15516PRTBurkholderia cenocepacia 155Gly Leu Asn Arg Arg Pro Ala Arg
Val Gly Arg Asn Pro Lys Ser Gly 1 5 10 15 15616PRTBurkholderia
pseudomallei 156Gly Leu Asn Arg Arg Pro Ala Arg Val Gly Arg Asn Pro
Lys Ser Gly 1 5 10 15 15716PRTBordetella pertussis 157Ser Leu Ser
Gln Arg Ser Pro Arg Ile Gly Arg Asn Pro Lys Ser Gly 1 5 10 15
15816PRTMoraxella catarrhalis 158Cys Leu His His Arg Ser Ala Arg
Ile Ala Arg Asn Pro Arg Thr Gly 1 5 10 15 15917PRTBorrelia
burgdorferi 159Glu Val Arg Lys Arg Lys Gly Arg Leu Asn Ala Arg Asn
Pro Gln Thr 1 5 10 15 Gly 1606PRTStreptococcus pyogeneses 160Ala
Phe Lys Ala Gly Lys 1 5 1617PRTStreptococcus pyogeneses 161Ala Leu
Lys Asp Ala Val Lys 1 5 16220PRTStreptococcus pyogeneses 162Ile Ala
Ala Ser Lys Val Pro Ala Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15
Asp Ala Val Lys 20 1636PRTStreptococcus gallolyticus 163Ala Phe Lys
Ala Gly Lys 1 5 1647PRTStreptococcus gallolyticus 164Ala Leu Lys
Asp Ala Val Lys 1 5 16520PRTStreptococcus gallolyticus 165Ile Ala
Ala Ser Lys Val Pro Ala Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15
Asp Ala Val Lys 20 1666PRTStreptococcus sobrinus 166Ala Phe Lys Ala
Gly Lys 1 5 1677PRTStreptococcus sobrinus 167Ala Leu Lys Asp Ala
Val Lys 1 5 16820PRTStreptococcus sobrinus 168Ile Ala Ala Ser Lys
Val Pro Ala Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val
Lys 20 1696PRTStreptococcus agalactiae 169Ala Phe Lys Ala Gly Lys 1
5 1707PRTStreptococcus agalactiae 170Ala Leu Lys Asp Ala Val Lys 1
5 17120PRTStreptococcus agalactiae 171Ile Ala Ala Ser Lys Val Pro
Ala Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1726PRTStreptococcus pneumoniae 172Ala Phe Lys Ala Gly Lys 1 5
1737PRTStreptococcus pneumoniae 173Ala Leu Lys Asp Ala Val Lys 1 5
17420PRTStreptococcus pneumoniae 174Ile Ala Ala Ser Lys Val Pro Ala
Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1756PRTStreptococcus gordonii 175Ala Phe Lys Ala Gly Lys 1 5
1767PRTStreptococcus gordonii 176Ala Leu Lys Asp Ala Val Lys 1 5
17720PRTStreptococcus gordonii 177Ile Ala Ala Ser Lys Val Pro Ala
Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1786PRTStreptococcus mutans 178Ala Phe Lys Ala Gly Lys 1 5
1797PRTStreptococcus mutans 179Ala Leu Lys Asp Ala Val Lys 1 5
18020PRTStreptococcus mutans 180Ile Lys Ala Ser Lys Val Pro Ala Phe
Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1816PRTEnterococcus faecalis 181Ala Phe Lys Pro Gly Lys 1 5
1827PRTEnterococcus faecalis 182Ala Leu Lys Asp Ala Val Lys 1 5
18320PRTEnterococcus faecalis 183Ile Ala Ala Ser Lys Val Pro Ala
Phe Lys Pro Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1846PRTStaphylococcus aureus 184Ala Phe Lys Ala Gly Lys 1 5
1857PRTStaphylococcus aureus 185Ala Leu Lys Asp Ala Val Lys 1 5
18620PRTStaphylococcus aureus 186Ile Pro Ala Ser Lys Val Pro Ala
Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1876PRTStaphylococcus epidermidis 187Ala Phe Lys Ala Gly Lys 1 5
1887PRTStaphylococcus epidermidis 188Ala Leu Lys Asp Ala Val Lys 1
5 18920PRTStaphylococcus epidermidis 189Ile Pro Ala Ser Lys Val Pro
Ala Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
1906PRTHaemophilus influenzae 190Ala Phe Val Ser Gly Lys 1 5
1917PRTHaemophilus influenzae 191Ala Leu Lys Asp Ala Ile Lys 1 5
19220PRTHaemophilus influenzae 192Ile Ala Ala Ser Lys Val Pro Ala
Phe Val Ser Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Ile Lys 20
1936PRTAggregatibacter actinomycetemcomitans 193Ala Phe Val Ser Gly
Lys 1 5 1947PRTAggregatibacter actinomycetemcomitans 194Ala Leu Lys
Asp Ala Val Lys 1 5 19520PRTAggregatibacter actinomycetemcomitans
195Ile Ala Ala Ser Lys Val Pro Ala Phe Val Ser Gly Lys Ala Leu Lys
1 5 10 15 Asp Ala Val Lys 20 1966PRTVibrio cholerae 196Ala Phe Val
Ala Gly Lys 1 5 1977PRTVibrio cholerae 197Ala Leu Lys Asp Ala Ile
Lys 1 5 19820PRTVibrio cholerae 198Ile Ala Ala Ala Asn Val Pro Ala
Phe Val Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Ile Lys 20
1996PRTEscherichia coli 199Ala Phe Val Ser Gly Lys 1 5
2007PRTEscherichia coli 200Ala Leu Lys Asp Ala Val Lys 1 5
20120PRTEscherichia coli 201Ile Ala Ala Ala Asn Val Pro Ala Phe Val
Ser Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Lys 20
2026PRTPseudomonas aeruginosa 202Gly Phe Lys Ala Gly Lys 1 5
2037PRTPseudomonas aeruginosa 203Ala Leu Lys Asp Ala Val Asn 1 5
20420PRTPseudomonas aeruginosa 204Ile Ala Ala Ala Lys Ile Pro Gly
Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Asn 20
2056PRTEscherichia coli 205Ser Phe Arg Ala Gly Lys 1 5
2067PRTEscherichia coli 206Ala Leu Lys Asp Ala Val Asn 1 5
20720PRTEscherichia coli 207Ile Ala Ala Ala Lys Val Pro Ser Phe Arg
Ala Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Asn 20 2086PRTVibrio
cholerae 208Ser Phe Lys Ala Gly Lys 1 5 2097PRTVibrio cholerae
209Ala Leu Lys Asp Ala Cys Asn 1 5 21020PRTVibrio cholerae 210Ile
Ala Glu Ala Lys Val Pro Ser Phe Lys Ala Gly Lys Ala Leu Lys 1 5 10
15 Asp Ala Cys Asn 20 2116PRTBordetella pertussis 211Lys Phe Arg
Pro Gly Lys 1 5 2127PRTBordetella pertussis 212Ala Leu Lys Asp Ala
Val Asn 1 5 21320PRTBordetella pertussis 213Ile Lys Lys Ala Lys Val
Pro Lys Phe Arg Pro Gly Lys Ala Leu Lys 1 5 10 15 Asp Ala Val Asn
20 2146PRTPrevotella melaninogenica 214Lys Phe
Lys Ala Gly Ala 1 5 2158PRTPrevotella melaninogenica 215Glu Leu Ala
Asp Ala Val Asn Lys 1 5 21620PRTPrevotella melaninogenica 216Ala
Ala Lys Lys Val Ala Lys Phe Lys Ala Gly Ala Glu Leu Ala Asp 1 5 10
15 Ala Val Asn Lys 20 2176PRTPrevotella intermedia 217Lys Phe Lys
Pro Gly Ala 1 5 2188PRTPrevotella intermedia 218Glu Leu Ala Asp Ala
Val Asn Ala 1 5 21920PRTPrevotella intermedia 219Ala Ala Lys Lys
Val Ala Lys Phe Lys Pro Gly Ala Glu Leu Ala Asp 1 5 10 15 Ala Val
Asn Ala 20 2206PRTMoraxella catarrhalis 220Ser Phe Lys Ala Gly Lys
1 5 2217PRTMoraxella catarrhalis 221Val Leu Lys Glu Ser Val Asn 1 5
22220PRTMoraxella catarrhalis 222Ile Ala Ala Ser Lys Val Pro Ser
Phe Lys Ala Gly Lys Val Leu Lys 1 5 10 15 Glu Ser Val Asn 20
2236PRTPorphyromonas gingivalis 223Arg Phe Lys Pro Gly Ser 1 5
2245PRTPorphyromonas gingivalis 224Thr Leu Glu Leu Lys 1 5
22520PRTPorphyromonas gingivalis 225Ile Ser Ile Pro Ala Arg Lys Val
Val Arg Phe Lys Pro Gly Ser Thr 1 5 10 15 Leu Glu Leu Lys 20
2266PRTHelicobacter pylori 226Lys Phe Lys Pro Gly Lys 1 5
22710PRTHelicobacter pylori 227Thr Leu Lys Gln Lys Val Glu Glu Gly
Lys 1 5 10 22820PRTHelicobacter pylori 228Lys Arg Val Pro Lys Phe
Lys Pro Gly Lys Thr Leu Lys Gln Lys Val 1 5 10 15 Glu Glu Gly Lys
20 2296PRTPrevotella melaninogenica 229Ser Phe Lys Pro Ala Lys 1 5
2308PRTPrevotella melaninogenica 230Thr Phe Ile Glu Asp Met Lys Lys
1 5 23120PRTPrevotella melaninogenica 231Pro Ala His Asp Phe Pro
Ser Phe Lys Pro Ala Lys Thr Phe Ile Glu 1 5 10 15 Asp Met Lys Lys
20 2326PRTPrevotella intermedia 232Ser Phe Lys Pro Ala Lys 1 5
2338PRTPrevotella intermedia 233Thr Phe Ile Glu Asp Met Lys Lys 1 5
23420PRTPrevotella intermedia 234Pro Ala His Asp Phe Pro Ser Phe
Lys Pro Ala Lys Thr Phe Ile Glu 1 5 10 15 Asp Met Lys Lys 20
2356PRTPorphyromonas gingivalis 235Ala Phe Lys Pro Ser Lys 1 5
2369PRTPorphyromonas gingivalis 236Ile Phe Met Ser Gln Met Lys Gln
Asp 1 5 23720PRTPorphyromonas gingivalis 237Lys Arg Asn Ile Pro Ala
Phe Lys Pro Ser Lys Ile Phe Met Ser Gln 1 5 10 15 Met Lys Gln Asp
20 2386PRTMycobacterium tuberculosis 238Ala Phe Arg Pro Gly Ala 1 5
23922PRTMycobacterium tuberculosis 239Gln Phe Lys Ala Val Val Ser
Gly Ala Gln Arg Leu Pro Ala Glu Gly 1 5 10 15 Pro Ala Val Lys Arg
Gly 20 24020PRTMycobacterium tuberculosis 240Ala Lys Arg Pro Ala
Thr Lys Ala Pro Ala Lys Lys Ala Thr Ala Arg 1 5 10 15 Arg Gly Arg
Lys 20 2416PRTMycobacterium smegmatis 241Ala Phe Arg Pro Gly Ala 1
5 24222PRTMycobacterium smegmatis 242Gln Phe Lys Ala Val Ile Ser
Gly Ala Gln Lys Leu Pro Ala Asp Gly 1 5 10 15 Pro Ala Val Lys Arg
Gly 20 24320PRTMycobacterium smegmatis 243Thr Lys Ala Pro Ala Lys
Lys Ala Ala Ala Lys Lys Ala Pro Ala Lys 1 5 10 15 Lys Gly Arg Arg
20 2446PRTPrevotella melaninogenica 244Asn Phe Lys Pro Ala Ala 1 5
24514PRTPrevotella melaninogenica 245Thr Ile Lys Gly His Val Arg
Lys Gly Gly Gln Asp Asn Gly 1 5 10 24620PRTPrevotella
melaninogenica 246Asn Phe Lys Pro Ala Ala Thr Ile Lys Gly His Val
Arg Lys Gly Gly 1 5 10 15 Gln Asp Asn Gly 20 2476PRTPrevotella
intermedia 247Asn Phe Arg Ala Thr Ala 1 5 24812PRTPrevotella
intermedia 248Ser Val Lys Glu Lys Leu Lys Lys Gly Gly Ala Glu 1 5
10 24920PRTPrevotella intermedia 249Val Leu Asn Phe Arg Ala Thr Ala
Ser Val Lys Glu Lys Leu Lys Lys 1 5 10 15 Gly Gly Ala Glu 20
2506PRTEscherichia coli 250Thr Phe Arg Pro Gly Gln 1 5
25114PRTEscherichia coli 251Lys Leu Lys Ser Arg Val Glu Asn Ala Ser
Pro Lys Asp Glu 1 5 10 25220PRTEscherichia coli 252Thr Phe Arg Pro
Gly Gln Lys Leu Lys Ser Arg Val Glu Asn Ala Ser 1 5 10 15 Pro Lys
Asp Glu 20 2536PRTSalmonella enterica 253Thr Phe Arg Pro Gly Gln 1
5 25414PRTSalmonella enterica 254Lys Leu Lys Ser Arg Val Glu Asn
Ala Ser Pro Lys Glu Glu 1 5 10 25520PRTSalmonella enterica 255Thr
Phe Arg Pro Gly Gln Lys Leu Lys Ser Arg Val Glu Asn Ala Ser 1 5 10
15 Pro Lys Glu Glu 20 2566PRTVibrio cholerae 256Thr Phe Arg Pro Gly
Gln 1 5 25713PRTVibrio cholerae 257Lys Leu Lys Ala Arg Val Glu Asn
Ile Lys Val Glu Lys 1 5 10 25820PRTVibrio cholerae 258Val Thr Phe
Arg Pro Gly Gln Lys Leu Lys Ala Arg Val Glu Asn Ile 1 5 10 15 Lys
Val Glu Lys 20 2596PRTPseudomonas aeruginosa 259Thr Phe Arg Pro Gly
Gln 1 5 26014PRTPseudomonas aeruginosa 260Lys Leu Lys Ala Arg Val
Glu Ala Tyr Ala Gly Thr Lys Ser 1 5 10 26120PRTPseudomonas
aeruginosa 261Thr Phe Arg Pro Gly Gln Lys Leu Lys Ala Arg Val Glu
Ala Tyr Ala 1 5 10 15 Gly Thr Lys Ser 20 2626PRTBurkholderia
cenocepacia 262Thr Phe His Ala Ser Gln 1 5 26311PRTBurkholderia
cenocepacia 263Lys Leu Lys Ala Leu Val Glu Asn Gly Ala Glu 1 5 10
26420PRTBurkholderia cenocepacia 264Arg Val Val Thr Phe His Ala Ser
Gln Lys Leu Lys Ala Leu Val Glu 1 5 10 15 Asn Gly Ala Glu 20
2656PRTBurkholderia pseudomallei 265Thr Phe His Ala Ser Gln 1 5
26616PRTBurkholderia pseudomallei 266Lys Leu Lys Ala Leu Val Glu
Asn Gly Ala Glu Pro Asp Leu Ala Arg 1 5 10 15 26720PRTBurkholderia
pseudomallei 267His Ala Ser Gln Lys Leu Lys Ala Leu Val Glu Asn Gly
Ala Glu Pro 1 5 10 15 Asp Leu Ala Arg 20 2686PRTBordetella
pertussis 268Thr Phe His Ala Ser Gln 1 5 26919PRTBordetella
pertussis 269Lys Leu Lys Ser Val Val Glu Gln Pro Asn Ser Pro Pro
Asp Pro Ala 1 5 10 15 Ser Ala Glu 27020PRTBordetella pertussis
270Gln Lys Leu Lys Ser Val Val Glu Gln Pro Asn Ser Pro Pro Asp Pro
1 5 10 15 Ala Ser Ala Glu 20 2716PRTNeisseria gonorrhoeae 271Thr
Phe His Ala Ser Gln 1 5 27214PRTNeisseria gonorrhoeae 272Lys Leu
Lys Gly Met Val Glu His Tyr Tyr Asp Lys Gln Arg 1 5 10
27320PRTNeisseria gonorrhoeae 273Thr Phe His Ala Ser Gln Lys Leu
Lys Gly Met Val Glu His Tyr Tyr 1 5 10 15 Asp Lys Gln Arg 20
2746PRTNeisseria meningitidis 274Thr Phe His Ala Ser Gln 1 5
27514PRTNeisseria meningitidis 275Lys Leu Lys Ser Met Val Glu His
Tyr Tyr Asp Lys Gln Arg 1 5 10 27620PRTNeisseria meningitidis
276Thr Phe His Ala Ser Gln Lys Leu Lys Ser Met Val Glu His Tyr Tyr
1 5 10 15 Asp Lys Gln Arg 20 2776PRTHaemophilus influenzae 277Thr
Phe Lys Pro Gly Gln 1 5 27810PRTHaemophilus influenzae 278Lys Leu
Arg Ala Arg Val Glu Lys Thr Lys 1 5 10 27920PRTHaemophilus
influenzae 279Arg Arg Val Val Thr Phe Lys Pro Gly Gln Lys Leu Arg
Ala Arg Val 1 5 10 15 Glu Lys Thr Lys 20 2806PRTAggregatibacter
actinomycetemcomitans 280Val Phe Lys Pro Gly Gln 1 5
28113PRTAggregatibacter actinomycetemcomitans 281Lys Leu Arg Asn
Arg Val Glu Lys Val Lys Pro Lys Ala 1 5 10 28220PRTAggregatibacter
actinomycetemcomitans 282Val Val Phe Lys Pro Gly Gln Lys Leu Arg
Asn Arg Val Glu Lys Val 1 5 10 15 Lys Pro Lys Ala 20
2836PRTMoraxella catarrhalis 283Thr Phe Lys Ala Gly Gln 1 5
28412PRTMoraxella catarrhalis 284Lys Leu Arg Gly Trp Ile Asp Ser
Gln Asn Glu Gly 1 5 10 28520PRTMoraxella catarrhalis 285Val Val Thr
Phe Lys Ala Gly Gln Lys Leu Arg Gly Trp Ile Asp Ser 1 5 10 15 Gln
Asn Glu Gly 20 2866PRTTreponema palladium 286Val Phe Arg Pro Ser
Lys 1 5 28717PRTTreponema palladium 287Arg Leu Lys Ser Ala Val Arg
Gly Tyr Arg Ser Gly Glu Val Gly Ala 1 5 10 15 Asp 28820PRTTreponema
palladium 288Pro Ser Lys Arg Leu Lys Ser Ala Val Arg Gly Tyr Arg
Ser Gly Glu 1 5 10 15 Val Gly Ala Asp 20 2896PRTPrevotella
melaninogenica 289Ser Phe Thr Pro Asp Thr 1 5 29022PRTPrevotella
melaninogenica 290Val Met Lys Glu Leu Val Asn Lys Pro Phe Ser Gln
Phe Glu Thr Val 1 5 10 15 Val Ile Asn Asp Gly Val 20
29120PRTPrevotella melaninogenica 291Met Gln Ala Gly Asp Thr Met
Lys Val Pro Lys Val Glu Leu Arg Pro 1 5 10 15 Glu Tyr Arg Lys 20
2926PRTPrevotella intermedia 292Ser Phe Thr Pro Asp Ala 1 5
29322PRTPrevotella intermedia 293Thr Met Lys Glu Leu Val Asn Lys
Pro Phe Ala Gln Phe Glu Thr Val 1 5 10 15 Val Leu Asn Asp Gly Val
20 29420PRTPrevotella intermedia 294Ser Ala Gly Asp Thr Met Lys Val
Pro Lys Val Glu Leu Arg Pro Gln 1 5 10 15 Tyr Arg Thr Lys 20
2956PRTEscherichia coli 295His Phe Lys Pro Gly Lys 1 5
29610PRTEscherichia coli 296Glu Leu Arg Asp Arg Ala Asn Ile Tyr Gly
1 5 10 29720PRTEscherichia coli 297Lys Tyr Val Pro His Phe Lys Pro
Gly Lys Glu Leu Arg Asp Arg Ala 1 5 10 15 Asn Ile Tyr Gly 20
2986PRTSalmonella enterica 298His Phe Lys Pro Gly Lys 1 5
29910PRTSalmonella enterica 299Glu Leu Arg Asp Arg Ala Asn Ile Tyr
Gly 1 5 10 30020PRTSalmonella enterica 300Lys Tyr Val Pro His Phe
Lys Pro Gly Lys Glu Leu Arg Asp Arg Ala 1 5 10 15 Asn Ile Tyr Gly
20 3016PRTVibrio cholerae 301His Phe Lys Pro Gly Lys 1 5
3028PRTVibrio cholerae 302Glu Leu Arg Glu Arg Val Asn Leu 1 5
30320PRTVibrio cholerae 303Glu Gly Lys Tyr Val Pro His Phe Lys Pro
Gly Lys Glu Leu Arg Glu 1 5 10 15 Arg Val Asn Leu 20
3046PRTPseudomonas aeruginosa 304His Phe Lys Pro Gly Lys 1 5
30510PRTPseudomonas aeruginosa 305Glu Leu Arg Asp Arg Val Asn Glu
Pro Glu 1 5 10 30620PRTPseudomonas aeruginosa 306Lys Phe Val Pro
His Phe Lys Pro Gly Lys Glu Leu Arg Asp Arg Val 1 5 10 15 Asn Glu
Pro Glu 20 3076PRTHaemophilus influenzae 307Tyr Phe Lys Ala Gly Lys
1 5 30810PRTHaemophilus influenzae 308Glu Leu Lys Ala Arg Val Asp
Val Gln Ala 1 5 10 30920PRTHaemophilus influenzae 309Lys Ser Val
Pro Tyr Phe Lys Ala Gly Lys Glu Leu Lys Ala Arg Val 1 5 10 15 Asp
Val Gln Ala 20 3106PRTAggregatibacter actinomycetemcomitans 310Tyr
Phe Lys Ala Gly Lys 1 5 31111PRTAggregatibacter
actinomycetemcomitans 311Glu Leu Arg Glu Arg Val Asp Val Tyr Ala
Ala 1 5 10 31220PRTAggregatibacter actinomycetemcomitans 312Cys Val
Pro Tyr Phe Lys Ala Gly Lys Glu Leu Arg Glu Arg Val Asp 1 5 10 15
Val Tyr Ala Ala 20 3136PRTNeisseria gonorrhoeae 313His Phe Lys Pro
Gly Lys 1 5 31415PRTNeisseria gonorrhoeae 314Glu Leu Arg Glu Arg
Val Asp Leu Ala Leu Lys Glu Asn Ala Asn 1 5 10 15 31520PRTNeisseria
gonorrhoeae 315Phe Lys Pro Gly Lys Glu Leu Arg Glu Arg Val Asp Leu
Ala Leu Lys 1 5 10 15 Glu Asn Ala Asn 20 3166PRTNeisseria
meningitidis 316His Phe Lys Pro Gly Lys 1 5 31715PRTNeisseria
meningitidis 317Glu Leu Arg Glu Arg Val Asp Leu Ala Leu Lys Glu Asn
Ala Asn 1 5 10 15 31820PRTNeisseria meningitidis 318Phe Lys Pro Gly
Lys Glu Leu Arg Glu Arg Val Asp Leu Ala Leu Lys 1 5 10 15 Glu Asn
Ala Asn 20 3196PRTBurkholderia cenocepacia 319His Phe Lys Pro Gly
Lys 1 5 32023PRTBurkholderia cenocepacia 320Glu Leu Arg Glu Arg Val
Asp Gly Arg Ala Gly Glu Pro Leu Lys Ala 1 5 10 15 Asp Asp Pro Asp
Asp Asp Arg 20 32120PRTBurkholderia cenocepacia 321Glu Arg Val Asp
Gly Arg Ala Gly Glu Pro Leu Lys Ala Asp Asp Pro 1 5 10 15 Asp Asp
Asp Arg 20 3226PRTBurkholderia pseudomallei 322His Phe Lys Pro Gly
Lys 1 5 32323PRTBurkholderia pseudomallei 323Glu Leu Arg Glu Arg
Val Asp Gly Arg Ala Gly Glu Pro Leu Lys Asn 1 5 10 15 Asp Glu Pro
Glu Asp Ala Gln 20 32420PRTBurkholderia pseudomallei 324Glu Arg Val
Asp Gly Arg Ala Gly Glu Pro Leu Lys Asn Asp Glu Pro 1 5 10 15 Glu
Asp Ala Gln 20 3256PRTBordetella pertussis 325His Phe Lys Ala Gly
Lys 1 5 32622PRTBordetella pertussis 326Glu Leu Arg Glu Trp Val Asp
Leu Val Gly Asn Asp Gln Gly Asp Asp 1 5 10 15 Ser Ser Asn Gly Ser
Ser 20 32720PRTBordetella pertussis 327Asp Ser Ser Asn Gly Ser Ser
Asp Pro Leu Gln Ser Val Met Asp Met 1 5 10 15 His Ala Met His 20
3286PRTMoraxella catarrhalis 328Tyr Phe Lys Pro Gly Lys 1 5
32911PRTMoraxella catarrhalis 329Ala Leu Arg Glu Ser Val Asn Leu
Val Asn Asp 1 5 10 33020PRTMoraxella catarrhalis 330Ala Thr Pro Tyr
Phe Lys Pro Gly Lys Ala Leu Arg Glu Ser Val Asn 1 5 10 15 Leu Val
Asn Asp 20 3316PRTBorrelia burgdorferi 331Tyr Phe Arg Pro Gly Lys 1
5 33211PRTBorrelia burgdorferi 332Asp Leu Lys Glu Arg Val Trp Gly
Ile Lys Gly 1 5 10 33320PRTBorrelia burgdorferi 333His Val Ala Tyr
Phe Arg Pro Gly Lys Asp Leu Lys Glu Arg Val Trp 1 5 10 15 Gly Ile
Lys Gly 20 3346PRTTreponema denticola 334Arg Phe Lys Pro Gly Lys 1
5 33517PRTTreponema denticola 335Glu Leu Lys Glu Ala Leu His Lys
Ile Asp Thr Gln Glu Leu Ile Glu 1 5 10 15 Ser 33620PRTTreponema
denticola 336Pro Gly Lys Glu Leu Lys Glu Ala Leu His Lys Ile Asp
Thr Gln Glu 1 5 10 15 Leu Ile Glu Ser 20 33712PRTEscherichia coli
337Gly Arg Asn Pro Lys Thr Gly Glu Asp Ile Pro Ile 1 5 10
33812PRTEscherichia coli 338Gly Arg Asn Pro Lys Thr Gly Asp Lys Val
Glu Leu 1 5 10 33912PRTEscherichia coli 339Gly Arg Asn Pro Gln Thr
Gly Lys Glu Ile Lys Ile 1 5 10 34012PRTEscherichia coli 340Gly Arg
Asn Pro Gln Thr Gly Lys Glu Ile Thr Ile 1 5 10
* * * * *