U.S. patent application number 15/370144 was filed with the patent office on 2017-08-31 for prevention, treatment and diagnosis of p.gingivalis infection.
This patent application is currently assigned to Oral Health Australia Pty Ltd.. The applicant listed for this patent is Oral Health Australia Pty Ltd.. Invention is credited to Keith J. Cross, Eric Charles Reynolds, Neil Martin O'Brien Simpson, Nada Slakeski.
Application Number | 20170247420 15/370144 |
Document ID | / |
Family ID | 41720713 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170247420 |
Kind Code |
A1 |
Reynolds; Eric Charles ; et
al. |
August 31, 2017 |
PREVENTION, TREATMENT AND DIAGNOSIS OF P.GINGIVALIS INFECTION
Abstract
The invention relates to generation and use of cellular and
humoral responses for the prevention and treatment of P. gingivalis
related conditions and diseases.
Inventors: |
Reynolds; Eric Charles;
(Melbourne, AU) ; Simpson; Neil Martin O'Brien;
(Melbourne, AU) ; Cross; Keith J.; (Melbourne,
AU) ; Slakeski; Nada; (Melbourne, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Oral Health Australia Pty Ltd. |
Carlton |
|
AU |
|
|
Assignee: |
Oral Health Australia Pty
Ltd.
Carlton
AU
|
Family ID: |
41720713 |
Appl. No.: |
15/370144 |
Filed: |
December 6, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14487461 |
Sep 16, 2014 |
9518109 |
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15370144 |
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13060653 |
Feb 24, 2011 |
8871213 |
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PCT/AU09/01112 |
Aug 28, 2009 |
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14487461 |
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61151132 |
Feb 9, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/02 20180101; C12Y
304/21 20130101; C07K 2317/34 20130101; A61P 43/00 20180101; A61K
2039/53 20130101; C12Y 304/21004 20130101; A61K 2039/505 20130101;
C12Y 306/05002 20130101; G01N 2333/95 20130101; A61K 39/0216
20130101; A61P 31/04 20180101; A61K 2039/55566 20130101; C07K
16/1257 20130101; C12N 9/52 20130101; C07K 2319/00 20130101; C07K
16/1203 20130101; C12N 9/14 20130101; G01N 33/56955 20130101; C07K
2317/76 20130101; C07K 14/195 20130101 |
International
Class: |
C07K 14/195 20060101
C07K014/195; C12N 9/52 20060101 C12N009/52; G01N 33/569 20060101
G01N033/569; A61K 39/02 20060101 A61K039/02; C07K 16/12 20060101
C07K016/12 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 29, 2008 |
AU |
2008904476 |
Oct 23, 2008 |
AU |
2008905483 |
Jun 30, 2009 |
AU |
2009903052 |
Claims
1.-70. (canceled)
71. A chimeric or fusion protein for inducing an immune response to
P. gingivalis, the protein comprising a first peptide joined
directly or through a linker to a second peptide or polypeptide,
wherein: (A) said first peptide comprises a region of a P.
gingivalis trypsin-like enzyme selected from the group consisting
of: (i) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:1; and (ii) part of,
or all of a sequence that is the same as, or homologous to the
sequence shown in SEQ ID No:2; and (B) said second peptide or
polypeptide comprises an adhesin domain of P. gingivalis, or
fragment thereof, selected from the group consisting of: (i) part
of, or all of a sequence that is the same as, or homologous to the
sequence of an adhesin domain of the Lys-X-proteinase of P.
gingivalis; (ii) part of, or all of a sequence that is the same as,
or homologous to the sequence of an adhesin domain of the
Arg-X-proteinase of P. gingivalis; and (iii) part of, or all of a
sequence that is the same as, or homologous to the sequence of a
HagA adhesin domain of P. gingivalis.
72. The chimeric or fusion protein according to claim 71, wherein
said first peptide comprises a sequence selected from the group
consisting of SEQ ID NOs: 3-34 and sequences at least 90%
homologous thereto.
73. The chimeric or fusion protein according to claim 71, wherein
said second peptide or polypeptide comprises a sequence selected
from the group consisting of SEQ ID NOs: 35-39.
74. The chimeric or fusion protein according to claim 71, wherein:
(a) said first peptide comprises a sequence that is the same as or
homologous to a sequence selected from the group consisting of SEQ
ID NOs: 27-30 and said second peptide or polypeptide comprises a
sequence that is the same as or homologous to a sequence selected
from the group consisting of SEQ ID NOs: 36-37; or (b) said first
peptide comprises a sequence that is the same as or homologous to a
sequence selected from the group consisting of SEQ ID NOs: 31-34
and said second peptide or polypeptide comprises a sequence that is
the same as or homologous to SEQ ID NO:39.
75. A chimeric or fusion protein according to claim 71, wherein:
the C-terminal residue of said first peptide is covalently linked
directly or through a linker that is either (i) up to 15 amino
acids in length, or (ii) less than 5 amino acids in length, to
either (a) the N-terminal residue or (b) the C-terminal residue of
said second peptide or polypeptide, or the N-terminal residue of
said first peptide is covalently linked directly or through a
linker that is either (i) up to 15 amino acids in length, or (ii)
less than 5 amino acids in length, to either (a) the N-terminal
residue or (b) the C-terminal residue of said second peptide or
polypeptide.
76. A composition comprising a chimeric or fusion protein according
to claim 71.
77. The composition according to claim 76, further comprising an
adjuvant.
78. A method of preventing or reducing the incidence or severity of
a P. gingivalis-related condition or disease in a subject,
comprising administering to the subject a chimeric or fusion
protein of claim 71.
79. An antibody raised against the chimeric or fusion protein of
claim 71.
80. The antibody according to claim 79, wherein the antibody is a
monoclonal antibody.
81. A method of preventing or reducing the severity of a P.
gingivalis-related disease or condition in a subject, comprising
administering to the subject the antibody of claim 79.
82. A nucleic acid molecule comprising a sequence encoding the
chimeric or fusion protein of claim 71.
83. A method for the diagnosis or monitoring of a P.
gingivalis-related condition or disease in a subject, which
comprises assaying a biological sample from said subject with the
chimeric or fusion protein of claim 71 to detect anti-P. gingivalis
antibodies.
84. A method for the diagnosis or monitoring of a P.
gingivalis-related condition or disease in a subject, which
comprises assaying a biological sample from said subject with the
antibody according to claim 79, to detect the presence of P.
gingivalis.
85. A chimeric or fusion protein according to claim 71 wherein the
second peptide or polypeptide comprises or consists of a sequence
shown in one or more of SEQ ID No: 69 to 79 or one or more of SEQ
ID No: 83 to 85.
86. A chimeric or fusion protein according to claim 90 wherein the
first peptide comprises or consists of a sequence shown in one of
more of SEQ ID No: 3 to 26 or one or more of SEQ ID No: 64 to
68.
87. A chimeric or fusion protein for inducing an immune response to
P. gingivalis, comprising: (A) a first peptide with a sequence from
a P. gingivalis trypsin-like enzyme selected from the group
consisting of KAS1 (SEQ ID NO: 27), KAS2 (SEQ ID NO: 28), KAS3 (SEQ
ID NO: 29), PAS1K (SEQ ID NO: 30), KAS4 (SEQ ID NO: 64), KAS5 (SEQ
ID NO: 65), KAS6 (SEQ ID NO: 66), RAS1 (SEQ ID NO: 31), RAS2 (SEQ
ID NO: 32), RAS3 (SEQ ID NO: 33), KAS4 (SEQ ID NO: 67), RAS5 (SEQ
ID NO: 68), and PAS1R (SEQ ID NO: 34), and (B) a second peptide or
polypeptide comprises an adhesin domain of P. gingivalis, or
fragment thereof, (i) selected from the group consisting of KA1
(SEQ ID NO: 35), KA2 (SEQ ID NO: 40), KA3 (SEQ ID NO: 41), KA4 (SEQ
ID NO: 42), KA5 (SEQ ID NO: 43), RA1 (SEQ ID NO: 38), RA2 (SEQ ID
NO: 44), RA3 (SEQ ID NO: 45), RA4 (SEQ ID NO: 46), HA1 (SEQ ID NO:
80), HA1* (SEQ ID NO: 81), HA1** (SEQ ID NO: 82), or (ii) selected
from the group consisting of SEQ ID NOs.: 69-79, or (iii) selected
from the group consisting of SEQ ID NOs.: 83-85.
88. A chimeric or fusion protein for inducing an immune response to
P. gingivalis, comprising at least two sequences that are the same
as, or homologous to a sequence from a P. gingivalis trypsin-like
enzyme selected from the group consisting of KAS1 (SEQ ID NO: 27),
KAS2 (SEQ ID NO: 28), KAS3 (SEQ ID NO: 29), PAS1K (SEQ ID NO: 30),
KAS4 (SEQ ID NO: 64), KAS5 (SEQ ID NO: 65), KAS6 (SEQ ID NO: 66),
RAS1 (SEQ ID NO: 31), KAS2 (SEQ ID NO: 32), RAS3 (SEQ ID NO: 33),
RAS4 (SEQ ID NO: 67), RAS5 (SEQ ID NO: 68), and PAS1R (SEQ ID NO:
34); and at least one sequence that comprises an adhesin domain of
P. gingivalis, or fragment thereof, selected from: (i) part of, or
all of a sequence that is the same as, or homologous to the
sequence of an adhesin domain of the Lys-X-proteinase of P.
gingivalis; and (ii) part of, or all of a sequence that is the same
as, or homologous to the sequence of an adhesin domain of the
Arg-X-proteinase of P. gingivalis; and (iii) part of, or all of a
sequence that is the same as, or homologous to the sequence of a
HagA adhesin domain of P. gingivalis.
89. The chimeric or fusion protein according to claim 74 wherein
said first peptide comprises a sequence that is the same as or
homologous to the sequence set out in SEQ ID NO:28 and said second
peptide or polypeptide comprises a sequence that is the same as or
homologous to the sequence shown in SEQ ID No: 37.
90. The chimeric or fusion protein according to claim 74 wherein
said first peptide comprises a sequence that is the same as or
homologous to the sequence set out in SEQ ID NO:27 and said second
peptide or polypeptide comprises a sequence that is the same as or
homologous to the sequence shown in SEQ ID No: 36.
Description
FIELD OF THE INVENTION
[0001] The invention relates to peptides and chimeric or fusion
proteins and to the use of these proteins to elicit cellular and
humoral responses for the prevention and treatment of P.
gingivalis-related conditions and diseases.
BACKGROUND OF THE INVENTION
[0002] Chronic periodontitis is an inflammatory disease of the
supporting tissues of the teeth leading to resorption of alveolar
bone and eventual tooth loss. The disease is a major public health
problem in all societies and is estimated to affect up to 15% of
the adult population with severe forms affecting 5-6%.
[0003] The development and progression of chronic periodontitis has
been associated with specific Gram-negative bacteria in subgingival
plaque. The presence of Porphyromonas gingivalis in subgingival
plaque has been strongly associated with disease.
[0004] The persistence of P. gingivalis in subgingival plaque from
periodontitis patients after treatment (scaling and root planing)
has been reported to be significantly associated with progressive
alveolar bone loss. Furthermore an increase in P. gingivalis cell
numbers in subgingival plaque has been shown to correlate with
disease severity as measured by attachment loss, periodontal pocket
depth and bleeding on probing.
[0005] Oral infection with P. gingivalis has been shown to induce
periodontal bone loss in mice, rats and non-human primates. In
addition, there has been increasing linkage of periodontal disease,
and of P. gingivalis infection, with cardiovascular diseases and
certain cancers.
[0006] A number of virulence factors have been reported to
contribute to the pathogenicity of P. gingivalis including; LPS,
fimbriae, hemagglutinin, hemolysin and extracellular hydrolytic
enzymes (especially the Arg-X and Lys-X specific proteinases),
otherwise known as "P. gingivalis trypsin-like enzymes".
[0007] The magnitude of the public health problem is such that
there is a need for an antiserum, particularly specific antibodies
that provide a strong protective response to P. gingivalis
infection and means for providing same.
[0008] One problem has been that it is not clear how to obtain a
strong protective response to P. gingivalis infection where there
are a plethora of virulence factors to select from.
[0009] The relative immunogenicity of epitopes amongst virulence
factors is not well understood, nor is the relative immunogenicity
of epitopes on a given factor, particularly where it is not clear
as to whether further epitopes remain to be identified.
[0010] One particular problem has been that many virulence factors
are formed from multiple domains and are difficult to express so as
to present a conformation approaching that found on P. gingivalis.
Further, when these domains are expressed as discrete units i.e. in
isolation of other virulence factor domains, they tend to fold into
a conformation distinguished from that found on P. gingivalis.
[0011] Further, of the many different options for modifying the
immunogenicity of a virulence factor it is not clear which would be
most likely to provide for a protective immune response.
[0012] In work leading to the present invention the inventors have
identified peptides having an amino acid sequence that is the same
as, or that shares homology with, an amino acid sequence that forms
a region of a P. gingivalis trypsin-like enzyme, said region
defining a site in said enzyme for cleavage of a peptide bond
located C-terminal to Lys or Arg in a peptide containing Lys or
Arg, and incorporated such a peptide into a chimeric or fusion
protein which, when used as a vaccine, provides better protection
against periodontal tissue destruction than purified
proteinase-adhesin complex formed from native P. gingivalis
trypsin-like enzyme or killed whole cells.
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention provides a chimeric or
fusion protein for inducing an immune response to P. gingivalis,
the protein including a first peptide joined directly or through a
linker to a second peptide, wherein: [0014] (A) said first peptide
includes: [0015] (i) part of, or all of a sequence that is the same
as, or homologous to the sequence shown in SEQ ID No:1; or [0016]
(ii) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:2; and [0017] (B)
said second peptide includes: [0018] (i) part of, or all of a
sequence that is the same as, or homologous to the sequence of an
adhesin domain of the Lys-X-proteinase of P. gingivalis; or [0019]
(ii) part of, or all of a sequence that is the same as, or
homologous to the sequence of an adhesin domain of the
Arg-X-proteinase of P. gingivalis; or [0020] (iii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
a HagA adhesin domain of P. gingivalis.
[0021] In another aspect, the invention provides a chimeric or
fusion protein for inducing an immune response to P. gingivalis,
the protein including a peptide joined directly or through a linker
to a polypeptide, wherein: [0022] (A) said peptide includes: [0023]
(i) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:1; or [0024] (ii)
part of, or all of a sequence that is the same as, or homologous to
the sequence shown in SEQ ID No:2; and [0025] (B) said polypeptide
includes: [0026] (i) part of, or all of a sequence that is the same
as, or homologous to the sequence of an adhesin domain of the
Lys-X-proteinase of P. gingivalis; or [0027] (ii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
an adhesin domain of the Arg-X-proteinase of P. gingivalis; or
[0028] (iii) part of, or all of a sequence that is the same as, or
homologous to the sequence of a HagA adhesin domain of P.
gingivalis.
[0029] In another aspect, the invention provides a peptide for
inducing an immune response to P. gingivalis the peptide having a
sequence: [0030] (i) that is the same as, or homologous to the
sequence shown in one of SEQ ID No: 64 to 66; and [0031] (ii) that
is the same as, or homologous to the sequence shown in SEQ ID No:
67 or 68.
[0032] In one aspect, the peptide having a sequence that is the
same as or homologous to sequence shown in one of SEQ ID No: 64 to
68 may be provided in the form of a chimeric or fusion protein in
which the peptide is joined directly or through a linker to a
second peptide, wherein the second peptide includes: [0033] (i)
part of, or all of a sequence that is the same as, or homologous to
the sequence of an adhesin domain of the Lys-X-proteinase of P.
gingivalis; or [0034] (ii) part of, or all of a sequence that is
the same as, or homologous to the sequence of an adhesin domain of
the Arg-X-proteinase of P. gingivalis; or [0035] (iii) part of, or
all of a sequence that is the same as, or homologous to the
sequence of a HagA adhesin domain of P. gingivalis.
[0036] In yet another aspect, the invention provides a composition
such as an antigenic composition, particularly a vaccine
composition, including a chimeric or fusion protein or peptide as
broadly described above, optionally in association with an
adjuvant.
[0037] In this aspect, the invention also provides a method of
preventing or reducing the incidence or severity of a P.
gingivalis-related condition or disease in a subject, which
comprises administering to the subject a chimeric or fusion protein
as described above, or a composition as described above.
[0038] In this aspect, the invention further provides the use of a
chimeric or fusion protein as described above, or a composition as
described above, in, or in the manufacture of a medicament for
preventing or reducing the incidence or severity of a P.
gingivalis-related condition or disease in a subject.
[0039] In another aspect, the invention provides an antibody,
particularly a monoclonal antibody, raised against a chimeric or
fusion protein or peptide as broadly described above.
[0040] In this aspect, the invention also provides a method of
preventing or reducing the severity of a P. gingivalis-related
disease or condition in a subject, which comprises administering to
the subject an antibody as described above.
[0041] In this aspect, the invention further provides the use of an
antibody as described above in, or in the manufacture of a
medicament for preventing or reducing the incidence or severity of
a P. gingivalis-related condition or disease in a subject.
[0042] In yet another aspect, the invention also provides a nucleic
acid molecule including a nucleotide sequence encoding a chimeric
or fusion protein as broadly described above, optionally
operatively linked to at least one regulatory element.
[0043] In this aspect, the invention further provides a vector
including such a nucleic acid molecule, as well as a prokaryotic or
eukaryotic cell including such a nucleic acid molecule.
[0044] In this aspect, the invention also provides a method of
preventing or reducing the incidence or severity of a P.
gingivalis-related condition or disease in a subject, which
comprises administering to the subject a nucleic acid molecule as
described above, a vector as described above, or a prokaryotic or
eukaryotic cell as described above.
[0045] In this aspect, the invention further provides the use of a
nucleic acid molecule as described above, a vector as described
above, or a prokaryotic or eukaryotic cell as described above, in,
or in the manufacture of a medicament for preventing or reducing
the severity of a P. gingivalis-related disease or condition in a
subject.
[0046] In a further aspect, the invention provides a method for the
diagnosis or monitoring of a P. gingivalis-related condition or
disease in a subject, which comprises use of a chimeric or fusion
protein as described above to detect anti-P. gingivalis antibodies
in a biological sample from said subject.
[0047] In this aspect, the invention also provides the use of a
chimeric or fusion protein as described above, to detect anti-P.
gingivalis antibodies in a biological sample from a subject.
[0048] In yet another aspect, the invention provides a method for
the diagnosis or monitoring of a P. gingivalis-related condition or
disease in a subject, which comprises use of an antibody as
described above, to detect the presence of P. gingivalis in a
biological sample from said subject.
[0049] In this aspect, the invention also provides the use of an
antibody as described above, to detect the presence of P.
gingivalis in a biological sample from a subject.
[0050] In another aspect, the invention provides a use of a peptide
having part of, or all of a sequence that is the same as, or
homologous to a sequence of a P. gingivalis Lys-X or Arg-X
proteinase, or a nucleic acid encoding said peptide for the
manufacture of a chimeric or fusion protein for inducing an immune
response to P. gingivalis. In this aspect the peptide may have a
sequence shown in one of SEQ ID No: 17, 18, 25 or 26.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIG. 1 shows a Coomassie blue stain of the SDS-PAGE gel of
recombinant Kgp Proteins. Lane 1=KAS2-KLA1, Lane 2=KLA1, Lane
3=KsA1, Lane 4=KAS1-KsA1. Molecular weight markers are indicated as
kDa.
[0052] FIGS. 2A-2B show antibody recognition of KAS2 peptide and
formalin killed P. gingivalis W50 cells. (FIG. 2A) KAS2 peptide was
probed with antisera raised to formalin killed P. gingivalis W50
cells (FK-W50), recombinant proteins KAS1-KsA1, KAS2-KLA1, and
synthetic KAS2-DT conjugate and PBS in an ELISA. (FIG. 2B) formalin
killed P. gingivalis W50 cells were probed with antisera raised to
formalin killed P. gingivalis W50 cells (FK-W50), recombinant
proteins KAS1-KsA1, KAS2-KLA1, KLA1 and PBS in an ELISA. Antibody
responses are expressed as the ELISA titre OD.sub.415 obtained
minus double the background level, with each titre representing the
mean.+-.standard deviation of three values.
[0053] FIG. 3 shows P. gingivalis-induced horizontal bone loss of
maxillae molars of mice immunised with the recombinant proteins and
recombinant chimera proteins, formalin-killed P. gingivalis and
adjuvant alone (PBS, IFA) or non-orally infected (non-challenged)
mice. In this figure KAS2-KLA1 is shown as AS2-LA1, KLA1 is shown
as LA1, KAS1-KsA1 is shown as AS1-sA1, KsA1 is shown as sA1.
Measurement of bone loss is the mean of the area measured in
millimeters squared (mm2) from the cementoenamel junction (CEJ) to
the alveolar bone crest (ABC) of the buccal side of each maxillary
molar of both the left and right maxillae. Data was normally
distributed as measured by Levene's homogeneity of variance and are
presented as mean (n=12) in mm2 and were analyzed using the One-Way
analysis of variance and Dunnett's T3 test. *, indicates group has
significantly (P<0.001) less bone loss than control (infected)
group. .dagger., indicates group has significantly (P<0.001)
more bone loss than the AS2-LA1 group.
[0054] FIGS. 4A-4B show serum antibody subclass responses of
immunised mice in the periodontitis model. Sera from mice; FIG. 4A
(pre-oral inoculation) and FIG. 4B (post-oral inoculation)
immunised with recombinant proteins KsA1, KLA1, KAS1-KsA1 and
KAS2-KLA1 and formalin killed P. gingivalis strain W50 were used in
the ELISA with the formalin killed P. gingivalis strain W50 as the
adsorbed antigen. Antibody responses IgG (black bars), IgG1 (grey
bars), IgG2a (white bars), IgG2b (horizontal striped bars), IgG3
(diagonal striped bars), are expressed as the ELISA titre (log 2)
obtained minus the background level, with each titre representing
the mean.+-.standard deviation of three values.
[0055] FIGS. 5A-5B show a PEPSCAN analysis of peptide-specific
antibody reactivity to overlapping peptides representing the KAS2
peptide sequence 433-468. (FIG. 5A) KAS2 overlapping peptides
(offset 1, overlap 7) probed with KAS1-KsA1 (white bars), KAS2-KLA1
(black bars) antisera. (FIG. 5B) KAS2 overlapping peptides (offset,
overlap 7) probed with KAS2-DT conjugate antisera. Each bar
displays the antibody reactivity (optical density [OD] at 415
nm).
[0056] FIGS. 6A-6C. Chimera AS2-LA1 induces an antibody response in
outbred mice that recognises P. gingivalis whole cells and the
RgpA-Kgp complex. CD1 outbred mice were immunised with chimera
AS2-LA1 (50 mg/mouse) and the collected sera used in ELISA with
AS2-LA1 (FIG. 6A), formalin killed P. gingivalis strain W50 (FIG.
6B) and RgpA-Kgp complex (FIG. 6C) as the absorbed antigens. In
this figure KAS2-KLA1 is shown as AS2-LA1. The titre for each
immunoglogulin isotype to each antigen was determined and the data
expressed as the ELISA titre ('000) obtained minus double the
background level, with each titre representing the mean.+-.standard
deviation of three values.
[0057] FIG. 7. Protein model of the Kgp proteinase. KAS2
[Asn433-Lys468]. (A) KAS4 [Asp388-Val395] (B), KAS5 [Asn510-Asp516]
(C) and KAS6 [Ile570-Tyr580] (D).
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0058] It will be understood that the invention disclosed and
defined in this specification extends to all alternative
combinations of two or more of the individual features mentioned or
evident from the text or drawings. All of these different
combinations constitute various alternative aspects of the
invention.
[0059] The inventors have found that the regions of P. gingivalis
trypsin-like enzymes that flank or otherwise define a catalytic or
active site for cleavage of a peptide bond are highly immunogenic
and indeed sufficient to provide for a humoral response to P.
gingivalis infection. In particular, it has been found that a
chimeric or fusion protein including one or more of these regions
provides protection against alveolar bone loss which is greater
than that seen for antisera raised against whole cells and other
immunogens. The finding is particularly surprising as, to date, the
catalytic domain of trypsin-like enzymes of P. gingivalis has been
found to be relatively weakly immunogenic.
[0060] In one aspect, the present invention provides a chimeric or
fusion protein for inducing an immune response to P. gingivalis,
the protein including a first peptide joined directly or through a
linker to a second peptide, wherein: [0061] (A) said first peptide
includes: [0062] (i) part of, or all of a sequence that is the same
as, or homologous to the sequence shown in SEQ ID No:1; or [0063]
(ii) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:2; and [0064] (B)
said second peptide includes: [0065] (i) part of, or all of a
sequence that is the same as, or homologous to the sequence of an
adhesin domain of the Lys-X-proteinase of P. gingivalis; or [0066]
(ii) part of, or all of a sequence that is the same as, or
homologous to the sequence of an adhesin domain of the
Arg-X-proteinase of P. gingivalis; or [0067] (iii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
a HagA adhesin domain of P. gingivalis.
[0068] As used herein, the term "peptide" is used to refer to an
amino acid sequence of up to about 40 amino acid residues,
preferably from 5 to 40 amino acid residues.
[0069] In one embodiment, a polypeptide is used in place of or in
other words instead of the "second peptide". The term "polypeptide"
is used to refer to an amino acid sequence of at least about 40
amino acid residues.
[0070] Thus, in another aspect there is provided a chimeric or
fusion protein for inducing an immune response to P. gingivalis,
the protein including a peptide joined directly or through a linker
to a polypeptide, wherein: [0071] (A) said peptide includes: [0072]
(i) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:1; or [0073] (ii)
part of, or all of a sequence that is the same as, or homologous to
the sequence shown in SEQ ID No:2; and [0074] (B) said polypeptide
includes: [0075] (i) part of, or all of a sequence that is the same
as, or homologous to the sequence of an adhesin domain of the
Lys-X-proteinase of P. gingivalis; or [0076] (ii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
an adhesin domain of the Arg-X-proteinase of P. gingivalis; or
[0077] (iii) part of, or all of a sequence that is the same as, or
homologous to the sequence of a HagA adhesin domain of P.
gingivalis.
[0078] In another aspect, the invention provides a peptide for
inducing an immune response to P. gingivalis selected from the
group consisting of: [0079] (i) a sequence that is the same as or
homologous to the sequence shown in one of SEQ ID No: 64 to 66; and
[0080] (ii) a sequence that is the same as or homologous to the
sequence shown in SEQ ID No: 67 or 68.
[0081] In an aspect of the invention, where the peptide has a
sequence of SEQ ID No: 64 to 68, the peptide may be provided in the
form of a chimeric or fusion protein in which the peptide is joined
directly or through a linker to a second peptide. In an embodiment,
the second peptide of the chimeric or fusion protein includes:
[0082] (i) part of, or all of a sequence that is the same as, or
homologous to the sequence of an adhesin domain of the
Lys-X-proteinase of P. gingivalis; or [0083] (ii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
an adhesin domain of the Arg-X-proteinase of P. gingivalis; or
[0084] (iii) part of, or all of a sequence that is the same as, or
homologous to the sequence of a HagA adhesin domain of P.
gingivalis.
[0085] In the above described embodiment a polypeptide is used in
place of, or in other words instead of the second peptide. Thus, in
another aspect there is provided a chimeric or fusion protein for
inducing an immune response to P. gingivalis, the protein including
a peptide joined directly or through a linker to a polypeptide,
wherein: [0086] (A) said peptide includes: [0087] (i) a sequence
that is the same as or homologous to the sequence shown in one of
SEQ ID No: 64 to 66; or [0088] (ii) a sequence that is the same as
or homologous to the sequence shown in SEQ ID No: 67 or 68; and
[0089] (B) said polypeptide includes: [0090] (i) part of, or all of
a sequence that is the same as, or homologous to the sequence of an
adhesin domain of the Lys-X-proteinase of P. gingivalis; or [0091]
(ii) part of, or all of a sequence that is the same as, or
homologous to the sequence of an adhesin domain of the
Arg-X-proteinase of P. gingivalis; or [0092] (iii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
a HagA adhesin domain of P. gingivalis.
[0093] As used herein, a reference to a "homologue" of a peptide or
polypeptide is a reference to a peptide or polypeptide having an
amino acid sequence that shares homology or that is homologous to,
or that has identity with the amino acid sequence of the
first-mentioned peptide or polypeptide, preferably at least 90%
sequence identity, more preferably at least 95% and even more
preferably at least 98% sequence identity when the comparison is
performed by a BLAST algorithm wherein the parameters of the
algorithm are selected to give the largest match between the
respective sequences over the entire length of the respective
reference sequences. Sequence identity refers to exact matches
between the amino acids of two sequences which are being compared.
Such a homologue may derive from a naturally occurring variant or
isolate of the Lys-X-proteinase or Arg-X-proteinase of P.
gingivalis. Alternatively, it may be a "conservative-substitution"
variant of a peptide or polypeptide from the Lys-X-proteinase or
Arg-X-proteinase of P. gingivalis in which one or more amino acid
residues have been changed without altering the overall
conformation and function of the peptide or polypeptide; including,
but by no means limited to, replacement of an amino acid with one
having similar properties. Amino acids with similar properties are
well known in the art. For example, polar/hydrophilic amino acids
which may be interchangeable include asparagine, glutamine, serine,
cysteine, threonine, lysine, arginine, histidine, aspartic acid and
glutamic acid; nonpolar/hydrophobic amino acids which may be
interchangeable include glycine, alanine, valine, leucine,
isoleucine, proline, tyrosine, phenylalanine, tryptophan and
methionine; acidic amino acids which may be interchangeable include
aspartic acid and glutamic acid and basic amino acids which may be
interchangeable include histidine, lysine and arginine. Preferably
such conservative-substitution variants have less than 20, more
preferably less than 15, more preferably less than 10, and most
preferably less than 5 amino acid changes.
[0094] A region of a P. gingivalis trypsin-like enzyme--especially
a Lys-X-proteinase (Kgp) or Arg-X-proteinase (RgpA)--that defines a
site in an enzyme for cleavage of a peptide bond can be determined
following the teaching of the specification herein, particularly in
relation to FIG. 7 and Example 9, which exemplify the process for
predicting three-dimensional conformation of the catalytic site as
it appears on P. gingivalis for Lys-X-proteinase. Example 10
provides methodology for modelling of the Arg-X-proteinase
three-dimensional conformation.
[0095] In certain embodiments, the chimeric or fusion protein, or
first or second peptide components thereof may be formed from a
peptidomimetic. A peptidomimetic is a molecule that mimics one or
more characteristics of a given peptide, for example conformation,
and that consists of amino acid residues, some of which may not be
naturally occurring.
[0096] Having identified the immunogenic regions of the catalytic
site, the inventors have determined the sequence of various peptide
immunogens against which a humoral response can be raised. In
particular, `six` regions that flank or otherwise define the
catalytic site have been defined as follows: KAS1/RAS1, KAS2/RAS2,
KAS3/RAS3, KAS4/RAS4, KAS5/RAS5 and KAS6 (see Table 1). With this
information, the inventors have been able to interrogate protein
sequence databases to determine peptides that share homology with
amino acid sequences that form regions that flank a catalytic site
and hence that represent immunogenic epitopes found on P.
gingivalis. The sequence of these peptides are identified by the
following structural formula:
TABLE-US-00001 TABLE 1 Sequences that flank the active site of Kgp
and RgpA. Kgp Lys-X RgpA Arg-X (numbering (numbering according to
Kgp Lys-X according to RgpA Arg-X Region SEQ ID No. 62) Consensus
No. 61) Consensus PAS1K/ PAS1K (432-453) LNTGVSFANYTAFIGS PAS1R
(426-446) FNGGISLANYTGHGSET PAS1R ETAWADP AWGT (SEQ ID NO: 34) (SEQ
ID NO: 30) KAS1/ KAS1 (432-454) LNTGV[G/S]FANYTAH RAS1 (426-448)
FNGGISL[V/A]NYTGHG RAS1 GSET[S/A]WADP[S/L] SETAWGTSH (SEQ ID NO:
27) (SEQ ID NO: 31) KAS2/ KAS2 (433-468) NTGV[G/S]FANYTAHG RAS2
(427-462) NGGISL[V/A]NYTGHGS RAS2 SET[S/A]WADP[S/L][L/V]
ETAWGTSHFGTTHVKQ T[A/T][T/S]Q[V/L]KAL LTNSNQ TNK[D/N]K (SEQ ID NO:
32) (SEQ ID NO: 28) KAS3/ KAS3 (436-455) V[G/S]FANYTAFIGSET RAS3
(430-449) ISL[V/A]NYTGHGSETA RAS3 [S/A]WADP[S/L][L/V] WGTSHF (SEQ
ID NO: 29) (SEQ ID NO: 33) KAS4/ KAS4 (388-395)
D[S/Y][Y/S]WN[P/S][K/Q] RAS4 (379-386) EGGPSADN RAS4 [I/V] (SEQ ID
NO: 64) (SEQ ID NO: 67) KA55/ KASS (510-516) NSYWGED RAS5 (508-514)
[N/D]Q[S/Y]WA[S/P]P RAS5 (SEQ ID NO: 65) (SEQ ID NO: 68) KAS6 KAS6
(570-580) IGN[V/I]THIGAHY (SEQ ID NO: 66)
[0097] The inventors have found that chimeric proteins including
these peptides have a number of utilities. For example, as
described herein, some produce a humoral response that is highly
protective for treatment or prevention of bone loss as observed in
chronic periodonitis. The peptides may also be used in a diagnostic
assay wherein they can detect or monitor specificities in an
individual's serum, thereby indicating whether or not the
individual is infected and if so, whether treatments are required
or if provided, whether they have been effective.
[0098] It will be understood that the region of a P. gingivalis
trypsin-like enzyme that defines a site in the enzyme for cleavage
of a peptide bond located C-terminal to Lys or Arg, does not
comprise a complete sequence of the Lys-X-proteinase or
Arg-X-proteinase.
[0099] As used herein, the terms "heterologous protein" or
"chimeric or fusion protein" are used to refer to a protein that is
composed of functional units, domains, sequences or regions of
amino acids derived from different sources or that are derived from
the same source and that have been assembled so as to have an
organisation that is distinguished from that observed in a molecule
from which the unit, domain, sequence or region is derived or
related to. A common feature of the chimeric or fusion proteins of
the invention is that they contain at least one peptide having an
amino acid sequence that is the same as or that shares homology
with a sequence of a P. gingivalis trypsin-like enzyme that defines
a catalytic site for cleavage of a peptide bond.
[0100] In a preferred embodiment, where the first peptide comprises
a peptide from the Kgp[432-468] region, it is preferably (i) a
peptide which comprises a sequence selected from VSFANYT and
VGFANYT, more preferably a sequence selected from GVSFANYT,
GVGFANYT, VSFANYTA and VGFANYTA; or (ii) a peptide which comprises
a sequence selected from ETAWAD, ETSWAD, TAWADP and TSWADP,
preferably a sequence selected from SETAWAD, SETSWAD, ETAWADP,
ETSWADP, TAWADPL and TSWADPL, more preferably a sequence selected
from GSETAWAD, GSETSWAD, SETAWADP, SETSWADP, ETAWADPL, ETSWADPL,
TAWADPLL and TSWADPLL. More preferably, this peptide is selected
from the KAS1[432-454], KAS2[433-468] and KAS3[436-455] peptides
shown in Table 1. Alternatively, the first peptide may be the
PAS1K[432-453] peptide, also known as PAS1(K48), disclosed in
International Patent Application No. PCT/AU98/00311 (WO 98/049192).
The sequence identifiers corresponding to these peptides are shown
in Table 3.
[0101] Similarly, in another preferred embodiment, where the first
peptide comprises a peptide from the RgpA[426-462] region, this
peptide is preferably selected from the RAS1[426-448],
RAS2[427-462] and RAS3[430-449] peptides shown in Table 1.
Alternatively, the first peptide may be the PAS1R[426-446] peptide,
also known as PAS1(R45), disclosed in International Patent
Application No. PCT/AU98/00311 (WO 98/049192).
[0102] In the chimeric or fusion protein of the invention, the
second peptide may be a peptide from an adhesin domain of a P.
gingivalis trypsin-like enzyme, such as Lys-X-proteinase (Kgp) or
Arg-X-proteinase (RgpA) or HagA (see Table 2). These domains are
sometimes also known as hemagglutinins. In the Lys-X-proteinase,
the preferred domains are KA1, KA2, KA3, KA4, KA5 as identified in
Table 2. In the Arg-X-proteinase, the preferred domains are RA1,
RA2, RA3 and RA4 as identified in Table 2. In HagA, the preferred
domains are HagA1, HagA1* and HagA1**.
TABLE-US-00002 TABLE 2 Adhesin domains of the Kgp and RgpA
proteinases. A1 sA1 LA1 A2 A3 A4 A5 Kgp Lys-X KA1 (738-1099) KsA1
(759-989) KLA1 (751-1056) KA2 (1157-1275) KA3 (1292-1424) KA4
(1427-1546) KA5 (1548-1732) proteinase SEQ ID NO: 35 SEQ ID NO: 36
SEQ ID NO: 37 SEQ ID NO: 40 SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO:
43 SEQ ID No. 62 RgpA Arg-X RA1 (720-1081) RsA1 (831-971) -- RA2
(1139-1257) RA3 (1274-1404) RA4 (1432-1706) -- proteinase SEQ ID
NO: 38 SEQ ID NO: 39 SEQ ID NO: 44 SEQ ID NO: 45 SEQ ID NO: 46 SEQ
ID No. 61 HagA HagA1 (26-351) SEQ ID NO. 63 (SEQ ID NO: 80), HagA1*
(366-625) (SEQ ID NO: 81), HagA1** (820-1077) (SEQ ID NO: 82) or
HagA1** (1272-1529) (SEQ ID NO: 82)
[0103] In addition to improving the humoral response to a peptide
of the invention such as KAS1, KAS2, KAS3, KAS4, KAS5 and KAS6 or
RAS1, RAS2 and RAS3, RAS4 and RAS5 when included with such a
peptide in a chimeric or fusion protein, the adhesin domain also
contains immunogenic epitopes, hence leading to the production of
multiple specificities to elicit a protective immunogenic response.
The finding that the immunogenic epitopes of the adhesin domain are
retained in a form approaching that in a P. gingivalis trypsin-like
enzyme when provided in the chimeric or fusion protein of the
invention is unanticipated.
[0104] It will be understood that in these embodiments of the
invention the chimeric or fusion protein may contain any one or
more of the peptides selected from KAS1/RAS1, KAS2/RAS2, KAS3/RAS3,
KAS4/RAS4, KAS5/RAS5 and KAS6/RAS6 together with any one or more
adhesin domains of a P. gingivalis trypsin-like enzyme, in
particular with any one or more of Lys-X-proteinase adhesin domains
(KA1, KA2, KA3, KA4 and KA5) or Arg-X-proteinase adhesin domains
(RA1, RA2, RA3 and RA4) or HagA domains HagA1, HagA1* and
HagA1**.
[0105] It will also be understood that it is not necessary for the
adhesin domain to be a complete domain as observed in a P.
gingivalis trypsin-like enzyme. For example the adhesin domain may
be a fragment of such a domain, in particular, preferred fragments
are the KsA1 and KLA1 domain fragments of the Lys-X-proteinase A1
domain (see Table 2). Where the domain is a fragment of an adhesin
domain it generally contains one or more adhesin domain specific
epitopes.
[0106] The sequence identifiers corresponding to the adhesin
related peptides are shown in Table 3.
[0107] In one embodiment the second peptide or polypeptide includes
a sequence shown in one or more of SEQ ID No: 69 to 79 or one or
more of 83 to 85.
[0108] The chimeric or fusion protein of the present invention may
also include one or more additional peptides selected from the
Kgp[432-468] region of the Lys-X-proteinase and/or one or more
additional peptides selected from the RgpA[426-462] region of the
Arg-X-proteinase.
[0109] In preferred embodiments of the present invention, the
chimeric or fusion protein includes one or more of KAS1, KAS2,
KAS3, KAS4, KAS5 and KAS6, or one or more of RAS1, RAS2, RAS3, RAS4
and RAS5, together with KsA1 or KLA1.
[0110] Thus in certain embodiments, the chimeric or fusion protein
may include at least one further peptide wherein said further
peptide includes:
(i) part of, or all of a sequence that is the same as, or
homologous to the sequence shown in SEQ ID No:1; or (ii) part of,
or all of a sequence that is the same as, or homologous to the
sequence shown in SEQ ID No:2; or (iii) part of, or all of a
sequence that is the same as, or homologous to the sequence of an
adhesin domain of the Lys-X-proteinase of P. gingivalis; or (iv)
part of, or all of a sequence that is the same as, or homologous to
the sequence of an adhesin domain of the Arg-X-proteinase of P.
gingivalis; or (v) part of, or all of a sequence that is the same
as, or homologous to the sequence of a HagA adhesin domain of P.
gingivalis.
[0111] Other examples of domains, units, sequences or regions that
may be included in a chimeric or fusion protein as described herein
include domains for binding to receptors or ligands such as Fc
binding regions or Fc receptors, domains for improving half-life
such as albumin or domains for facilitating expression or
purification of the chimeric or fusion protein.
[0112] In the chimeric or fusion proteins of the present invention,
the C-terminal residue of the first peptide may be covalently
linked to the N-terminal residue of an adhesin domain polypeptide,
or the N-terminal residue of the first peptide may be covalently
linked to the C-terminal residue of an adhesin domain polypeptide.
In this arrangement, the first peptide and adhesin domain
polypeptide, are said to be "directly linked" or "adjacent".
[0113] In other embodiments, the chimeric or fusion protein
includes a linker for linking the first peptide to an adhesin
domain polypeptide. The linker may be any linker able to join a
peptide to a polypeptide, including both amino acid and non-amino
acid linkers. Preferably, the linker is non-immunogenic. Suitable
linkers may be up to 15 amino acids in length, although less than
five amino acids is preferred. The linker may function to bring the
first peptide and adhesin domain polypeptide into a closer spatial
arrangement than normally observed in a P. gingivalis trypsin-like
enzyme. Alternatively, it may space the first peptide and adhesin
domain polypeptide apart.
[0114] The chimeric or fusion proteins of the invention may be
produced by recombinant expression systems (such as recombinant DNA
technology) or by chemical synthesis (such as solid phase peptide
synthesis). These techniques are well known in the art.
[0115] The heterologous or chimeric protein is particularly
advantageous because it improves the humoral response obtained over
that obtained using the first or second peptide components of the
chimeric or fusion protein alone.
[0116] The inventors have found that chimeric proteins including
these peptides have a number of utilities. For example, as
described herein, some produce a humoral response that is highly
protective for treatment or prevention of bone loss as observed in
chronic periodontitis. The peptides may also be used in a
diagnostic assay wherein they can detect or monitor specificities
in an individual's serum, thereby indicating whether or not the
individual is infected and if so, whether treatments are required
or if provided, whether they have been effective.
[0117] In one embodiment, the chimeric or fusion protein induces a
protective immune response, typically a response that at least
minimises or limits connective tissue damage otherwise associated
with P. gingivalis infection. In one embodiment the protective
response at least minimises or limits P. gingivalis induced bone
loss. A model system for measuring bone loss mediated by P.
gingivalis infection is discussed herein. Typically the protective
immune response is predominantly a humoral response. In certain
embodiments the protective immune response also includes a cellular
response.
[0118] The present invention also provides a composition including
a chimeric or fusion protein as broadly described above. Typically
the composition is antigenic or immunogenic. More particularly, the
invention provides a composition suitable for eliciting a
protective or therapeutic immune response against P. gingivalis
infection, including the chimeric or fusion protein, optionally in
association with an adjuvant. Such a composition may also include
another component for modulating or potentiating the immune
response. One embodiment, the composition takes the form of a
vaccine.
[0119] Various adjuvants are known for use in conjunction with
vaccine compositions. The adjuvants aid by modulating the immune
response and in attaining a more durable and higher level of
immunity using smaller amounts of vaccine antigen or fewer doses
than if the vaccine antigen were administered alone. Examples of
adjuvants include incomplete Freund's adjuvant (IFA), Adjuvant 65
(containing peanut oil, mannide monooleate and aluminium
monostearate), oil emulsions, Ribi adjuvant, the pluronic polyols,
polyamines, Avridine, Quil A, saponin, MPL, QS-21, mineral gels
such as aluminium salts and calcium salts, nanoparticles such as
hydroxyapatite, calcium phosphate, aluminium salts, sugar oligomers
and polymers such as mannan, chitosan. Other examples include oil
in water emulsions such as SAF-1, SAF-0, MF59, Seppic ISA720, and
other particulate adjuvants such ISCOMs.TM. and ISCOM Matrix.TM..
An extensive but not exhaustive list of other examples of adjuvants
are listed in Cox and Coulter 1992 [In: Wong WK (ed.) Animals
parasite control utilising technology. Bocca Raton; CRC press,
1992; 49-112]. In addition to the adjuvant, the vaccine composition
may include conventional pharmaceutically acceptable carriers,
excipients, fillers, buffers or diluents as appropriate. One or
more doses of the vaccine composition containing adjuvant may be
administered prophylactically to prevent periodontitis or
therapeutically to treat already present periodontitis.
[0120] In a preferred composition, the chimeric or fusion protein
is combined with a mucosal adjuvant and administered via the oral,
buccal or nasal route. Examples of mucosal adjuvants are
nanoparticles, cholera toxin and heat labile E. coli toxin, the
non-toxic B subunits of these toxins, genetic mutants of these
toxins which have a reduced toxicity. Other methods which may be
utilised to deliver the antigenic protein orally/buccally/nasally
include incorporation or absorption of the protein into or onto
particles of biodegradable polymer (such as acrylates or
polyesters) or nanoparticles (such as hydroxyapatite) by
microencapsulation to aid uptake of the microspheres from the
gastrointestinal tract or other mucosal surfaces and to protect
degradation of the proteins. Liposomes, ISCOMs.TM., hydrogels are
examples of other potential methods which may be further enhanced
by the incorporation of targeting molecules such as LTB, CTB or
lectins for delivery of the antigenic protein to the mucosal immune
system. In addition to the antigenic protein and the mucosal
adjuvant or delivery system, the vaccine composition may include
conventional pharmaceutically acceptable carriers, excipients,
fillers, coatings, dispersion media, antibacterial or antifungal
agents, and buffers or diluents as appropriate.
[0121] In this aspect, the invention also provides a method of
preventing or reducing the incidence or severity of a P.
gingivalis-related condition or disease in a subject, which
comprises administering to the subject a chimeric or fusion protein
as described above, or an composition as described above.
[0122] The subject may be a human or other animal subject, and is
preferably a human.
[0123] Typically, the P. gingivalis-related condition or disease is
chronic periodontis, however it may also be bone loss, especially
alveolar bone loss, or coronary artery disease.
[0124] Many methods are known for administration of a vaccine
composition to a human or animal subject, including but not limited
to intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous, intranasal, sub-lingual, buccal and oral
administration. These routes of administration are particularly
useful for vaccination.
[0125] In another aspect, the invention provides an antibody,
preferably a monoclonal antibody, raised against a chimeric or
fusion protein as broadly described above.
[0126] These antibodies may be produced by standard techniques, and
may be used in passive immunisation of a subject. Accordingly, in
this aspect, the invention also provides a method of preventing or
reducing the severity of a P. gingivalis-related disease or
condition in a subject, which comprises administering to the
subject an antibody as described above.
[0127] In a further aspect, the present invention provides a
nucleic acid molecule including a nucleotide sequence encoding a
chimeric or fusion protein as broadly described above, optionally
operatively linked to at least one regulatory element. In one
embodiment the nucleic acid is provided in isolated or
substantially purified form.
[0128] The nucleic acid molecule may, for example, be inserted into
a suitable expression vector for production of the chimeric protein
as a recombinant protein by insertion of the expression vector into
a prokaryotic or eukaryotic host cell. Successful expression of the
recombinant protein requires that the expression vector contains
the necessary regulatory elements for transcription and translation
which are compatible with, and recognised by the particular host
cell system used for expression. A variety of host cell systems may
be utilized to express the recombinant protein, which include, but
are not limited to bacteria transformed with a bacteriophage
vector, plasmid vector, or cosmid DNA; yeast containing yeast
vectors; fungi containing fungal vectors; insect cell lines
infected with virus (e.g. baculovirus); and mammalian cell lines
transfected with plasmid or viral expression vectors, or infected
with recombinant virus (e.g. vaccinia virus, adenovirus,
adeno-associated virus, retrovirus, etc).
[0129] Using methods known in the art of molecular biology, various
promoters and enhancers can be incorporated into the expression
vector, to increase the expression of the recombinant protein,
provided that the increased expression of the amino acid sequences
is compatible with (for example, non-toxic to) the particular host
cell system used.
[0130] The selection of the promoter will depend on the expression
system used. Promoters vary in strength, i.e. ability to facilitate
transcription. Generally, it is desirable to use a strong promoter
in order to obtain a high level of transcription of the coding
nucleotide sequence and expression into recombinant protein. For
example, bacterial, phage, or plasmid promoters known in the art
from which a high level of transcription have been observed in a
host cell system including E. coli include the lac promoter, trp
promoter, recA promoter, ribosomal RNA promoter, the P.sub.R and
P.sub.L promoters, lacUV5, ompF, bla, Ipp, and the like, may be
used to provide transcription of the inserted nucleotide sequence
encoding amino acid sequences.
[0131] Other control elements for efficient transcription or
translation include enhancers, and regulatory signals. Enhancer
sequences are DNA elements that appear to increase transcriptional
efficiency in a manner relatively independent of their position and
orientation with respect to a nearby coding nucleotide sequence.
Thus, depending on the host cell expression vector system used, an
enhancer may be placed either upstream or downstream from the
inserted coding sequences to increase transcriptional efficiency.
Other regulatory sites, such as transcription or translation
initiation signals, can be used to regulate the expression of the
coding sequence.
[0132] In another embodiment, the vector may be a viral or
bacterial vaccine vector, and used to provide a recombinant viral
vaccine, a recombinant bacterial vaccine, a recombinant attenuated
bacterial vaccine, or an inactivated recombinant viral vaccine.
Vaccinia virus is the best known example, in the art, of an
infectious virus that is engineered to express vaccine antigens
derived from other organisms. The recombinant live vaccinia virus,
which is attenuated or otherwise treated so that it does not cause
disease by itself, is used to immunize the host. Subsequent
replication of the recombinant virus within the host provides a
continual stimulation of the immune system with the vaccine
antigens thereby providing long lasting immunity.
[0133] Other live vaccine vectors include: adenovirus,
cytomegalovirus, and preferably the poxviruses such as vaccinia
[Paoletti and Panicali, U.S. Pat. No. 4,603,112] and attenuated
Salmonella strains [Stocker et al., U.S. Pat. Nos. 5,210,035;
4,837,151; and 4,735,801; and Curtiss et al., 1988, Vaccine
6:155-160]. Live vaccines are particularly advantageous because
they continually stimulate the immune system which can confer
substantially long-lasting immunity. When the immune response is
protective against subsequent P. gingivalis infection, the live
vaccine itself may be used in a preventive vaccine against P.
gingivalis. In particular, the live vaccine can be based on a
bacterium that is a commensal inhabitant of the oral cavity. This
bacterium can be transformed with a vector carrying a recombinant
chimeric protein and then used to colonise the oral cavity, in
particular the oral mucosa. Once colonised in the oral mucosa, the
expression of the recombinant protein will stimulate the mucosal
associated lymphoid tissue to produce neutralising antibodies. To
further illustrate this embodiment, using molecular biological
techniques well known in the art, nucleotide sequences encoding the
chimeric proteins of this invention may be inserted into the
vaccinia virus genomic DNA at a site which allows for expression of
epitopes but does not negatively affect the growth or replication
of the vaccinia virus vector. The resultant recombinant virus can
be used as the immunogen in a vaccine formulation. The same methods
can be used to construct an inactivated recombinant viral vaccine
formulation except that the recombinant virus is inactivated, such
as by chemical means known in the art, prior to use as an immunogen
and without substantially affecting the immunogenicity of the
expressed immunogen. The inactivated recombinant-vaccine may be
formulated with a suitable adjuvant in order to enhance the
immunological response to the vaccine antigens.
[0134] The invention also provides for the use of a nucleic acid
molecule including a nucleotide sequence encoding a chimeric or
fusion protein of this invention directly as the vaccine
formulation. Nucleotide sequences encoding the chimeric proteins,
operatively linked to one or more regulatory elements, can be
introduced directly to vaccinate an individual ("direct gene
transfer") against pathogenic strains of P. gingivalis. Direct gene
transfer into a vaccinated individual, resulting in expression of
the genetic material by the vaccinated individual's cells such as
vascular endothelial cells as well as the tissue of the major
organs, has been demonstrated by techniques in the art such as by
injecting intravenously an expression plasmid:cationic liposome
complex [Zhu et al., 1993, Science 261:209-211]. Other effective
methods for delivering vector DNA into a target cell are known in
the art. In one example, purified recombinant plasmid DNA
containing viral genes has been used to inoculate (whether
parenterally, mucosally, or via gene-gun immunization) vaccines to
induce a protective immune response [Fynan et al. 1993, Proc Natl
Acad Sci USA 90:11478-11482]. In another example, cells removed
from an individual can be transfected or electroporated by standard
procedures known in the art, resulting in the introduction of the
recombinant vector DNA intro the target cell. Cells containing the
recombinant vector DNA may then be selected for using methods known
in the art, such as by use of a selection marker expressed in the
vector, and the selected cells may then be re-introduced into the
individual to express the recombinant protein.
[0135] In this aspect, the invention further provides a method of
preventing or reducing the incidence or severity of a P.
gingivalis-related condition or disease in a subject, which
comprises administering to the subject a nucleic acid molecule as
described above, a vector as described above, or a prokaryotic or
eukaryotic cell as described above.
[0136] In other embodiments there is provided a pharmaceutical
composition including a chimeric or fusion protein or an antibody
as described above. The composition may further include diluent,
excipient, or carrier or chemotherapeutic agent for treatment of a
P. gingivalis-related condition or disease and may be adapted for
oral administration. The compositions of this invention may be
incorporated in lozenges, or in chewing gum or other products, e.g.
by stirring into a warm gum base or coating the outer surface of a
gum base, illustrative of which are jelutong, rubber latex,
vinylite resins, etc., desirably with conventional plasticizers or
softeners, sugar or other sweeteners or such as glucose, sorbitol
and the like.
[0137] An oral composition of this invention which contains the
above-mentioned pharmaceutical composition may be prepared and used
in various forms applicable to the mouth such as dentifrice
including toothpastes, toothpowders and liquid dentifrices,
mouthwashes, troches, chewing gums, dental pastes, gingival massage
creams, gargle tablets, dairy products and other foodstuffs. An
oral composition according to this invention may further include
additional well known ingredients depending on the type and form of
a particular oral composition.
[0138] In certain preferred forms of the invention the oral
composition may be substantially liquid in character, such as a
mouthwash or rinse. In such a preparation the vehicle is typically
a water-alcohol mixture desirably including a humectant as
described below. Generally, the weight ratio of water to alcohol is
in the range of from about 1:1 to about 20:1. The total amount of
water-alcohol mixture in this type of preparation is typically in
the range of from about 70 to about 99.9% by weight of the
preparation. The alcohol is typically ethanol or isopropanol.
Ethanol is preferred.
[0139] The pH of such liquid and other preparations of the
invention is generally in the range of from about 5 to about 9 and
typically from about 5.0 to 7.0. The pH can be controlled with acid
(e.g. citric acid or benzoic acid) or base (e.g. sodium hydroxide)
or buffered (as with sodium citrate, benzoate, carbonate, or
bicarbonate, disodium hydrogen phosphate, sodium dihydrogen
phosphate, etc).
[0140] In other desirable forms of this invention, the
pharmaceutical composition may be substantially solid or pasty in
character, such as toothpowder, a dental tablet or a toothpaste
(dental cream) or gel dentifrice. The vehicle of such solid or
pasty oral preparations generally contains dentally acceptable
polishing material.
[0141] In a toothpaste, the liquid vehicle may comprise water and
humectant typically in an amount ranging from about 10% to about
80% by weight of the preparation. Glycerine, propylene glycol,
sorbitol and polypropylene glycol exemplify suitable
humectants/carriers. Also advantageous are liquid mixtures of
water, glycerine and sorbitol. In clear gels where the refractive
index is an important consideration, about 2.5-30% w/w of water, 0
to about 70% w/w of glycerine and about 20-80% w/w of sorbitol are
preferably employed.
[0142] Toothpaste, creams and gels typically contain a natural or
synthetic thickener or gelling agent in proportions of about 0.1 to
about 10, preferably about 0.5 to about 5% w/w. A suitable
thickener is synthetic hectorite, a synthetic colloidal magnesium
alkali metal silicate complex clay available for example as
Laponite (e.g. CP, SP 2002, D) marketed by Laporte Industries
Limited. Laponite D is, approximately by weight 58.00% SiO.sub.2,
25.40% MgO, 3.05% Na.sub.2O, 0.98% Li.sub.2O, and some water and
trace metals. Its true specific gravity is 2.53 and it has an
apparent bulk density of 1.0 g/ml at 8% moisture.
[0143] Other suitable thickeners include Irish moss, iota
carrageenan, gum tragacanth, starch, polyvinylpyrrolidone,
hydroxyethylpropylcellulose, hydroxybutyl methyl cellulose,
hydroxypropyl methyl cellulose, hydroxyethyl cellulose (e.g.
available as Natrosol), sodium carboxymethyl cellulose, and
colloidal silica such as finely ground Syloid (e.g. 244).
Solubilizing agents may also be included such as humectant polyols
such propylene glycol, dipropylene glycol and hexylene glycol,
cellosolves such as methyl cellosolve and ethyl cellosolve,
vegetable oils and waxes containing at least about 12 carbons in a
straight chain such as olive oil, castor oil and petrolatum and
esters such as amyl acetate, ethyl acetate and benzyl benzoate.
[0144] It will be understood that, as is conventional, the oral
preparations will usually be sold or otherwise distributed in
suitable labelled packages. Thus, a bottle of mouth rinse will have
a label describing it, in substance, as a mouth rinse or mouthwash
and having directions for its use; and a toothpaste, cream or gel
will usually be in a collapsible tube, typically aluminium, lined
lead or plastic, or other squeeze, pump or pressurized dispenser
for metering out the contents, having a label describing it, in
substance, as a toothpaste, gel or dental cream.
[0145] Organic surface-active agents may be used in the
compositions of the present invention to achieve increased
prophylactic action, assist in achieving thorough and complete
dispersion of the active agent throughout the oral cavity, and
render the instant compositions more cosmetically acceptable. The
organic surface-active material is preferably anionic, non-ionic or
ampholytic in nature and preferably does not interact with the
active agent. It is preferred to employ as the surface-active agent
a detersive material which imparts to the composition detersive and
foaming properties. Suitable examples of anionic surfactants are
water-soluble salts of higher fatty acid monoglyceride
monosulfates, such as the sodium salt of the monosulfated
monoglyceride of hydrogenated coconut oil fatty acids, higher alkyl
sulfates such as sodium lauryl sulfate, alkyl aryl sulfonates such
as sodium dodecyl benzene sulfonate, higher alkylsulfo-acetates,
higher fatty acid esters of 1,2-dihydroxy propane sulfonate, and
the substantially saturated higher aliphatic acyl amides of lower
aliphatic amino carboxylic acid compounds, such as those having 12
to 16 carbons in the fatty acid, alkyl or acyl radicals, and the
like. Examples of the last mentioned amides are N-lauroyl
sarcosine, and the sodium, potassium, and ethanolamine salts of
N-lauroyl, N-myristoyl, or N-palmitoyl sarcosine which should be
substantially free from soap or similar higher fatty acid material.
Examples of water-soluble non-ionic surfactants suitable for use
are condensation products of ethylene oxide with various reactive
hydrogen-containing compounds reactive therewith having long
hydrophobic chains (e.g. aliphatic chains of about 12 to 20 carbon
atoms), which condensation products ("ethoxamers") contain
hydrophilic polyoxyethylene moieties, such as condensation products
of poly (ethylene oxide) with fatty acids, fatty alcohols, fatty
amides, polyhydric alcohols (e.g. sorbitan monostearate) and
polypropyleneoxide (e.g. Pluronic materials).
[0146] The surface active agent is typically present in amount of
about 0.1-5% by weight. It is noteworthy, that the surface active
agent may assist in the dissolving of the active agent of the
invention and thereby diminish the amount of solubilizing humectant
needed.
[0147] Various other materials may be incorporated in the oral
preparations of this invention such as whitening agents,
preservatives, silicones, chlorophyll compounds and/or ammoniated
material such as urea, diammonium phosphate, and mixtures thereof.
These adjuvants, where present, are incorporated in the
preparations in amounts which do not substantially adversely affect
the properties and characteristics desired.
[0148] Any suitable flavouring or sweetening material may also be
employed. Examples of suitable flavouring constituents are
flavouring oils, e.g. oil of spearmint, peppermint, wintergreen,
sassafras, clove, sage, eucalyptus, marjoram, cinnamon, lemon, and
orange, and methyl salicylate. Suitable sweetening agents include
sucrose, lactose, maltose, sorbitol, xylitol, sodium cyclamate,
perillartine, AMP (aspartyl phenyl alanine, methyl ester),
saccharine, and the like. Suitably, flavour and sweetening agents
may each or together comprise from about 0.1% to 5% more of the
preparation.
[0149] Compositions intended for oral use may be prepared according
to any method known in the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavouring agents, colouring agents and preserving agents
in order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient in admixture
with non-toxic pharmaceutically acceptable excipients which are
suitable for the manufacture of tablets. These excipients may be
for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract or periodontal pocket and thereby
provide a sustained action over a longer period. For example, a
time delay material such as glyceryl monostearate or glyceryl
distearate may be employed.
[0150] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0151] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose, hydropropyl
methylcellulose, sodium alginate, polyvinylpyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a
naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate.
[0152] The aqueous suspensions may also contain one or more
preservatives or antimicrobial agents, for example benzoates, such
as ethyl, or n-propyl p-hydroxybenzoate another example is
chlorhexidine gluconate, one or more colouring agents, one or more
flavouring agents, and one or more sweetening agents, such as
sucrose or saccharin.
[0153] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavouring agents may be added
to provide palatable oral preparations. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0154] In a further aspect, the present invention provides a method
for the diagnosis or monitoring of a P. gingivalis-related
condition or disease in a subject, which comprises use of a
chimeric or fusion protein as described above to detect anti-P.
gingivalis antibodies in a biological sample from said subject.
[0155] In yet another aspect, the invention provides a method for
the diagnosis or monitoring of a P. gingivalis-related condition or
disease in a subject, which comprises use of an antibody as
described above, to detect the presence of P. gingivalis in a
biological sample from said subject.
[0156] In yet another aspect, the invention provides a peptide for
inducing an immune response to P. gingivalis including the sequence
shown in one of SEQ ID No: 17, 18, 25 and 26. In one embodiment,
the peptide has a sequence that is homologous to one of SEQ ID No:
17, 18, 25 and 26. The peptide may have a length of 5 to 40 amino
acids.
[0157] In yet another aspect, the invention provides a nucleic acid
encoding a peptide having a sequence shown in one of SEQ ID No: 17,
18, 25 and 26.
[0158] In yet another aspect, the invention provides a use of a
peptide having a sequence shown in one of SEQ ID No: 17, 18, 25 and
26, or a nucleic acid encoding a peptide having a sequence shown in
one of SEQ ID No: 17, 18, 25 and 26, for the manufacture of a
chimeric or fusion protein for inducing an immune response to P.
gingivalis.
[0159] In yet another aspect, the invention provides a use of a
peptide having a sequence shown in one of SEQ ID No: 17, 18, 25 and
26, or a nucleic acid encoding a peptide having a sequence shown in
one of SEQ ID No: 17, 18, 25 and 26, for inducing an immune
response to P. gingivalis. In one embodiment, the peptide is
administered simultaneously or sequentially with a second peptide
including: [0160] (i) part of, or all of a sequence that is the
same as, or homologous to the sequence of an adhesin domain of the
Lys-X-proteinase of P. gingivalis; or [0161] (ii) part of, or all
of a sequence that is the same as, or homologous to the sequence of
an adhesin domain of the Arg-X-proteinase of P. gingivalis; or
[0162] (iii) part of, or all of a sequence that is the same as, or
homologous to the sequence of a HagA adhesin domain of P.
gingivalis.
TABLE-US-00003 [0162] TABLE 3 SEQ ID NO: Amino acid sequence
Fragment 1 LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]T Kgp [432-468]
[A/T][T/S]Q[V/L]KALTNK[D/N]K 2
FNGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSN RgpA [426-462] Q 3 VSFANYT
4 VGFANYT 5 GVSFANYT 6 GVGFANYT 7 VSFANYTA 8 VGFANYTA 9 ETAWAD 10
ETSWAD 11 TAWADP 12 TSWADP 13 SETAWAD 14 SETSWAD 15 ETAWADP 16
ETSWADP 17 TAWADPL 18 TSWADPL 19 GSETAWAD 20 GSETSWAD 21 SETAWADP
22 SETSWADP 23 ETAWADPL 24 ETSWADPL 25 TAWADPLL 26 TSWADPLL 27
LNTGV[G/S]FANYTAHGSET[S/A]WADP[S/L] KAS1 28
NTGV[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V]T KAS2
[A/T][T/S]Q[V/L]KALTNK[D/N]K 29
V[G/S]FANYTAHGSET[S/A]WADP[S/L][L/V] KAS3 30 LNTGVSFANYTAHGSETAWADP
PAS1K 31 FNGGISL[V/A]NYTGHGSETAWGTSH RAS1 32
NGGISL[V/A]NYTGHGSETAWGTSHFGTTHVKQLTNSNQ RAS2 33
ISL[V/A]NYTGHGSETAWGTSHF RAS3 34 FNGGISLANYTGHGSETAWGT PAS1R 35
ANEAKVVLAADNVVVGDNTGYQFLLDADHNTFGSVIPATG KA1
PLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEV
VIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTF
EAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRD
GTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV
CKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTP
NPNPNPNPNPGTTLSESFENGIPASWKTIDADGDGHGW
KPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPA
LDLPNGGKLTFWVCAQDANYASEHYAVYASSTGNDASN FTNALLEETITA 36
FLLDADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPAN KsA1
ADPVVIT0NIIVTGQGEVVIPGGVYDYCITNPEPASGKMW
IAGDGGNQPARYDDFTFEAGKKYTFTMRRAGMGDGTDM
EVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGN
HEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGS
SVGQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESF 37
WGDNTGYQFLLDADHNTFGSVIPATGPLFTGTASSNLYS KLA1
ANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGVYDYCITN
PEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRR
AGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTF
EEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEF
APVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT
TLSESFENGIPASWKTIDADGDGHGWKPGNAPGIAGYNS
NGCVYSESFGLGGIGVLTPDNYLITPALDLPNGG 38
SGQAEIVLEAHDVVVNDGSGYQILLDADHDQYGQVIPSDT RAI
HTLWPNCSVPANLFAPFEYTVPENADPSCSPTNMIMDGT
ASVNIPAGTYDFAIAAPQANAKIWIAGQGPTKEDDYVFEA
GKKYHFLMKKMGSGDGTELTISEGGGSDYTYTVYRDGT
KIKEGLTATTFEEDGVATGNHEYCVEVKYTAGVSPKVCK
DVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTPNP
NPNPNPNPNPGTTTLSESFENGIPASWKTIDADGDGHG
WKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLIT
PALDLPNGGKLTFWVCAQDANYASEHYAVYASSTGNDA SNFTNALLEETITA 39
DDYVFEAGKKYHFLMKKMGSGDGTELTISEGGGSDYTYT RsA1
VYRDGTKIKEGLTATTFEEDGVATGNHEYCVEVKYTAGV
SPKVCKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAP NGTPNPNPNPNPNPNPGTTTLSESF 40
ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQ KA2
LDWLTAHGGSNVVSSFSWNGMALNPDNYLISKDVTGAT
KVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETP NGIN 41
PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT KA3
MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGN
HEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVQNLTAEQ APNSMDAILKWNAPAS 42
AEVLNEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSF KA4
AGHNSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGTL
TFWVCAQDANYASEHYAVYASSTGNDASNFANALLEEVL TA 43
TVVTAPEAIRGTRAQGTWYQKTVQLPAGTKYVAFRHFGC KA5
TDFFWINLDDVVITSGNAPSYTYTIYRNNTQIASGVTETTY
RDPDLATGFYTYGVKVVYPNGESAIETATLNITSLADVTA
QKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGRNTVV YTAQGGHYAVMVVVDGKSYVEKLAVK
44 ADFTETFESSTHGEAPAEWTTIDADGDGQGWLCLSSGQ RA2
LDWLTAHGGTNVVSSFSWNGMALNPDNYLISKDVTGAT
KVKYYYAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETP NGIN 45
PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT RA3
MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGN
HEYCVEVKYTAGVSPKKCVNVTVNSTQFNPVKNLKAQP DGGDVVLKWEAPSA 46
ANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPATG RA4
PLFTGTASSDLYSANFESLIPANADPVVTTQNIIVTGQGEV
VIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTF
EAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRD
GTKIKEGLTETTYRDAGMSAQSHEYCVEVKYTAGVSPKV
CVDYIPDGVADVTAQKPYTLTVVGKTITVTCQGEAMIYDM
NGRRLAAGRNTVVYTAQGGYYAVMVVVDGKSYVEKLAI K 47
GACCATGGCTCATCACCATCACCATCACAATACCGG KAS2- AGTCAGCTTTGCA FOR 48
GACTCGAGTTATTTGTCCTTATTAGTGAGTGCTTTC KAS2- REV 49
GACCATGGCTTGGGGAGACAATACGGGTTAC KLA1- FOR 50
GACTCGAGACCTCCGTTAGGCAAATCC KLA1- REV 51
CCGTATTGTCTCCCCATTTGTCCTTATTAGTGAGTGC KAS2- TTTC KLA1-REV 52
CACTAATAAGGACAAATGGGGAGACAATACGGGTTA KAS2- C KLA1-FOR 53
CATGGATCTGAGACCGCATGGGCTGATCCACTTTTC KAS1- TTGTTGGATGCCGAT KsA1-
FOR1 54 CCATGGCTTTGAATACCGGAGTCAGCTTTGCAAACT KAS1-
ATACAGCGCATGGATCTGAGACCGCA KsA1- FOR2 55 CTCGAGGAATGATTCGGAAAGTGTT
KAS1- KsA1- REV 56 CCATGGCTGATTATAGCTGGAATTCCCAGGTAGTCA multi-
GCTTTGCAAACTATACA FOR1 57 CTTTGCAAACTATACAGCGCATGGATCTGAGACCGC
multi- ATGGGCTGATCCACTT FOR2 58
ATGGGCTGATCCACTTCTGAATTCTTATTGGGGCGA multi- GATCGGCAATATTACC FOR3
59 GATCGGCAATATTACCCATATTGGTGCTCATTACGC multi- TTGGGGAGACAATACG
FOR4 60 CTCGAGACCTCCGTTAGGCAAATCCAATGCCGGTGT multi-REV
TATCAGATAGTTGTCA 61 MKNLNKFVSIALCSSLLGGMAFAQQTELGRNPNVRLLES RgpA
TQQSVTKVQFRMDNLKFTEVQTPKGIGQVPTYTEGVNL
SEKGMPTLPILSRSLAVSDTREMKVEVVSSKFIEKKNVLI
APSKGMIMRNEDPKKIPYVYGKTYSQNKFFPGEIATLDD
PFILRDVRGQVVNFAPLQYNPVTKTLRIYTEITVAVSETSE
QGKNILNKKGTFAGFEDTYKRMFMNYEPGRYTPVEEKQ
NGRMIVIVAKKYEGDIKDFVDWKNQRGLRTEVKVAEDIA
SPVTANAIQQFVKQEYEKEGNDLTYVLLIGDHKDIPAKITP
GIKSDQVYGQIVGNDHYNEVFIGRFSCESKEDLKTQIDRT
IHYERNITTEDKWLGQALCIASAEGGPSADNGESDIQHE
NVIANLLTQYGYTKIIKCYDPGVTPKNIIDAFNGGISLANYT
GHGSETAWGTSHFGTTHVKQLTNSNQLPFIFDVACVNG
DFLFSMPCFAEALMRAQKDGKPTGTVAIIASTINQSWAS
PMRGQDEMNEILCEKHPNNIKRTFGGVTMNGMFAMVEK
YKKDGEKMLDTWTVFGDPSLLVRTLVPTKMQVTAPAQI
NLTDASVNVSCDYNGAIATISANGKMFGSAVVENGTATI
NLTGLTNESTLTLTVVGYNKETVIKTINTNGEPNPYQPVS
NLTATTQGQKVTLKWDAPSTKTNATTNTARSVDGIRELV
LLSVSDAPELLRSGQAEIVLEAHDVVVNDGSGYQILLDAD
HDQYGQVIPSDTHTLWPNCSVPANLFAPFEYTVPENAD
PSCSPTNMIMDGTASVNIPAGTYDFAIAAPQANAKIWIAG
QGPTKEDDYVFEAGKKYHFLMKKMGSGDGTELTISEGG
GSDYTYTVYRDGTKIKEGLTATTFEEDGVATGNHEYCVE
VKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSAVGQKV
TLKWDAPNGTPNPNPNPNPNPNPGTTTLSESFENGIPA
SWKTIDADGDGHGWKPGNAPGIAGYNSNGCVYSESFG
LGGIGVLTPDNYLITPALDLPNGGKLTFWVCAQDANYAS
EHYAVYASSTGNDASNFTNALLEETITAKGVRSPEAMRG
RIQGTWRQKTVDLPAGTKYVAFRHFQSTDMFYIDLDEVE
IKANGKRADFTETFESSTHGEAPAEWTTIDADGDGQGW
LCLSSGQLDWLTAHGGTNVVSSFSWNGMALNPDNYLIS
KDVTGATKVKYYYAVNDGFPGDHYAVMISKTGTNAGDF
TVVFEETPNGINKGGARFGLSTEADGAKPQSVWIERTVD
LPAGTKYVAFRHYNCSDLNYILLDDIQFTMGGSPTPTDY
TYTVYRDGTKIKEGLTETTFEEDGVATGNHEYCVEVKYT
AGVSPKKCVNVTVNSTQFNPVKNLKAQPDGGDVVLKW
EAPSAKKTEGSREVKRIGDGLFVTIEPANDVRANEAKVV
LAADNVWGDNTGYQFLLDADHNTFGSVIPATGPLFTGTA
SSDLYSANFESLIPANADPVVTTQNIIVTGQGEVVIPGGV
YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKK
YTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIK
EGLTETTYRDAGMSAQSHEYCVEVKYTAGVSPKVCVDY
IPDGVADVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGR
RLAAGRNTVVYTAQGGYYAVMVVVDGKSYVEKLAIK 62
MRKLLLLIAASLLGVGLYAQSAKIKLDAPTTRTTCTNNSF Kgp
KQFDASFSFNEVELTKVETKGGTFASVSIPGAFPTGEVG
SPEVPAVRKLIAVPVGATPVVRVKSFTEQVYSLNQYGSE
KLMPHQPSMSKSDDPEKVPFVYNAAAYARKGFVGQELT
QVEMLGTMRGVRIAALTINPVQYDVVANQLKVRNNIElEV
SFQGADEVATQRLYDASFSPYFETAYKQLFNRDVYTDH
GDLYNTPVRMLVVAGAKFKEALKPWLTWKAQKGFYLDV
HYTDEAEVGTTNASIKAFIHKKYNDGLAASAAPVFLALVG
DTDVISGEKGKKTKKVTDLYYSAVDGDYFPEMYTFRMS
ASSPEELTNIIDKVLMYEKATMPDKSYLEKVLLIAGADYS
WNSQVGQPTIKYGMQYYYNQEHGYTDVYNYLKAPYTG
CYSHLNTGVSFANYTAFIGSETAWADPLLTTSQLKALTNK
DKYFLAIGNCCITAQFDYVQPCFGEVITRVKEKGAYAYIG
SSPNSYWGEDYYVVSVGANAVFGVQPTFEGTSMGSYDA
TFLEDSYNTVNSIMWAGNLAATHAGNIGNITHIGAHYYW
EAYHVLGDGSVMPYRAMPKTNTYTLPASLPQNQASYSI
QASAGSYVAISKDGVLYGTGVANASGVATVSMTKQITEN
GNYDVVITRSNYLPVIKQIQVGEPSPYQPVSNLTATTQG
QKVTLKWEAPSAKKAEGSREVKRIGDGLFVTIEPANDVR
ANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIPAT
GPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQG
EVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDD
FTFEAGKKYTFTMRRAGMGDGTDMEVEDDSPASYTYTV
YRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVS
PKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPN
GTPNPNPNPNPNPGTTLSESFENGIPASWKTIDADGDG
HGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNY
LITPALDLPNGGKLTFWVCAQDANYASEHYAVYASSTGN
DASNFTNALLEETITAKGVRSPKAIRGRIQGTWRQKTVDL
PAGTKYVAFRHFQSTDMFYIDLDEVEIKANGKRADFTET
FESSTHGEAPAEWTTIDADGDGQGWLCLSSGQLDWLT
AHGGSNVVSSFSWNGMALNPDNYLISKDVTGATKVKYY
YAVNDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINK
GGARFGLSTEANGAKPQSVVVIERTVDLPAGTKYVAFRH
YNCSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKE
GLTETTFEEDGVATGNHEYCVEVKYTAGVSPKKCVNVT
VNSTQFNPVQNLTAEQAPNSMDAILKWNAPASKRAEVL
NEDFENGIPASWKTIDADGDGNNWTTTPPPGGSSFAGH
NSAICVSSASYINFEGPQNPDNYLVTPELSLPGGGTLTF
WVCAQDANYASEHYAVYASSTGNDASNFANALLEEVLT
AKTVVTAPEAIRGTRAQGTWYQKTVQLPAGTKYVAFRH
FGCTDFFWINLDDVVITSGNAPSYTYTIYRNNTQIASGVT
ETTYRDPDLATGFYTYGVKVVYPNGESAIETATLNITSLA
DVTAQKPYTLTVVGKTITVTCQGEAMIYDMNGRRLAAGR
NTVVYTAQGGHYAVMVVVDGKSYVEKLAVK 63
MRKLNSLFSLAVLLSLLCWGQTAAAQGGPKTAPSVTHQ HagA
AVQKGIRTSKAKDLRDPIPAGMARIILEAHDVWEDGTGy
QMLWDADHNQYGASIPEESFWFANGTIPAGLYDPFEYK
VPVNADASFSPTNFVLDGTASADIPAGTYDYVIINPNPGII
YIVGEGVSKGNDYVVEAGKTYHFTVQRQGPGDAASVVV
TGEGGNEFAPVQNLQWSVSGQTVTLTWQAPASDKRTY
VLNESFDTQTLPNGWTMIDADGDGHNWLSTINVYNTAT
HTGDGAMFSKSWTASSGAKIDLSPDNYLVTPKFTVPEN
GKLSYWVSSQEPWTNEHYGVFLSTTGNEAANFTIKLLEE
TLGSGKPAPMNLVKSEGVKAPAPYQERTIDLSAYAGQQ
VYLAFRHFGCTGIFRLYLDDVAVSGEGSSNDYTYTVYRD
NVVIAQNLTATTFNQENVAPGQYNYCVEVKYTAGVSPKV
CKDVTVEGSNEFAPVQNLTGSAVGQKVTLKWDAPNGTP
NPNPGTTTLSESFENGIPASWKTIDADGDGNNWTTTPPP
GGSSFAGHNSAICVSSASYINFEGPQNPDNyLVTPELSL
PNGGTLTFWVCAQDANYASEHYAVYASSTGNDASNFA
NALLEEVLTAKTVVTAPEAIRGTRVQGTVVYQKTVQLPAG
TKYVAFRHFGCTDFFWINLDDVEIKANGKRADFTETFES
STHGEAPAEWTTIDADGDGQGWLCLSSGQLGWLTAHG
GTNVVASFSWNGMALNPDNYLISKDVTGATKVKYYYAV
NDGFPGDHYAVMISKTGTNAGDFTVVFEETPNGINKGG
ARFGLSTEANGAKPQSVWIERTVDLPAGTKYVAFRHYN
CSDLNYILLDDIQFTMGGSPTPTDYTYTVYRDGTKIKEGL
TETTFEEDGVATGNHEYCVEVKYTAGVSPKECVNVTVD
PVQFNPVQNLTGSAVGQKVTLKWDAPNGTPNPNPGTTT
LSESFENGIPASWKTIDADGDGNNWTTTpppGGTSFAG
HNSAICVSSASYINFEGPQNPDNYLVTPELSLPNGGTLTF
WVCAQDANYASEHYAVYASSTGNDASNFANALLEEVLT
AKTVVTAPEAIRGTRVQGTVVYQKTVQLPAGTKYVAFRH
FGCTDFFWINLDDVEIKANGKRADFTETFESSTHGEAPA
EWTTIDADGDGQGWLCLSSGQLDWLTAHGGTNVVASF
SWNGMALNPDNYLISKDVTGATKVKYYYAVNDGFPGDH
YAVMISKTGTNAGDFTVVFEETPNGINKGGARFGLSTEA
NGAKPQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLD
DIQFTMGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDG
VATGNHEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQN
LTGSAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIP
ASWKTIDADGDGNNWTTTPPPGGTSFAGHNSAICVSSA
SYINFEGPQNPDNYLVTPELSLPNGGTLTFWVCAQDAN
YASEHYAVYASSTGNDASNFANALLEEVLTAKTVVTAPE
AIRGTRVQGTWYQKTVQLPAGTKYVAFRHFGCTDFFWI
NLDDVEIKANGKRADFTETFESSTHGEAPAEWTTIDADG
DGQGWLCLSSGQLGWLTAHGGTNVVASFSWNGMALN
PDNYLISKDVTGATKVKYYYAVNDGFPGDHYAVMISKTG
TNAGDFTVVFEETPNGINKGGARFGLSTEANGAKPQSV
WIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFTMGG
SPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGNHEY
CVEVKYTAGVSPKECVNVTINPTQFNPVQNLTAEQAPNS
MDAILKWNAPASKRAEVLNEDFENGIPASWKTIDADGDG
NNWTTTPPPGGSSFAGHNSAICVSSASYINFEGPQNPD
NYLVTPELSLPGGGTLTFWVCAQDANYASEHYAVYASS
TGNDASNFANALLEEVLTAKTVVTAPEAIRGTRVOGIVVY
QKTVQLPAGTKYVAFRHFGCTDFFWINLDDVVITSGNAP
SYTYTIYRNNTQIASGVTETTYRDPDLATGFYTYGVKVVY
PNGESAIETATLNITSLADVTAQKPYTLTVVGKTITVTCQG
EAMIYDMNGRRLAAGRNTVVYTAQGGHYAVMVVVDGK SYVEKLAVK 64
D[S/Y][Y/S]WN[P/S][K/Q][I/V] KAS4 65 NSYWGED KAS5 66
IGN[V/I]IHIGAHY KAS6 67 EGGPSADN RAS4 68 [N/D]Q[S/Y]WA[S/P]P RAS5
69 PVSNLTATTQGQKVTLKWDAPST ABM1- RgpA.sub.cat 70
PVSNLTATTQGQKVTLKWEAPSA ABM1- Kgp.sub.cat 71
PVQNLTGSSVGQKVTLKWDAPST ABM1- KgpA1 72 PVQNLTGSAVGQKVTLKWDAPNG
ABM1- RgpA1 & RgpAA3 73 PVKNLKAQPDGGDVVLKWEAPSA ABM1-
HagAA1*/** 74 PVQNLTAEQAPNSMDAILKWNAP ABM1- KgpA3 HagAA3 75
PVQNLTQWSVSGQTVTLTWQAPAS ABM2- HagAA1 76
YTYTVYRDGTKIKEGLTETTFEEDGVA ABM2- ABM2- RgpAA4 77
YTYTVYRDNVVIAQNLTATTFNQENVA ABM2- HagA1* 78
YTYTVYRDGTKIKEGLTA/ETTFEEDGVA ABM2 AH other adhesins 79
PNGTP(NP).sub.1-6GTT(T)LSESF ABM3-All adhesins 80
GGPKTAPSVTHQAVQKGIRTSKAKDLRDPIPAGMARIILE HagA1
AHDVWEDGTGYQMLWDADHNQYGASIPEESFWFANGTI [26-351]
PAGLYDPFEYKVPVNADASFSPTNFVLDGTASADIPAGTY
DYVIINPNPGIIYIVGEGVSKGNDYVVEAGKTYHFTVQRQ
GPGDAASVVVTGEGGNEFAPVQNLQWSVSGQTVTLTW
QAPASDKRTYVLNESFDTQTLPNGWTMIDADGDGHNWL
STINVYNTATHTGDGAMFSKSWTASSGAKIDLSPDNYLVT
PKFTVPENGKLSYWVSSQEPWTNEHYGVFLSTTGNEAA NFTIKLLEETLGSG 81
APAPYQERTIDLSAYAGQQVYLAFRHFGCTGIFRLYLDDV HagA1*
AVSGEGSSNDYTYTVYRDNVVIAQNLTATTFNQENVAPG [366-625]
QYNYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTG
SAVGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASW
KTIDADGDGNNWTTTPPPGGSSFAGHNSAICVSSASYIN
FEGPQNPDNYLVTPELSLPNGGTLTFWVCAQDANYASE HYAVYASSTGNDASNFANALLEEVLTA
82 PQSVWIERTVDLPAGTKYVAFRHYNCSDLNYILLDDIQFT HagA1**
MGGSPTPTDYTYTVYRDGTKIKEGLTETTFEEDGVATGN [820-
HEYCVEVKYTAGVSPKECVNVTVDPVQFNPVQNLTGSA 1077] or
VGQKVTLKWDAPNGTPNPNPGTTTLSESFENGIPASWKT HagA1**
IDADGDGNNWTTTPPPGGTSFAGHNSAICVSSASYINFE [1272-
GPQNPDNYLVTPELSLPNGGTLTFWVCAQDANYASEHY 1529]
AVYASSTGNDASNFANALLEEVLTA 83 PYQPVSNLTATTQGQ ABM1[436 -450] 84
EGLTATTFEEDGVAA ABM2 [672-686] 85 GTPNPNPNPNPNPNPGT ABM3
[455-471]
[0163] The invention is further illustrated by the following
Examples which are included by way of exemplification and not
limitation of the invention.
Example 1
Methods and Materials.
Bacterial Strains and Growth Conditions.
[0164] Lyophilised cultures of Porphyromonas gingivalis W50 were
grown anaerobically at 37.degree. C. on lysed horse blood agar
plates supplemented with 5 .mu.g/ml haemin, 0.5 .mu.g/ml cysteine
(HB agar, <10 passages). After 3-4 days colonies were used to
inoculate brain heart infusion medium containing 5 .mu.g/ml haemin,
0.5 .mu.g/ml cysteine (1). Batch cultures were grown anaerobically
in a MK3 Anaerobic Workstation (Don Whitley Scientific Ltd.,
Adelaide, Australia). Cells were harvested during exponential
growth phase by centrifugation (7500 g, 30 min, 4.degree. C.) and
washed twice with PG buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM
CaCl.sub.2, and 5 mM cysteine-HCl, pH 8.0) in the anaerobic
workstation. Growth of batch cultures was monitored at 650 nm using
a spectrophotometer (model 295E, Perkin-Elmer). Culture purity was
checked routinely by Gram stain, microscopic examination and using
a variety of biochemical tests according to Slots (2).
[0165] Construction of pET28 Constructs Containing Adhesin
Sequences and Adhesin Sequences with N-Terminal Addition of Kgp
Proteinase Sequences.
[0166] Kgp residues representing peptides and chimeric peptides of
the active site (AS) and KgpA1 adhesin (A1) domains were
over-expressed in E. coli as recombinant (r) proteins with hexa-His
tags using pET expression vectors (Novagen). The r-proteins
expressed were rKAS2, and rKLA1 and the r-chimeric proteins were
rKAS2-KLA1, rKAS1-KsA1 and rKAS4-KAS3-KAS5-KAS6-KLA1 (also referred
to as multiKAS-KLA1). The amino acid sequences representing the
various A1 and AS domains are described in Tables 1 and 2.
[0167] The various KAS and KA1 domains of the kgp gene were
amplified from pNS1 (3.5 kb BamHI lys fragment in pUC18) or P.
gingivalis genomic DNA respectively using primers listed in Table
4, Taq DNA polymerase (Invitrogen) and a PC-960 thermal cycler
(Corbett Research Technologies). Primer pairs KAS2-FOR and KAS2-REV
and KLA1-FOR and KLA1-REV were used to generate PCR fragments
encoding KAS2 and KLA1 respectively using the following reaction
conditions: 94.degree. C., 3 minutes, followed by 28 cycles of
94.degree. C., 45 sec (denaturing); 62.degree. C., 40 seconds
(annealing) and 72.degree. C., 20 seconds (extension) followed by a
final cycle of 72.degree. C., 5 min.
[0168] The KAS2-KLA1 chimeric PCR product was produced by gene
splicing by overlap extension (SOEing) as follows: PCR products
were produced using primer pairs KAS2-FOR and KAS2-KLA1-chimera-REV
and KAS2-KLA1-chimera-FOR and KLA1-REV using the conditions
described above. The PCR products were then annealed and a final
PCR was performed with primers KAS2-FOR and KLA1-REV (94.degree.
C., 2 minutes, followed by 28 cycles of 94.degree. C., 30 sec;
50.degree. C., 30 seconds and 72.degree. C., 40 seconds followed by
a final cycle of 72.degree. C., 5 min.
[0169] For the preparation of the KAS1-KsA1 PCR product, two
successive PCR's were conducted using the KAS1-KsA1-REV primer with
each of the KAS1-KsA1-FOR primers 1 and, 2 in succession (reaction
conditions 94.degree. C. for 2 minutes followed by 35 cycles of
94.degree. C., 15 seconds; 63.degree. C., 30 seconds and 72.degree.
C., 2 minutes) to produce the KAS1-KsA1 PCR product. The
KAS1-KsA1-FOR1 and KAS1-KsA1-FOR2 primers contain an 3'extension
overlapping the 5' of the previous PCR product.
[0170] For the preparation of the multiKAS-KLA1 PCR fragment, four
successive PCR's were conducted using the multi-REV primer with
each of the multi-FOR primers 1, 2, 3 and 4 in succession (reaction
conditions were 95.degree. C., 2 minutes followed by 35 cycles of
95.degree. C., 20 seconds; 68.degree. C., 1.5 minutes) to produce
the multiKAS-KLA1 PCR product. Each multi-FOR primer contains a
3'extension overlapping the 5' of the previous PCR product.
[0171] All of the PCR fragments encoding KAS2, KLA1, KAS2-KLA1,
KAS1-KsA1 and multiKAS-KLA1. were purified using PCR purification
columns (Qiagen), ligated into the TA cloning vector, pGem-T Easy
(Promega) and transformed into E. coli JM109 following the
manufacturer's protocol. Purified recombinant pGemT-Easy constructs
were digested with Ncol and Xhol and directionally cloned into
Ncol/Xhol digested pET28b (Novagen) and transformed into the
non-expression host, E. coli JM109 [DH5.alpha.]. The recombinant
pET28 constructs were purified and transformed into the E. coli
expression host, BL21 (DE3) [HMS174(DE3)] (Novagen) and selected on
LB containing 50 .mu.g kanamycin following the manufacturer's
instructions. The integrity of each insert was confirmed by DNA
sequence analysis.
[0172] The oligonucleotide primers (Table 4) have been designed to
incorporate restriction enzyme sites, stop codons and hexa-His Tags
where necessary. The primers used for the rKAS2, rKLA1 and
rKAS2-KLA1 were designed to limit the inclusion of extraneous
coding sequence to no more than three amino acids plus the hexa-his
tag in r-proteins. The rKAS1 and the rKLA1 were designed to contain
a hexa-His tag at the N-terminal and C-terminal ends respectively,
so that they may be directly compared to the rKAS2-KLA1 which has a
hexa-his tag at both N- and C-termini. In rKAS1-KsA1 and
rmultiKAS-KLA1 the His Tags are found at the C-termini.
TABLE-US-00004 TABLE 4 Oligonucleotide primers used for the
amplification of the nucleotide sequences encoding the various
fragments and chimeras of Kgp A1 and AS Recombinant (r) protein Oil
Sequence (5'-3') Characteristics* (5%31 rKAS2 KAS2-
GACCATGGCTCATCACCATCACC GA buffer-NcoI (including ATG FOR
ATCACAATACCGGAGTCAGCTTT start)-CT-(His).sub.6-AS (nt 1992-2012) GCA
(SEQ ID NO: 47) KAS2- GACTCGAGTTATTTGTCCTTATTA GA buffer-XhoI-TTA
Stop-KAS1 REV GTGAGTGCTTTC (nt 2099-2075) (SEQ ID NO: 48) rKLA1
KLA1- GACCATGGCTTGGGGAGACAATA GA buffer-NcoI (including ATG FOR
CGGGTTAC (SEQ ID NO: 49) start)-CT-A1 (nt 2946-2966) KLA1-
GACTCGAGACCTCCGTTAGGCAA GA buffer-XhoI-A1 (nt 3863- REV ATCC (SEQ
ID NO: 50) 3845) rKAS2-KLA1 KAS2- CCGTATTGTCTCCCCATTTGTCCT A1 (nt
2961-2946)-KAS1 (nt KLA1- TATTAGTGAGTGCTTTC 2099-2075) REV (SEQ ID
NO: 51) KAS2- CACTAATAAGGACAAATGGGGAG KAS1 (nt 2084-2099)-A1 (nt
KLA1- ACAATACGGGTTAC 2946-2966) FOR (SEQ ID NO: 52) rKAS1-KsA1
KAS1- CATGGATCTGAGACCGCATGGG AS (nt 2025-2057)-A1 (nt 2970- KsA1-
CTGATCCACTTTTCTTGTTGGATG 2987)- FOR1 CCGAT (SEQ ID NO: 53) KAS1-
CCATGGCTTTGAATACCGGAGTC NcoI-CT-AS (nt 1989-2042) KsA1-
AGCTTTGCAAACTATACAGCGCA FOR2 TGGATCTGAGACCGCA SEQ ID NO: 54) KAS1-
CTCGAGGAATGATTCGGAAAGTG XhoI-A1 (nt 3663-3644) KsA1- TT (SEQ ID NO:
55) REV rmultiKAS- multi- CCATGGCTGATTATAGCTGGAAT NcoI-CT-KAS4 (nt
1857-1880)- KLA1 FOR1 TCCCAGGTAGTCAGCTTTGCAAA KAS3 (nt 2001-2021)
CTATACA (SEQ ID NO: 56) multi- CTTTGCAAACTATACAGCGCATG KAS3 (nt
2006-2057) FOR2 GATCTGAGACCGCATGGGCTGAT CCACTT (SEQ ID NO: 57)
multi- ATGGGCTGATCCACTTCTGAATT KAS3 (nt 2042-2060)-KAS5 (nt FOR3
CTTATTGGGGCGAGATCGGCAAT 2223-2240)-KAS6 (nt 2403-2417) ATTACC (SEQ
ID NO: 58) multi- GATCGGCAATATTACCCATATTG G-KAS6 2403-2435)-GCT
FOR4 GTGCTCATTACGCTTGGGGAGAC (Ala spacer)-A1 (nt 2946-2960) AATACG
(SEQ ID NO: 59) multi- CTCGAGACCTCCGTTAGGCAAAT Xho-A1 (nt
3863-3818) REV CCAATGCCGGTGTTATCAGATAG TTGTCA (SEQ ID NO: 60)
*nucleotide (nt) sequence numbers from lysine-specific cysteine
proteinase gene sequence accession number U75366
Expression and Purification of Recombinant Proteins.
[0173] Recombinant proteins were expressed from pET28::KLA1(KAS2,
KAS2-LA1, KAS1-SA1, multiKAS-KLA1) constructs by induction with
isopropyl .beta.-D-thiogalactosidase (IPTG). All recombinant
proteins were produced as 6-His Tag fusion proteins and purified
with NI-NTA purification system (Invitrogen) under denaturing
conditions. Briefly, E. coli (DE3) single colony transformants were
used to inoculate 20 mL of Luria-Bertani (LB) broth containing 50
.mu.g/ml kanamycin at 37.degree. C. on an orbital shaker overnight.
This inoculum was then used to inoculate 1 L of LB containing 50
.mu.g/ml kanamycin. The OD.sub.600 of this culture was allowed to
reach 0.5-07 (mid-log phase) before inducing protein expression
with isopropyl IPTG at 0.1 mM for 2 hours at 37.degree. C. with
shaking of 200 rpm. Cells were harvested (7,500 g) and resuspended
in a denaturing binding buffer (8M Urea, 20 mM Sodium Phosphate pH
8.0 & 500 mM NaCl) and sonicated on ice for 3.times.15 s bursts
at 30 s intervals using a Branson Sonifer 250 Cell disrupter
(Branson Ultronics Corporation, Danbury, Conn.) with the microtip
on setting 3, then centrifuged at 39,000 g for 30 min at 4.degree.
C. Recombinant proteins were purified from the supernatant by
loading onto a pre-equilibrated Ni-NTA Agarose column and then
washing with denaturing washing buffer (8M Urea, 20 mM Sodium
Phosphate pH 6.0 & 500 mM NaCl) to elute unbound proteins. The
column was then washed using 10 volumes of binding buffer B and the
recombinant protein was eluted with denaturing elution buffer (8M
Urea, 20 mM Sodium Phosphate pH 6.0, 500 mM NaCl & 0.5 M
Imidazole). Purified protein was dialyzed against 2M Urea-PBS and
stored at -80.degree. C.
[0174] Recombinant protein samples were analysed by SDS-PAGE and
their molecular masses determined using ProtParam on-line
(http://au.expasy.org/tools/protparam.html). Protein concentration
of all samples was determined by the Bio-Rad Protein Assay using
BSA as a standard.
Immunisation and the Mouse Periodontitis Model.
[0175] The mouse periodontitis experiments were performed as
described previously (3) and were approved by the University of
Melbourne Ethics Committee for Animal Experimentation. BALB/c mice
6-8 weeks old (12 mice per group) housed in microisolators were
immunized subcutaneously (s.c. 100 .mu.L) with either 50 .mu.g of
one of the recombinant proteins or RgpA-Kgp complex,
2.times.10.sup.9 formalin killed cells of P. gingivalis strain W50
or PBS; each antigen was emulsified in incomplete Freund's adjuvant
(IFA). After 30 days the mice were boosted with antigen (s.c.
injection, emulsified in IFA) and then bled from the retrobulbar
plexus 12 days later. Four days after the second immunisation mice
were given kanamycin (Sigma-Aldrich, New South Wales, Australia) at
1 mg/ml in deionized water ad libitum for 7 days. Three days after
the antibiotic treatment (2 days after bleeding), mice were orally
inoculated four times 2 days apart with 1.times.10.sup.10 viable P.
gingivalis W50 (25 .mu.l) in PG buffer (50 mM Tris-HCl, 150 mM
NaCl, 5 mM CaCl.sub.2, and 5 mM cysteine-HCl, pH 8.0) containing 2%
(wt/vol) carboxymethyl cellulose (CMC; Sigma-Aldrich, New South
Wales, Australia), and a control group was sham infected with PG
buffer containing 2% (wt/vol) CMC alone. The inocula were prepared
in the anaerobic chamber and then immediately applied to the
gingival margin of the maxillary molar teeth. Two weeks later, mice
received another four doses (2 days apart) of 1.times.10.sup.10
cells of viable P. gingivalis W50 (25 .mu.l) in PG buffer
containing 2% (wt/vol) CMC. The number of viable bacteria in each
inoculum was verified by enumeration on blood agar. Mice were fed a
soft powdered diet (Barastock, Australia) and housed in cages
fitted with a raised wire mesh bottom to prevent access to bedding.
Four weeks after the last dose, mice were bled from the retrobulbar
plexus and killed, and the maxillae were removed and cut in half
with one half (right) used for alveolar bone loss measurement and
the other half (left) used for real-time PCR.
[0176] The right half maxillae were boiled (1 min) in deionized
water, mechanically defleshed, and immersed in 2% (wt/vol)
potassium hydroxide (16 h, 25.degree. C.). The half maxillae were
then washed (two times with deionized water) and immersed in 3%
(wt/vol) hydrogen peroxide (6 h, 25.degree. C.). After the half
maxillae were washed (two times with deionized water), they were
stained with 0.1% (wt/vol) aqueous methylene blue, and a digital
image of the buccal aspect of each half maxilla was captured with
an Olympus DP12 digital camera mounted on a dissecting microscope,
using OLYSIA BioReport software version 3.2 (Olympus Australia Pty
Ltd., New South Wales, Australia) to assess horizontal bone loss.
Horizontal bone loss is loss occurring in a horizontal plane,
perpendicular to the alveolar bone crest (ABC) that results in a
reduction of the crest height. Each half maxilla was aligned so
that the molar buccal and lingual cusps of each tooth image were
superimposed, and the image was captured with a micrometer scale in
frame, so that measurements could be standardized for each image.
The area from the cementoenamel junction to the ABC for each molar
tooth was measured using OLYSIA BioReport software version 3.2
imaging software. Bone loss measurements were determined twice by a
single examiner using a randomized and blinded protocol.
Determination of Subclass Antibody by an ELISA.
[0177] To determine the subclass antibody responses of mouse sera,
enzyme-linked immunosorbent assays (ELISAs) were performed in
triplicate using a 5-.mu.g/ml solution of formalin killed P.
gingivalis W50 in phosphate-buffered saline (PBS) (0.01 M
Na.sub.2HPO.sub.4, 1.5 mM KH.sub.2PO.sub.4, 0.15 M NaCl), pH 7.0,
containing 0.1% (vol/vol) Tween 20 (PBST) to coat wells of
flat-bottom polyvinyl microtiter plates (Dynatech Laboratories,
McLean, Va.). After removal of the coating solution, PBST
containing 2% (wt/vol) skim milk powder was added to wells to block
the uncoated plastic for 1 h at room temperature. After the wells
were washed four times with PBST, serial dilutions of mouse sera in
PBST containing 0.5% (wt/vol) skim milk (SK-PBST) were added to
each well and incubated for 16 h at room temperature. After the
wells were washed six times with PBST, a 1/2,000 dilution of goat
IgG to mouse IgM, IgA, IgG1, IgG2a, IgG2b, or IgG3 (Sigma, New
South Wales, Australia) was added in SK-PBST and allowed to bind
for 2 h at room temperature. Plates were washed six times in PBST,
and a 1/5,000 dilution of horseradish peroxidase-conjugated rabbit
anti-goat immunoglobulin (Sigma, New South Wales, Australia) in
SK-PBST was added to each well and incubated for 1 h at room
temperature. After the wells were washed six times with PBST, bound
antibody was detected by the addition of 100 .mu.l of ABTS
substrate [0.9 mM 2,2'-azino-bis(3-ethylbenz-thiazoline-6) sulfonic
acid in 80 mM citric acid containing 0.005% (vol/vol) hydrogen
peroxide, pH 4.0] to each well. The optical density at 415 nm was
measured using a microplate reader (Bio-Rad microplate reader,
model 450).
SDS-PAGE Gel Electrophoresis and Western Blotting.
[0178] Recombinant proteins (10 .mu.g) were analysed using the
XCell surelock Mini-Cell electrophoresis system. Recombinant
proteins were mixed in 20 .mu.l of reducing sample buffer (10%
[wt/vol] SDS, 0.05% [wt/vol] bromophenol blue, 25% [vol/vol]
glycerol, and 0.05% [vol/vol] 2-mercaptoethanol). The pH was
adjusted to pH 8.0 with 1.5 M Tris-HCl, and then the solution was
heated for 5 min at 100.degree. C. Recombinant proteins (10
.mu.g/lane) were loaded onto Novex 12% (wt/vol) Tris-glycine
precast mini gels, and electrophoresis was performed using a
current of 30 to 50 mA and a potential difference of 125 V using a
Novex electrophoresis system (Novex, San Diego, Calif.). Proteins
were visualized using 0.25% w/v Coomassie blue R250.
Epitope Analysis of the Kgp Proteinase Active Site Peptide (KAS-2)
Sequence.
[0179] The antibody binding sites for the Lys-specific proteinase
active site peptide KAS2 (433-468 SEQ ID No: 28) was determined by
synthesising N-terminally biotinylated overlapping eight residue
peptides (offset by one, overlapping by seven residues) on a
multipin peptide synthesis system (Chiron Technologies, Melbourne,
Australia) using standard solid-phase peptide synthesis protocols
for Fmoc chemistry. Biotinylated peptides (5 .mu.g/mL) in 0.1 M
PBS, pH 7.4 were bound to strepavidin coated plates, overnight at
4.degree. C. (Nunc, NSW Australia). After the wells were washed
four times with PBST epitope mapping of the plate-bound peptides
was carried out by ELISA as per Chiron Technologies instructions
using mouse sera at a dilution of 1:1000 in 1% w/v non-fat skim
milk powder in 0.1 M PBS, pH 7.4, containing 0.1% v/v Tween 20
(SK-PBST). After the wells were washed six times with PBST, a
1/2,000 dilution of goat IgG to mouse IgG (Sigma, New South Wales,
Australia) was added in SK-PBST and allowed to bind for 2 h at room
temperature. Plates were washed six times in PBST, and a 1/5,000
dilution of horseradish peroxidase-conjugated rabbit anti-goat
immunoglobulin (Sigma, New South Wales, Australia) in SK-PBST was
added to each well and incubated for 1 h at room temperature. After
the wells were washed six times with PBST, bound antibody was
detected by the addition of 100 .mu.l of ABTS substrate [0.9 mM
2,2'-azino-bis(3-ethylbenz-thiazoline-6) sulfonic acid in 80 mM
citric acid containing 0.005% (vol/vol) hydrogen peroxide, pH 4.0]
to each well. The optical density at 415 nm was measured using a
microplate reader (Bio-Rad microplate reader, model 450).
Statistical Analysis.
[0180] The bone loss data were statistically analyzed using a
one-way analysis of variance (ANOVA) and Dunnett's T3 test (SPSS
for Windows, version 12). The IgA, IgM, and IgG subclass antibody
titers were statistically analyzed using Student's t test using
SPSS software (SPSS for Windows, version 12).
Example 2
Characterisation and Purification of the Recombinant Proteins
(KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1).
[0181] In order to characterise the ability of Kgp adhesin A1
domain fragments and chimera Kgp proteinase and Kgp adhesin A1
domain fragments to protect against P. gingivalis infection, we
expressed and purified the recombinant proteins:--KsA1, KLA1,
KAS1-KsA1 and KAS2-KLA1. Recombinant proteins (KsA1 and KLA1) and
recombinant chimera proteins (KAS1-KsA1 and KAS2-KLA1) were
purified from inclusion bodies using nickel chelate affinity
chromatography and the purified proteins analysed by SDS-PAGE (FIG.
1). Each of the purified recombinant proteins consisted of one
major protein band with molecular weights of 40, 36, 31 and 32 kDa
corresponding to KAS2-KLA1, KLA1, KsA1 and KAS1-KsA1, and these
weights corresponded to the calculated molecular masses of the
His-tag recombinant proteins using ProtParam. To characterize the
immunogenicity of the recombinant proteins KsA1, KLA1, KAS1-KsA1
and KAS2-KLA1 were used to immunize mice and the sera was used to
probe KAS2 peptide coated plates and formalin killed P. gingivalis
W50 cells coated plates (FIG. 2). Recombinant chimera proteins
KAS1-KsA1 and KAS2-KLA1 antisera were found to recognize KAS2
peptide (FIG. 2A) at a similar level to KAS2 specific antisera
(KAS2-diptheria toxoid conjugate) as well as formalin killed P.
gingivalis W50 cells (FIG. 2B). However, antisera against the
recombinant protein KLA1 only recognized killed P. gingivalis W50
cells (FIG. 2B).
Example 3
[0182] Effect of Immunization with the Recombinant Proteins (KsA1,
KLA1, KAS1-KsA1 and KAS2-KLA1) on P. gingivalis Induced Alveolar
Bone Loss in the Mouse Periodontitis Model.
[0183] The recombinant proteins KsA1, KLA1, KAS1-KsA1 and
KAS2-KLA1, formalin killed P. gingivalis strain W50 and the
RgpA-Kgp complex were used to determine and compare the protection
induced against P. gingivalis induced alveolar bone loss using a
modified mouse model of periodontal bone loss based on that
reported by Baker et al (4). Mice were immunized (days 0 and 30)
with either recombinant proteins KsA1, KLA1, KAS1-KsA1 or
KAS2-KLA1, RgpA-Kgp complex or formalin killed P. gingivalis strain
W50 (FK-W50) cells or PBS adjuvant alone and were then orally
challenged with viable P. gingivalis W50. Immunization with all of
the recombinant antigens, RgpA-Kgp complex and FK-W50 cells
protected BALB/c mice against P. gingivalis-induced alveolar bone
loss as these animals exhibited significantly (p<0.001) less
bone loss compared to the PBS immunized group (FIG. 3). However the
KAS2-KLA1 immunised mice had significantly less bone loss than mice
immunised with KLA1 (p<0.01); KsA1 (p<0.001), RgpA-Kgp
complex (p<0.001), FK-W50 cells (p<0.001) and non-challenged
mice (p<0.001). There was no significant difference in bone loss
between the KAS2-KLA1 and KAS1-KsA1 immunised mice. Furthermore,
KAS1-KsA1 immunised mice exhibited significantly less bone loss
than non-challenged mice (p<0.01) and RgpA-Kgp complex immunised
mice (p<0.05), but were not significantly different from KsA1,
KLA1, and FK-W50 immunised mice. There was no significant
difference in bone loss between the KsA1, KLA1, RgpA-Kgp complex
and FK-W50 immunised mice.
Example 4
[0184] Antibody Subclass Responses Induced by Immunization with the
Recombinant Proteins (KsA1, KLA1, KAS1-KsA1 and KAS2-KLA1) in the
Mouse Periodontitis Models.
[0185] Prior and post to oral inoculation challenge with viable P.
gingivalis cells mice were bled and the sera collected by
centrifugation. FIG. 4 shows the antibody subclass reactivity to
formalin-killed P. gingivalis W50 cells for each immunogen (KsA1,
KLA1, KAS1-KsA1 or KAS2-KLA1 or formalin killed P. gingivalis
strain W50 (FK-W50) cells) in the mouse periodontitis model. All of
the protective immunogens induced a high IgG antibody titre to
FK-W50. Furthermore, the predominant antibody subclass each
protective immunogen induced was IgG1 with only weakly
immunoreactive IgG2a, IgG2b and IgG3 FK-W50-specific antibodies
(FIG. 4). The predominant antibody subclass induced by each
immunogen both pre (FIG. 4A) and post-oral inoculation (FIG. 4B)
was IgG1.
Example 5
Epitope Mapping of KAS2 (433-468).
[0186] Overlapping biotinylated eight residue peptides (offset by
one, overlap by seven) for KAS2 (433-468) were synthesised and used
to coat streptavidin coated plates. The antibody binding epitopes
were then identified using antisera from mice immunized with
KAS1-KsA1, KAS2-KLA1 and KAS2-diphtheria toxoid conjugate (FIG. 5).
A two fold increase in optical density (415 nm) above background
was considered as a positive antibody response (threshold OD). The
antisera recognised the following peptide sequences derived from
SEQ ID No.28 viz. KAS1-KsA1 recognised peptides 435-442, 436-443,
445-452, 446-453 and 447-454 (threshold OD=0.07, FIG. 5A) whereas
KAS2-KLA1 recognised peptides 435-442, 447-454 and 448-455
(threshold ID=0.07, FIG. 5A). This suggests recognition of a number
of minimal epitopes viz. peptide 436-442 (VSFANYT and its variant
VGFANYT), peptide 447-452 (ETAWAD and its variant ETSWAD), and
peptide 448-453 (TAWADP and its variant TSWADP). Peptides which
include the peptide 436-442 epitope include GVSFANYT, GVGFANYT,
VSFANYTA and VGFANYTA. Peptides which include the peptide 447-452
and/or 448-453 epitopes include SETAWAD, SETSWAD, ETAWADP, ETSWADP,
TAWADPL and TSWADPL, more particularly GSETAWAD, GSETSWAD,
SETAWADP, SETSWADP, ETAWADPL, ETSWADPL, TAWADPLL and TSWADPLL.
Example 6
Synthesis of KAS and RAS Peptides for Conjugation to a Protein.
[0187] Peptides were synthesized manually or using a CEM Microwave
peptide synthesizer. Standard solid-phase peptide synthesis
protocols for Fmoc chemistry were used throughout. Peptides were
assembled as the carboxyamide form using Rink-linker derived
AM-sure resin (AAPPTEC, KY, USA). Coupling was accomplished with
HBTU/HOBt activation using 4 equiv of Fmoc-amino acid and 6 equiv
of DIPEA. The Fmoc group was removed by 20% piperidine in 1M
HOBt/DMF.
[0188] Resins bearing KAS or RAS peptides were swollen in DMF and
the N-terminal Fmoc group removed by 2% v/v DBU in DMF containing
2% v/v piperidine. The N-terminal amino group was then derivatised
with S-Acetylmercaptoacetic acid (SAMA) group using 5 equiv of
SAMA-OPfp and 5 equiv of HOBt. The reaction was monitored by the
trinitrobenzene sulphonic acid (TNBSA) test. When a negative TNBSA
test was returned the resin was washed (5.times.DMF, 3.times.DCM
and 3.times.diethyl ether). The resin was then dried under vacuum.
Cleavage of peptides from the resin support was performed using
TFA:phenol:TIPS:EDT:water (92:2:2:2:2) cleavage cocktail for 2.5
hours or 4 hours depending on the arginine content of the peptide.
After cleavage the resin was removed by filtration and the filtrate
concentrated to approximately 1 mL under a stream of nitrogen.
After the peptide products were precipitated in cold ether, they
were centrifuged and washed three times. The peptide precipitates
were dissolved in 5 to 10 mL of water containing 0.1% v/v TFA and
insoluble residue removed by centrifugation. Peptides were purified
by RP-HPLC.
[0189] A number of different chemical moieties can be used for
derivatising peptides for conjugation to proteins, these would
introduced reactive groups such as; halides (bromo, chloro and
iodo), maleimido, succinimidyl, hydrazinyl, oxime, thiol, which
would then be used conjugate the derivatised peptide to a protein
such as KgpA1 through its native cysteine residues or has been
derivatised with the complementary reactive group that allows the
chemical ligation to proceed to form a peptide-protein
conjugate.
Conjugation of SAMA-Peptides to KA1.
[0190] To a solution, containing 10 mg/mL of recombinant KA1 or
other adhesin domain of the RgpA-Kgp complex in phosphate-buffered
saline (0.1M sodium phosphate, 0.9% NaCl, pH 7.4) was added 0.1 mL
of a 1% w/v solution of m-maleimido benzoyl-N-hydroxysuccinimide
ester (MBS) in DMF. After 30 min unreacted MBS was removed and
MBS-modified KA1 collected by gel filtration using a PD10 column
(Pharmacia, NSW, Australia) equilibrated in conjugation buffer
(0.1M sodium phosphate, 5 mM EDTA; pH 6.0). Purified SAMA-peptide
(1.3 .mu.mole) was dissolved in 200 .mu.L 6M guanidine HCl
containing 0.5 M Tris; 2 mM EDTA, pH 6.0 and diluted with 800 .mu.L
MilliQ water and deprotected in-situ by addition of 25 .mu.L of 2M
NH.sub.2OH (40 equiv) dissolved in MilliQ water. The collected
MBS-KA1 was immediately reacted with deprotected SAMA-peptide and
stirred for one hour at room temperature. The peptide-KA1 conjugate
was separated from unreacted peptide by gel filtration using a PD10
column equilibrated in PBS pH 7.4 and lyophilized. The reaction was
monitored using the Ellmans test.
Example 7
Preparation of Antibodies.
[0191] Polyclonal antiserum to recombinant proteins are raised in
mice by immunising with the proteins subcutaneously. The mice are
immunised at day 0 with 25 .mu.g of protein in incomplete Freund's
adjuvant and day 30 with 25 .mu.g of protein in incomplete Freund's
adjuvant. Immunisations are carried out using standard procedures.
Polyclonal antisera having a high titre against the proteins are
obtained. If desired monoclonal antibodies directed specifically
against recombinant proteins are obtained using standard
procedures.
Example 8
Immunization for the Generation of Antibodies.
[0192] BALB/c mice or CD1 (Swiss out bred mices) 6-8 weeks old (10
mice per group) were immunized subcutaneously (s.c. 100 .mu.L) with
either 50 .mu.g of the KAS2-LA1 chimera and the antigen emulsified
in incomplete Freund's adjuvant (IFA). After 30 days the mice were
boosted with antigen (s.c. injection, emulsified in IFA) and 12
days later the mice were killed and cardiac bled to collect
sera.
Determination of Subclass Antibody by an ELISA.
[0193] To determine the subclass antibody responses of mouse sera,
enzyme-linked immunosorbent assays (ELISAs) were performed in
triplicate using a 5-.mu.g/ml solution of KAS2-LA1 chimera or
formalin killed P. gingivalis W50 or the RgpA-Kgp complex in
phosphate-buffered saline (PBS) (0.01 M Na.sub.2HPO.sub.4, 1.5 mM
KH.sub.2PO.sub.4, 0.15 M NaCl), pH 7.0, containing 0.1% (vol/vol)
Tween 20 (PBST) to coat wells of flat-bottom polyvinyl microtiter
plates (Dynatech Laboratories, McLean, Va.). After removal of the
coating solution, PBST containing 2% (wt/vol) skim milk powder was
added to wells to block the uncoated plastic for 1 h at room
temperature. After the wells were washed four times with PBST,
serial dilutions of mouse sera in PBST containing 0.5% (wt/vol)
skim milk (SK-PBST) were added to each well and incubated for 16 h
at room temperature. After the wells were washed six times with
PBST, a 1/2,000 dilution of goat IgG to mouse IgM, IgA, IgG1,
IgG2a, IgG2b, or IgG3 (Sigma, New South Wales, Australia) was added
in SK-PBST and allowed to bind for 2 h at room temperature. Plates
were washed six times in PBST, and a 1/5,000 dilution of
horseradish peroxidase-conjugated rabbit anti-goat immunoglobulin
(Sigma, New South Wales, Australia) in SK-PBST was added to each
well and incubated for 1 h at room temperature. After the wells
were washed six times with PBST, bound antibody was detected by the
addition of 100 .mu.l of ABTS substrate [0.9 mM
2,2'-azino-bis(3-ethylbenz-thiazoline-6) sulfonic acid in 80 mM
citric acid containing 0.005% (vol/vol) hydrogen peroxide, pH 4.0]
to each well. The optical density at 415 nm was measured using a
microplate reader (Bio-Rad microplate reader, model 450).
Antibody Subclass Responses Induced by Immunization with the
Recombinant Protein KAS2-KLA1 in Outbred (CD1, Swiss) Mice.
[0194] CD1 (Swiss) mice were immunised with the KAS2-LA1 chimera,
bled and the sera collected by centrifugation. FIG. 6 shows the
antibody subclass reactivity to KAS2-LA1 chimera, formalin-killed
P. gingivalis W50 cells and the RgpA-Kgp complex. The KAS2-LA1
chimera induced a strong IgG antibody with a predominant IgG1
antibody response that recognised the KAS2-LA1 chimera and cross
reacted strongly with FK P. gingivalis W50 cells and the RgpA-Kgp
complex (FIG. 6). Furthermore, the KAS2-LA1 chimera induced only
weak immunoreactive IgG2a, IgG2b and IgG3 antigen-specific
antibodies (FIG. 6).
Example 9
Development of a Kgp Structural Model and Identification of Active
Site Surface Accessible Sequences.
[0195] Our work has shown that Kgp proteinase active site peptides
are highly immunogenic and induce high levels of protection against
P. gingivalis-induced bone loss. In an attempt to identify further
proteinase active site peptides as vaccine candidates a model of
the catalytic domain of Kgp was developed using the Orchestrar
suite of programs within Sybyl7.3 (FIG. 7). The model is based on
PDB structure 1 crv of the RgpB protease from P. gingivalis, the
proteins have a 23.58% pairwise identity and the Z-score is 25.09
(a high-confidence model). The Meta-PPisp protein interaction
server predicts two protein-protein interaction surfaces for Kgp:
the substrate binding surface (as in RgpB), and a second surface
unique to Kgp. The major differences between the RgpB and Kgp
models are in the loops that frame the second interaction surface
and a 19-residue gap (Val526 to Phe545) that couldn't be modeled in
Kgp that falls within the second interaction surface. FIG. 7 shows
the Kgp model with the thicker ribbons showing surface accessible
sequences around the proteinase active site of Kgp, the surface
accessible sequences were found to be Asp388-Gln394, Leu421-Ala423,
Ala443-Glu447 with Ala451, Asn510-Trp513, and Ile570-Gly577 with
Tyr580. From the model (FIG. 6) it is evident that along with KAS2
(A) three other sequences KAS4 (Asp388-Val395) (B), KAS5
(Asn510-Asp516) (C) and KAS6 (Ile570-Tyr580) (D) are prominent and
of sufficient length to be vaccine targets. Thus a recombinant
chimera protein can be produced that has each of these peptides in
sequence and joined on to the N-terminus of KLA1 to produce
multiKAS-KLA1, that can be used to induce an immune response and
hence to protect against P. gingivalis related diseases or
conditions.
Example 10
Process for Modelling Arg-X-Proteinase to Identify Immunogenic
Regions Flanking the Catalytic Site.
[0196] The Arg-X proteinase three dimensional structure was
determined according to the methods of Eichinger A, Beisel H G,
Jacob U, Huber R, Medrano F J, Banbula A, Potempa J, Travis J, Bode
W. Crystal structure of gingipain R: an Arg-specific bacterial
cysteine proteinase with a caspase-like fold. EMBO J. 1999 Oct. 15;
18(20):5453-62
Example 11
[0197] The following is an example of a toothpaste formulation
containing antibodies.
TABLE-US-00005 Ingredient % w/w Dicalcium phosphate dihydrate 50.0
Glycerol 20.0 Sodium carboxymethyl cellulose 1.0 Sodium lauryl
sulphate 1.5 Sodium lauroyl sarconisate 0.5 Flavour 1.0 Sodium
saccharin 0.1 Chlorhexidine gluconate 0.01 Dextranase 0.01 Goat
serum containing specific antibodies 0.2 Water balance
Example 12
[0198] The following is an example of a toothpaste formulation.
TABLE-US-00006 Ingredient % w/w Dicalcium phosphate dihydrate 50.0
Sorbitol 10.0 Glycerol 10.0 Sodium carboxymethyl cellulose 1.0
Sodium lauryl sulphate 1.5 Sodium lauroyl sarconisate 0.5 Flavour
1.0 Sodium saccharin 0.1 Sodium monofluorophosphate 0.3
Chlorhexidine gluconate 0.01 Dextranase 0.01 Bovine serum
containing specific antibodies 0.2 Water balance
Example 13
[0199] The following is an example of a toothpaste formulation.
TABLE-US-00007 Ingredient % w/w Dicalcium phosphate dihydrate 50.0
Sorbitol 10.0 Glycerol 10.0 Sodium carboxymethyl cellulose 1.0
Lauroyl diethanolamide 1.0 Sucrose monolaurate 2.0 Flavour 1.0
Sodium saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine
gluconate 0.01 Dextranase 0.01 Bovine milk Ig containing specific
antibodies 0.1 Water balance
Example 14
[0200] The following is an example of a toothpaste formulation.
TABLE-US-00008 Ingredient % w/w Sorbitol 22.0 Irish moss 1.0 Sodium
Hydroxide (50%) 1.0 Gantrez 19.0 Water (deionised) 2.69 Sodium
Monofluorophosphate 0.76 Sodium saccharine 0.3 Pyrophosphate 2.0
Hydrated alumina 48.0 Flavour oil 0.95 Mouse monoclonal antibodies
0.3 sodium lauryl sulphate 2.00
Example 15
[0201] The following is an example of a liquid toothpaste
formulation.
TABLE-US-00009 Ingredient % w/w Sodium polyacrylate 50.0 Sorbitol
10.0 Glycerol 20.0 Flavour 1.0 Sodium saccharin 0.1 Sodium
monofluorophosphate 0.3 Chlorhexidine gluconate 0.01 Ethanol 3.0
Equine Ig containing specific antibodies 0.2 Linolic acid 0.05
Water balance
Example 16
[0202] The following is an example of a mouthwash formulation.
TABLE-US-00010 Ingredient % w/w Ethanol 20.0 Flavour 1.0 Sodium
saccharin 0.1 Sodium monofluorophosphate 0.3 Chlorhexidine
gluconate 0.01 Lauroyl diethanolamide 0.3 Rabbit Ig containing
specific antibodies 0.2 Water balance
Example 17
[0203] The following is an example of a mouthwash formulation.
TABLE-US-00011 Ingredient % w/w Gantrez S-97 2.5 Glycerine 10.0
Flavour oil 0.4 Sodium monofluorophosphate 0.05 Chlorhexidine
gluconate 0.01 Lauroyl diethanolamide 0.2 Mouse monoclonal
antibodies 0.3 Water balance
Example 18
[0204] The following is an example of a lozenge formulation.
TABLE-US-00012 Ingredient % w/w Sugar 75-80 Corn syrup 1-20 Flavour
oil 1-2 NaF 0.01-0.05 Mouse monoclonal antibodies 0.3 Mg stearate
1-5 Water balance
Example 19
[0205] The following is an example of a gingival massage cream
formulation.
TABLE-US-00013 Ingredient % w/w White petrolatum 8.0 Propylene
glycol 4.0 Stearyl alcohol 8.0 Polyethylene Glycol 4000 25.0
Polyethylene Glycol 400 37.0 Sucrose monostearate 0.5
Chlorohexidine gluconate 0.1 Mouse monoclonal antibodies 0.3 Water
balance
Example 20
[0206] The following is an example of a chewing gum
formulation.
TABLE-US-00014 Ingredient % w/w Gum base 30.0 Calcium carbonate 2.0
Crystalline sorbitol 53.0 Glycerine 0.5 Flavour oil 0.1 Mouse
monoclonal antibodies 0.3 Water balance
Example 21
[0207] The following is an example of a pharmaceutical
formulation
TABLE-US-00015 Ingredient % w/w Humanised specific monoclonal
antibodies 10 Sterile phosphate buffered saline 90
Example 22
[0208] The following is an example of a periodontal gel
formulation.
TABLE-US-00016 Ingredient % w/w Pluronic F127 20.0 Stearyl alcohol
8.0 Specific antibodies 3.0 Colloidal silicon dioxide (Aerosil 200)
1.0 Chlorhexidine gluconate 0.1 Water balance
[0209] It should be understood that while the invention has been
described in details herein, the examples are for illustrative
purposes only. Other modifications of the embodiments of the
present invention that are obvious to those skilled in the art of
molecular biology, dental diagnostics, and related disciplines are
intended to be within the scope of the invention.
REFERENCES
[0210] 1. McKee, A. S., A. S. McDermid, A. Baskerville, A. B.
Dowsett, D. C. Ellwood, and P. D. Marsh. 1986. Effect of hemin on
the physiology and virulence of Bacteroides gingivalis W50. Infect.
Immun. 52:349-355. [0211] 2. Slots, J. 1982. Importance of
black-pigmented Bacteroides in human periodontal disease. Host
parasite interactions in periodontal diseases. American Society for
Microbiology. [0212] 3. O'Brien-Simpson, N. M., R. Pathirana, R. A.
Paolini, Y.-Y. Chen, P. D. Veith, T. V., R. N. Pike, N. Alley, and
E. C. Reynolds. 2005. An immune response directed to proteinase and
adhesin functional epitopes protects against Porphyromonas
gingivalis-induced bone loss. Journal of Immunology 175:3980-3989.
[0213] 4. Baker, P. J., R. T. Evans, and D. C. Roopenian. 1994.
Oral infection with Porphyromonas gingivalis and induced alveolar
bone loss in immunocompetent and severe combined immunodeficient
mice. Arch Oral Biol 39:1035-1040.
Sequence CWU 1
1
85137PRTPorphyromonas gingivalisMISC_FEATURE(6)..(6)X can be either
G or SMISC_FEATURE(18)..(18)X can be either S or
AMISC_FEATURE(23)..(23)X can be either S or
LMISC_FEATURE(24)..(24)X can be either L or
VMISC_FEATURE(26)..(26)X can be either A or
TMISC_FEATURE(27)..(27)X can be either T or
SMISC_FEATURE(29)..(29)X can be either V or
LMISC_FEATURE(36)..(36)X can be either D or N 1Leu Asn Thr Gly Val
Xaa Phe Ala Asn Tyr Thr Ala His Gly Ser Glu 1 5 10 15 Thr Xaa Trp
Ala Asp Pro Xaa Xaa Thr Xaa Xaa Gln Xaa Lys Ala Leu 20 25 30 Thr
Asn Lys Xaa Lys 35 237PRTPorphyromonas
gingivalisMISC_FEATURE(8)..(8)X can be either V or A 2Phe Asn Gly
Gly Ile Ser Leu Xaa Asn Tyr Thr Gly His Gly Ser Glu 1 5 10 15 Thr
Ala Trp Gly Thr Ser His Phe Gly Thr Thr His Val Lys Gln Leu 20 25
30 Thr Asn Ser Asn Gln 35 37PRTPorphyromonas gingivalis 3Val Ser
Phe Ala Asn Tyr Thr 1 5 47PRTPorphyromonas gingivalis 4Val Gly Phe
Ala Asn Tyr Thr 1 5 58PRTPorphyromonas gingivalis 5Gly Val Ser Phe
Ala Asn Tyr Thr 1 5 68PRTPorphyromonas gingivalis 6Gly Val Gly Phe
Ala Asn Tyr Thr 1 5 78PRTPorphyromonas gingivalis 7Val Ser Phe Ala
Asn Tyr Thr Ala 1 5 88PRTPorphyromonas gingivalis 8Val Gly Phe Ala
Asn Tyr Thr Ala 1 5 96PRTPorphyromonas gingivalis 9Glu Thr Ala Trp
Ala Asp 1 5 106PRTPorphyromonas gingivalis 10Glu Thr Ser Trp Ala
Asp 1 5 116PRTPorphyromonas gingivalis 11Thr Ala Trp Ala Asp Pro 1
5 126PRTPorphyromonas gingivalis 12Thr Ser Trp Ala Asp Pro 1 5
137PRTPorphyromonas gingivalis 13Ser Glu Thr Ala Trp Ala Asp 1 5
147PRTPorphyromonas gingivalis 14Ser Glu Thr Ser Trp Ala Asp 1 5
157PRTPorphyromonas gingivalis 15Glu Thr Ala Trp Ala Asp Pro 1 5
167PRTPorphyromonas gingivalis 16Glu Thr Ser Trp Ala Asp Pro 1 5
177PRTPorphyromonas gingivalis 17Thr Ala Trp Ala Asp Pro Leu 1 5
187PRTPorphyromonas gingivalis 18Thr Ser Trp Ala Asp Pro Leu 1 5
198PRTPorphyromonas gingivalis 19Gly Ser Glu Thr Ala Trp Ala Asp 1
5 208PRTPorphyromonas gingivalis 20Gly Ser Glu Thr Ser Trp Ala Asp
1 5 218PRTPorphyromonas gingivalis 21Ser Glu Thr Ala Trp Ala Asp
Pro 1 5 228PRTPorphyromonas gingivalis 22Ser Glu Thr Ser Trp Ala
Asp Pro 1 5 238PRTPorphyromonas gingivalis 23Glu Thr Ala Trp Ala
Asp Pro Leu 1 5 248PRTPorphyromonas gingivalis 24Glu Thr Ser Trp
Ala Asp Pro Leu 1 5 258PRTPorphyromonas gingivalis 25Thr Ala Trp
Ala Asp Pro Leu Leu 1 5 268PRTPorphyromonas gingivalis 26Thr Ser
Trp Ala Asp Pro Leu Leu 1 5 2723PRTPorphyromonas
gingivalisMISC_FEATURE(6)..(6)X can be either G or
SMISC_FEATURE(18)..(18)X can be either S or
AMISC_FEATURE(23)..(23)X can be either S or L 27Leu Asn Thr Gly Val
Xaa Phe Ala Asn Tyr Thr Ala His Gly Ser Glu 1 5 10 15 Thr Xaa Trp
Ala Asp Pro Xaa 20 2836PRTPorphyromonas
gingivalisMISC_FEATURE(5)..(5)X can be either G or
SMISC_FEATURE(17)..(17)X can be either S or
AMISC_FEATURE(22)..(22)X can be either S or
LMISC_FEATURE(23)..(23)X can be either L or
VMISC_FEATURE(25)..(25)X can be either A or
TMISC_FEATURE(26)..(26)X can be either T or
SMISC_FEATURE(28)..(28)X can be either V or
LMISC_FEATURE(35)..(35)X can be either D or N 28Asn Thr Gly Val Xaa
Phe Ala Asn Tyr Thr Ala His Gly Ser Glu Thr 1 5 10 15 Xaa Trp Ala
Asp Pro Xaa Xaa Thr Xaa Xaa Gln Xaa Lys Ala Leu Thr 20 25 30 Asn
Lys Xaa Lys 35 2920PRTPorphyromonas gingivalisMISC_FEATURE(2)..(2)X
can be either G or SMISC_FEATURE(14)..(14)X can be either S or
AMISC_FEATURE(19)..(19)X can be either S or
LMISC_FEATURE(20)..(20)X can be either L or V 29Val Xaa Phe Ala Asn
Tyr Thr Ala His Gly Ser Glu Thr Xaa Trp Ala 1 5 10 15 Asp Pro Xaa
Xaa 20 3022PRTPorphyromonas gingivalis 30Leu Asn Thr Gly Val Ser
Phe Ala Asn Tyr Thr Ala His Gly Ser Glu 1 5 10 15 Thr Ala Trp Ala
Asp Pro 20 3123PRTPorphyromonas gingivalisMISC_FEATURE(8)..(8)X can
be either V or A 31Phe Asn Gly Gly Ile Ser Leu Xaa Asn Tyr Thr Gly
His Gly Ser Glu 1 5 10 15 Thr Ala Trp Gly Thr Ser His 20
3236PRTPorphyromonas gingivalisMISC_FEATURE(7)..(7)X can be either
V or A 32Asn Gly Gly Ile Ser Leu Xaa Asn Tyr Thr Gly His Gly Ser
Glu Thr 1 5 10 15 Ala Trp Gly Thr Ser His Phe Gly Thr Thr His Val
Lys Gln Leu Thr 20 25 30 Asn Ser Asn Gln 35 3320PRTPorphyromonas
gingivalisMISC_FEATURE(4)..(4)X can be either V or A 33Ile Ser Leu
Xaa Asn Tyr Thr Gly His Gly Ser Glu Thr Ala Trp Gly 1 5 10 15 Thr
Ser His Phe 20 3421PRTPorphyromonas gingivalis 34Phe Asn Gly Gly
Ile Ser Leu Ala Asn Tyr Thr Gly His Gly Ser Glu 1 5 10 15 Thr Ala
Trp Gly Thr 20 35362PRTPorphyromonas gingivalis 35Ala Asn Glu Ala
Lys Val Val Leu Ala Ala Asp Asn Val Trp Gly Asp 1 5 10 15 Asn Thr
Gly Tyr Gln Phe Leu Leu Asp Ala Asp His Asn Thr Phe Gly 20 25 30
Ser Val Ile Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala Ser Ser 35
40 45 Asn Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro Ala Asn Ala
Asp 50 55 60 Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln
Gly Glu Val 65 70 75 80 Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile
Thr Asn Pro Glu Pro 85 90 95 Ala Ser Gly Lys Met Trp Ile Ala Gly
Asp Gly Gly Asn Gln Pro Ala 100 105 110 Arg Tyr Asp Asp Phe Thr Phe
Glu Ala Gly Lys Lys Tyr Thr Phe Thr 115 120 125 Met Arg Arg Ala Gly
Met Gly Asp Gly Thr Asp Met Glu Val Glu Asp 130 135 140 Asp Ser Pro
Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys 145 150 155 160
Ile Lys Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala 165
170 175 Ala Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly
Val 180 185 190 Ser Pro Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser
Asn Glu Phe 195 200 205 Ala Pro Val Gln Asn Leu Thr Gly Ser Ser Val
Gly Gln Lys Val Thr 210 215 220 Leu Lys Trp Asp Ala Pro Asn Gly Thr
Pro Asn Pro Asn Pro Asn Pro 225 230 235 240 Asn Pro Asn Pro Gly Thr
Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile 245 250 255 Pro Ala Ser Trp
Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly Trp 260 265 270 Lys Pro
Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn Gly Cys 275 280 285
Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val Leu Thr Pro 290
295 300 Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn Gly Gly
Lys 305 310 315 320 Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr
Ala Ser Glu His 325 330 335 Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn
Asp Ala Ser Asn Phe Thr 340 345 350 Asn Ala Leu Leu Glu Glu Thr Ile
Thr Ala 355 360 36231PRTPorphyromonas gingivalis 36Phe Leu Leu Asp
Ala Asp His Asn Thr Phe Gly Ser Val Ile Pro Ala 1 5 10 15 Thr Gly
Pro Leu Phe Thr Gly Thr Ala Ser Ser Asn Leu Tyr Ser Ala 20 25 30
Asn Phe Glu Tyr Leu Ile Pro Ala Asn Ala Asp Pro Val Val Thr Thr 35
40 45 Gln Asn Ile Ile Val Thr Gly Gln Gly Glu Val Val Ile Pro Gly
Gly 50 55 60 Val Tyr Asp Tyr Cys Ile Thr Asn Pro Glu Pro Ala Ser
Gly Lys Met 65 70 75 80 Trp Ile Ala Gly Asp Gly Gly Asn Gln Pro Ala
Arg Tyr Asp Asp Phe 85 90 95 Thr Phe Glu Ala Gly Lys Lys Tyr Thr
Phe Thr Met Arg Arg Ala Gly 100 105 110 Met Gly Asp Gly Thr Asp Met
Glu Val Glu Asp Asp Ser Pro Ala Ser 115 120 125 Tyr Thr Tyr Thr Val
Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu 130 135 140 Thr Ala Thr
Thr Phe Glu Glu Asp Gly Val Ala Ala Gly Asn His Glu 145 150 155 160
Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Val Cys 165
170 175 Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val Gln
Asn 180 185 190 Leu Thr Gly Ser Ser Val Gly Gln Lys Val Thr Leu Lys
Trp Asp Ala 195 200 205 Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn Pro
Asn Pro Asn Pro Gly 210 215 220 Thr Thr Leu Ser Glu Ser Phe 225 230
37306PRTPorphyromonas gingivalis 37Trp Gly Asp Asn Thr Gly Tyr Gln
Phe Leu Leu Asp Ala Asp His Asn 1 5 10 15 Thr Phe Gly Ser Val Ile
Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr 20 25 30 Ala Ser Ser Asn
Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro Ala 35 40 45 Asn Ala
Asp Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln 50 55 60
Gly Glu Val Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile Thr Asn 65
70 75 80 Pro Glu Pro Ala Ser Gly Lys Met Trp Ile Ala Gly Asp Gly
Gly Asn 85 90 95 Gln Pro Ala Arg Tyr Asp Asp Phe Thr Phe Glu Ala
Gly Lys Lys Tyr 100 105 110 Thr Phe Thr Met Arg Arg Ala Gly Met Gly
Asp Gly Thr Asp Met Glu 115 120 125 Val Glu Asp Asp Ser Pro Ala Ser
Tyr Thr Tyr Thr Val Tyr Arg Asp 130 135 140 Gly Thr Lys Ile Lys Glu
Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp 145 150 155 160 Gly Val Ala
Ala Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr 165 170 175 Ala
Gly Val Ser Pro Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser 180 185
190 Asn Glu Phe Ala Pro Val Gln Asn Leu Thr Gly Ser Ser Val Gly Gln
195 200 205 Lys Val Thr Leu Lys Trp Asp Ala Pro Asn Gly Thr Pro Asn
Pro Asn 210 215 220 Pro Asn Pro Asn Pro Asn Pro Gly Thr Thr Leu Ser
Glu Ser Phe Glu 225 230 235 240 Asn Gly Ile Pro Ala Ser Trp Lys Thr
Ile Asp Ala Asp Gly Asp Gly 245 250 255 His Gly Trp Lys Pro Gly Asn
Ala Pro Gly Ile Ala Gly Tyr Asn Ser 260 265 270 Asn Gly Cys Val Tyr
Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val 275 280 285 Leu Thr Pro
Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn 290 295 300 Gly
Gly 305 38362PRTPorphyromonas gingivalis 38Ser Gly Gln Ala Glu Ile
Val Leu Glu Ala His Asp Val Trp Asn Asp 1 5 10 15 Gly Ser Gly Tyr
Gln Ile Leu Leu Asp Ala Asp His Asp Gln Tyr Gly 20 25 30 Gln Val
Ile Pro Ser Asp Thr His Thr Leu Trp Pro Asn Cys Ser Val 35 40 45
Pro Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Val Pro Glu Asn Ala 50
55 60 Asp Pro Ser Cys Ser Pro Thr Asn Met Ile Met Asp Gly Thr Ala
Ser 65 70 75 80 Val Asn Ile Pro Ala Gly Thr Tyr Asp Phe Ala Ile Ala
Ala Pro Gln 85 90 95 Ala Asn Ala Lys Ile Trp Ile Ala Gly Gln Gly
Pro Thr Lys Glu Asp 100 105 110 Asp Tyr Val Phe Glu Ala Gly Lys Lys
Tyr His Phe Leu Met Lys Lys 115 120 125 Met Gly Ser Gly Asp Gly Thr
Glu Leu Thr Ile Ser Glu Gly Gly Gly 130 135 140 Ser Asp Tyr Thr Tyr
Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu 145 150 155 160 Gly Leu
Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn 165 170 175
His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys 180
185 190 Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu Phe Ala Pro
Val 195 200 205 Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr
Leu Lys Trp 210 215 220 Asp Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro
Asn Pro Asn Pro Asn 225 230 235 240 Pro Asn Pro Gly Thr Thr Thr Leu
Ser Glu Ser Phe Glu Asn Gly Ile 245 250 255 Pro Ala Ser Trp Lys Thr
Ile Asp Ala Asp Gly Asp Gly His Gly Trp 260 265 270 Lys Pro Gly Asn
Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn Gly Cys 275 280 285 Val Tyr
Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly Val Leu Thr Pro 290 295 300
Asp Asn Tyr Leu Ile Thr Pro Ala Leu Asp Leu Pro Asn Gly Gly Lys 305
310 315 320 Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser
Glu His 325 330 335 Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala
Ser Asn Phe Thr 340 345 350 Asn Ala Leu Leu Glu Glu Thr Ile Thr Ala
355 360 39141PRTPorphyromonas gingivalis 39Asp Asp Tyr Val Phe Glu
Ala Gly Lys Lys Tyr His Phe Leu Met Lys 1 5 10 15 Lys Met Gly Ser
Gly Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gly 20 25 30 Gly Ser
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys 35 40 45
Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly 50
55 60 Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser
Pro 65 70 75 80 Lys Val Cys Lys Asp Val Thr Val Glu Gly Ser Asn Glu
Phe Ala Pro 85 90 95 Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln
Lys Val Thr Leu Lys 100 105 110 Trp Asp Ala Pro Asn Gly Thr Pro Asn
Pro Asn Pro Asn Pro Asn Pro 115 120 125 Asn Pro Asn Pro Gly Thr Thr
Thr Leu Ser Glu Ser Phe 130 135 140 40119PRTPorphyromonas
gingivalis 40Ala Asp Phe Thr Glu Thr Phe Glu Ser Ser Thr His Gly
Glu Ala Pro 1 5 10 15 Ala Glu Trp Thr Thr Ile Asp Ala Asp Gly Asp
Gly Gln Gly Trp Leu 20 25 30 Cys Leu Ser Ser Gly Gln Leu Asp Trp
Leu Thr Ala His Gly Gly Ser 35 40 45 Asn Val Val Ser Ser Phe Ser
Trp Asn Gly Met Ala Leu Asn Pro Asp 50 55 60 Asn Tyr Leu Ile Ser
Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr 65 70 75 80 Tyr Tyr Ala
Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met 85 90
95 Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu
100 105 110 Glu Thr Pro Asn Gly Ile Asn 115 41133PRTPorphyromonas
gingivalis 41Pro Gln Ser Val Trp Ile Glu Arg Thr Val Asp Leu Pro
Ala Gly Thr 1 5 10 15 Lys Tyr Val Ala Phe Arg His Tyr Asn Cys Ser
Asp Leu Asn Tyr Ile 20 25 30 Leu Leu Asp Asp Ile Gln Phe Thr Met
Gly Gly Ser Pro Thr Pro Thr 35 40 45 Asp Tyr Thr Tyr Thr Val Tyr
Arg Asp Gly Thr Lys Ile Lys Glu Gly 50 55 60 Leu Thr Glu Thr Thr
Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His 65 70 75 80 Glu Tyr Cys
Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Lys 85 90 95 Cys
Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn Pro Val Gln Asn 100 105
110 Leu Thr Ala Glu Gln Ala Pro Asn Ser Met Asp Ala Ile Leu Lys Trp
115 120 125 Asn Ala Pro Ala Ser 130 42120PRTPorphyromonas
gingivalis 42Ala Glu Val Leu Asn Glu Asp Phe Glu Asn Gly Ile Pro
Ala Ser Trp 1 5 10 15 Lys Thr Ile Asp Ala Asp Gly Asp Gly Asn Asn
Trp Thr Thr Thr Pro 20 25 30 Pro Pro Gly Gly Ser Ser Phe Ala Gly
His Asn Ser Ala Ile Cys Val 35 40 45 Ser Ser Ala Ser Tyr Ile Asn
Phe Glu Gly Pro Gln Asn Pro Asp Asn 50 55 60 Tyr Leu Val Thr Pro
Glu Leu Ser Leu Pro Gly Gly Gly Thr Leu Thr 65 70 75 80 Phe Trp Val
Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr Ala 85 90 95 Val
Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala Asn Ala 100 105
110 Leu Leu Glu Glu Val Leu Thr Ala 115 120 43185PRTPorphyromonas
gingivalis 43Thr Val Val Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg
Ala Gln Gly 1 5 10 15 Thr Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala
Gly Thr Lys Tyr Val 20 25 30 Ala Phe Arg His Phe Gly Cys Thr Asp
Phe Phe Trp Ile Asn Leu Asp 35 40 45 Asp Val Val Ile Thr Ser Gly
Asn Ala Pro Ser Tyr Thr Tyr Thr Ile 50 55 60 Tyr Arg Asn Asn Thr
Gln Ile Ala Ser Gly Val Thr Glu Thr Thr Tyr 65 70 75 80 Arg Asp Pro
Asp Leu Ala Thr Gly Phe Tyr Thr Tyr Gly Val Lys Val 85 90 95 Val
Tyr Pro Asn Gly Glu Ser Ala Ile Glu Thr Ala Thr Leu Asn Ile 100 105
110 Thr Ser Leu Ala Asp Val Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val
115 120 125 Val Gly Lys Thr Ile Thr Val Thr Cys Gln Gly Glu Ala Met
Ile Tyr 130 135 140 Asp Met Asn Gly Arg Arg Leu Ala Ala Gly Arg Asn
Thr Val Val Tyr 145 150 155 160 Thr Ala Gln Gly Gly His Tyr Ala Val
Met Val Val Val Asp Gly Lys 165 170 175 Ser Tyr Val Glu Lys Leu Ala
Val Lys 180 185 44119PRTPorphyromonas gingivalis 44Ala Asp Phe Thr
Glu Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro 1 5 10 15 Ala Glu
Trp Thr Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu 20 25 30
Cys Leu Ser Ser Gly Gln Leu Asp Trp Leu Thr Ala His Gly Gly Thr 35
40 45 Asn Val Val Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn Pro
Asp 50 55 60 Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys
Val Lys Tyr 65 70 75 80 Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp
His Tyr Ala Val Met 85 90 95 Ile Ser Lys Thr Gly Thr Asn Ala Gly
Asp Phe Thr Val Val Phe Glu 100 105 110 Glu Thr Pro Asn Gly Ile Asn
115 45131PRTPorphyromonas gingivalis 45Pro Gln Ser Val Trp Ile Glu
Arg Thr Val Asp Leu Pro Ala Gly Thr 1 5 10 15 Lys Tyr Val Ala Phe
Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile 20 25 30 Leu Leu Asp
Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr 35 40 45 Asp
Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly 50 55
60 Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His
65 70 75 80 Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro
Lys Lys 85 90 95 Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn
Pro Val Lys Asn 100 105 110 Leu Lys Ala Gln Pro Asp Gly Gly Asp Val
Val Leu Lys Trp Glu Ala 115 120 125 Pro Ser Ala 130
46275PRTPorphyromonas gingivalis 46Ala Asn Glu Ala Lys Val Val Leu
Ala Ala Asp Asn Val Trp Gly Asp 1 5 10 15 Asn Thr Gly Tyr Gln Phe
Leu Leu Asp Ala Asp His Asn Thr Phe Gly 20 25 30 Ser Val Ile Pro
Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala Ser Ser 35 40 45 Asp Leu
Tyr Ser Ala Asn Phe Glu Ser Leu Ile Pro Ala Asn Ala Asp 50 55 60
Pro Val Val Thr Thr Gln Asn Ile Ile Val Thr Gly Gln Gly Glu Val 65
70 75 80 Val Ile Pro Gly Gly Val Tyr Asp Tyr Cys Ile Thr Asn Pro
Glu Pro 85 90 95 Ala Ser Gly Lys Met Trp Ile Ala Gly Asp Gly Gly
Asn Gln Pro Ala 100 105 110 Arg Tyr Asp Asp Phe Thr Phe Glu Ala Gly
Lys Lys Tyr Thr Phe Thr 115 120 125 Met Arg Arg Ala Gly Met Gly Asp
Gly Thr Asp Met Glu Val Glu Asp 130 135 140 Asp Ser Pro Ala Ser Tyr
Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys 145 150 155 160 Ile Lys Glu
Gly Leu Thr Glu Thr Thr Tyr Arg Asp Ala Gly Met Ser 165 170 175 Ala
Gln Ser His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val 180 185
190 Ser Pro Lys Val Cys Val Asp Tyr Ile Pro Asp Gly Val Ala Asp Val
195 200 205 Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val Val Gly Lys Thr
Ile Thr 210 215 220 Val Thr Cys Gln Gly Glu Ala Met Ile Tyr Asp Met
Asn Gly Arg Arg 225 230 235 240 Leu Ala Ala Gly Arg Asn Thr Val Val
Tyr Thr Ala Gln Gly Gly Tyr 245 250 255 Tyr Ala Val Met Val Val Val
Asp Gly Lys Ser Tyr Val Glu Lys Leu 260 265 270 Ala Ile Lys 275
4749DNAPorphyromonas gingivalis 47gaccatggct catcaccatc accatcacaa
taccggagtc agctttgca 494836DNAPorphyromonas gingivalis 48gactcgagtt
atttgtcctt attagtgagt gctttc 364931DNAPorphyromonas gingivalis
49gaccatggct tggggagaca atacgggtta c 315027DNAPorphyromonas
gingivalis 50gactcgagac ctccgttagg caaatcc 275141DNAPorphyromonas
gingivalis 51ccgtattgtc tccccatttg tccttattag tgagtgcttt c
415237DNAPorphyromonas gingivalis 52cactaataag gacaaatggg
gagacaatac gggttac 375351DNAPorphyromonas gingivalis 53catggatctg
agaccgcatg ggctgatcca cttttcttgt tggatgccga t
515462DNAPorphyromonas gingivalis 54ccatggcttt gaataccgga
gtcagctttg caaactatac agcgcatgga tctgagaccg 60ca
625525DNAPorphyromonas gingivalis 55ctcgaggaat gattcggaaa gtgtt
255653DNAPorphyromonas gingivalis 56ccatggctga ttatagctgg
aattcccagg tagtcagctt tgcaaactat aca 535752DNAPorphyromonas
gingivalis 57ctttgcaaac tatacagcgc atggatctga gaccgcatgg gctgatccac
tt 525852DNAPorphyromonas gingivalis 58atgggctgat ccacttctga
attcttattg gggcgagatc ggcaatatta cc 525952DNAPorphyromonas
gingivalis 59gatcggcaat attacccata ttggtgctca ttacgcttgg ggagacaata
cg 526052DNAPorphyromonas gingivalis 60ctcgagacct ccgttaggca
aatccaatgc cggtgttatc agatagttgt ca 52611706PRTPorphyromonas
gingivalis 61Met Lys Asn Leu Asn Lys Phe Val Ser Ile Ala Leu Cys
Ser Ser Leu 1 5 10 15 Leu Gly Gly Met Ala Phe Ala Gln Gln Thr Glu
Leu Gly Arg Asn Pro 20 25 30 Asn Val Arg Leu Leu Glu Ser Thr Gln
Gln Ser Val Thr Lys Val Gln 35 40 45 Phe Arg Met Asp Asn Leu Lys
Phe Thr Glu Val Gln Thr Pro Lys Gly 50 55 60 Ile Gly Gln Val Pro
Thr Tyr Thr Glu Gly Val Asn Leu Ser Glu Lys 65 70 75 80 Gly Met Pro
Thr Leu Pro Ile Leu Ser Arg Ser Leu Ala Val Ser Asp 85 90 95 Thr
Arg Glu Met Lys Val Glu Val Val Ser Ser Lys Phe Ile Glu Lys 100 105
110 Lys Asn Val Leu Ile Ala Pro Ser Lys Gly Met Ile Met Arg Asn Glu
115 120 125 Asp Pro Lys Lys Ile Pro Tyr Val Tyr Gly Lys Thr Tyr Ser
Gln Asn 130 135 140 Lys Phe Phe Pro Gly Glu Ile Ala Thr Leu Asp Asp
Pro Phe Ile Leu 145 150 155 160 Arg Asp Val Arg Gly Gln Val Val Asn
Phe Ala Pro Leu Gln Tyr Asn 165 170 175 Pro Val Thr Lys Thr Leu Arg
Ile Tyr Thr Glu Ile Thr Val Ala Val 180 185 190 Ser Glu Thr Ser Glu
Gln Gly Lys Asn Ile Leu Asn Lys Lys Gly Thr 195 200 205 Phe Ala Gly
Phe Glu Asp Thr Tyr Lys Arg Met Phe Met Asn Tyr Glu 210 215 220 Pro
Gly Arg Tyr Thr Pro Val Glu Glu Lys Gln Asn Gly Arg Met Ile 225 230
235 240 Val Ile Val Ala Lys Lys Tyr Glu Gly Asp Ile Lys Asp Phe Val
Asp 245 250 255 Trp Lys Asn Gln Arg Gly Leu Arg Thr Glu Val Lys Val
Ala Glu Asp 260 265 270 Ile Ala Ser Pro Val Thr Ala Asn Ala Ile Gln
Gln Phe Val Lys Gln 275 280 285 Glu Tyr Glu Lys Glu Gly Asn Asp Leu
Thr Tyr Val Leu Leu Ile Gly 290 295 300 Asp His Lys Asp Ile Pro Ala
Lys Ile Thr Pro Gly Ile Lys Ser Asp 305 310 315 320 Gln Val Tyr Gly
Gln Ile Val Gly Asn Asp His Tyr Asn Glu Val Phe 325 330 335 Ile Gly
Arg Phe Ser Cys Glu Ser Lys Glu Asp Leu Lys Thr Gln Ile 340 345 350
Asp Arg Thr Ile His Tyr Glu Arg Asn Ile Thr Thr Glu Asp Lys Trp 355
360 365 Leu Gly Gln Ala Leu Cys Ile Ala Ser Ala Glu Gly Gly Pro Ser
Ala 370 375 380 Asp Asn Gly Glu Ser Asp Ile Gln His Glu Asn Val Ile
Ala Asn Leu 385 390 395 400 Leu Thr Gln Tyr Gly Tyr Thr Lys Ile Ile
Lys Cys Tyr Asp Pro Gly 405 410 415 Val Thr Pro Lys Asn Ile Ile Asp
Ala Phe Asn Gly Gly Ile Ser Leu 420 425 430 Ala Asn Tyr Thr Gly His
Gly Ser Glu Thr Ala Trp Gly Thr Ser His 435 440 445 Phe Gly Thr Thr
His Val Lys Gln Leu Thr Asn Ser Asn Gln Leu Pro 450 455 460 Phe Ile
Phe Asp Val Ala Cys Val Asn Gly Asp Phe Leu Phe Ser Met 465 470 475
480 Pro Cys Phe Ala Glu Ala Leu Met Arg Ala Gln Lys Asp Gly Lys Pro
485 490 495 Thr Gly Thr Val Ala Ile Ile Ala Ser Thr Ile Asn Gln Ser
Trp Ala 500 505 510 Ser Pro Met Arg Gly Gln Asp Glu Met Asn Glu Ile
Leu Cys Glu Lys 515 520 525 His Pro Asn Asn Ile Lys Arg Thr Phe Gly
Gly Val Thr Met Asn Gly 530 535 540 Met Phe Ala Met Val Glu Lys Tyr
Lys Lys Asp Gly Glu Lys Met Leu 545 550 555 560 Asp Thr Trp Thr Val
Phe Gly Asp Pro Ser Leu Leu Val Arg Thr Leu 565 570 575 Val Pro Thr
Lys Met Gln Val Thr Ala Pro Ala Gln Ile Asn Leu Thr 580 585 590 Asp
Ala Ser Val Asn Val Ser Cys Asp Tyr Asn Gly Ala Ile Ala Thr 595 600
605 Ile Ser Ala Asn Gly Lys Met Phe Gly Ser Ala Val Val Glu Asn Gly
610 615 620 Thr Ala Thr Ile Asn Leu Thr Gly Leu Thr Asn Glu Ser Thr
Leu Thr 625 630 635 640 Leu Thr Val Val Gly Tyr Asn Lys Glu Thr Val
Ile Lys Thr Ile Asn 645 650 655 Thr Asn Gly Glu Pro Asn Pro Tyr Gln
Pro Val Ser Asn Leu Thr Ala 660 665 670 Thr Thr Gln Gly Gln Lys Val
Thr Leu Lys Trp Asp Ala Pro Ser Thr 675 680 685 Lys Thr Asn Ala Thr
Thr Asn Thr Ala Arg Ser Val Asp Gly Ile Arg 690 695 700 Glu Leu Val
Leu Leu Ser Val Ser Asp Ala Pro Glu Leu Leu Arg Ser 705 710 715 720
Gly Gln Ala Glu Ile Val Leu Glu Ala His Asp Val Trp Asn Asp Gly 725
730 735 Ser Gly Tyr Gln Ile Leu Leu Asp Ala Asp His Asp Gln Tyr Gly
Gln 740 745 750 Val Ile Pro Ser Asp Thr His Thr Leu Trp Pro Asn Cys
Ser Val Pro 755 760 765 Ala Asn Leu Phe Ala Pro Phe Glu Tyr Thr Val
Pro Glu Asn Ala Asp 770 775 780 Pro Ser Cys Ser Pro Thr Asn Met Ile
Met Asp Gly Thr Ala Ser Val 785 790 795 800 Asn Ile Pro Ala Gly Thr
Tyr Asp Phe Ala Ile Ala Ala Pro Gln Ala 805 810 815 Asn Ala Lys Ile
Trp Ile Ala Gly Gln Gly Pro Thr Lys Glu Asp Asp 820 825 830 Tyr Val
Phe Glu Ala Gly Lys Lys Tyr His Phe Leu Met Lys Lys Met 835 840 845
Gly Ser Gly Asp Gly Thr Glu Leu Thr Ile Ser Glu Gly Gly Gly Ser 850
855 860 Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys Glu
Gly 865 870 875 880 Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val Ala
Thr Gly Asn His 885 890 895 Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala
Gly Val Ser Pro Lys Val 900 905 910 Cys Lys Asp Val Thr Val Glu Gly
Ser Asn Glu Phe Ala Pro Val Gln 915 920 925 Asn Leu Thr Gly Ser Ala
Val Gly Gln Lys Val Thr Leu Lys Trp Asp 930 935 940 Ala Pro Asn Gly
Thr Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro 945 950 955 960 Asn
Pro Gly Thr Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly Ile Pro 965 970
975 Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly Trp Lys
980 985 990 Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser Asn Gly
Cys Val 995 1000 1005 Tyr Ser Glu Ser Phe Gly Leu Gly Gly Ile Gly
Val Leu Thr Pro 1010 1015 1020 Asp Asn Tyr Leu Ile Thr Pro Ala Leu
Asp Leu Pro Asn Gly Gly 1025 1030 1035 Lys Leu Thr Phe Trp Val Cys
Ala Gln Asp Ala Asn Tyr Ala Ser 1040 1045 1050 Glu His Tyr Ala Val
Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser 1055 1060 1065 Asn Phe Thr
Asn Ala Leu Leu
Glu Glu Thr Ile Thr Ala Lys Gly 1070 1075 1080 Val Arg Ser Pro Glu
Ala Met Arg Gly Arg Ile Gln Gly Thr Trp 1085 1090 1095 Arg Gln Lys
Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala 1100 1105 1110 Phe
Arg His Phe Gln Ser Thr Asp Met Phe Tyr Ile Asp Leu Asp 1115 1120
1125 Glu Val Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu
1130 1135 1140 Thr Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu
Trp Thr 1145 1150 1155 Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp
Leu Cys Leu Ser 1160 1165 1170 Ser Gly Gln Leu Asp Trp Leu Thr Ala
His Gly Gly Thr Asn Val 1175 1180 1185 Val Ser Ser Phe Ser Trp Asn
Gly Met Ala Leu Asn Pro Asp Asn 1190 1195 1200 Tyr Leu Ile Ser Lys
Asp Val Thr Gly Ala Thr Lys Val Lys Tyr 1205 1210 1215 Tyr Tyr Ala
Val Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val 1220 1225 1230 Met
Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val 1235 1240
1245 Phe Glu Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe
1250 1255 1260 Gly Leu Ser Thr Glu Ala Asp Gly Ala Lys Pro Gln Ser
Val Trp 1265 1270 1275 Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr
Lys Tyr Val Ala 1280 1285 1290 Phe Arg His Tyr Asn Cys Ser Asp Leu
Asn Tyr Ile Leu Leu Asp 1295 1300 1305 Asp Ile Gln Phe Thr Met Gly
Gly Ser Pro Thr Pro Thr Asp Tyr 1310 1315 1320 Thr Tyr Thr Val Tyr
Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu 1325 1330 1335 Thr Glu Thr
Thr Phe Glu Glu Asp Gly Val Ala Thr Gly Asn His 1340 1345 1350 Glu
Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys 1355 1360
1365 Lys Cys Val Asn Val Thr Val Asn Ser Thr Gln Phe Asn Pro Val
1370 1375 1380 Lys Asn Leu Lys Ala Gln Pro Asp Gly Gly Asp Val Val
Leu Lys 1385 1390 1395 Trp Glu Ala Pro Ser Ala Lys Lys Thr Glu Gly
Ser Arg Glu Val 1400 1405 1410 Lys Arg Ile Gly Asp Gly Leu Phe Val
Thr Ile Glu Pro Ala Asn 1415 1420 1425 Asp Val Arg Ala Asn Glu Ala
Lys Val Val Leu Ala Ala Asp Asn 1430 1435 1440 Val Trp Gly Asp Asn
Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp 1445 1450 1455 His Asn Thr
Phe Gly Ser Val Ile Pro Ala Thr Gly Pro Leu Phe 1460 1465 1470 Thr
Gly Thr Ala Ser Ser Asp Leu Tyr Ser Ala Asn Phe Glu Ser 1475 1480
1485 Leu Ile Pro Ala Asn Ala Asp Pro Val Val Thr Thr Gln Asn Ile
1490 1495 1500 Ile Val Thr Gly Gln Gly Glu Val Val Ile Pro Gly Gly
Val Tyr 1505 1510 1515 Asp Tyr Cys Ile Thr Asn Pro Glu Pro Ala Ser
Gly Lys Met Trp 1520 1525 1530 Ile Ala Gly Asp Gly Gly Asn Gln Pro
Ala Arg Tyr Asp Asp Phe 1535 1540 1545 Thr Phe Glu Ala Gly Lys Lys
Tyr Thr Phe Thr Met Arg Arg Ala 1550 1555 1560 Gly Met Gly Asp Gly
Thr Asp Met Glu Val Glu Asp Asp Ser Pro 1565 1570 1575 Ala Ser Tyr
Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys 1580 1585 1590 Glu
Gly Leu Thr Glu Thr Thr Tyr Arg Asp Ala Gly Met Ser Ala 1595 1600
1605 Gln Ser His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val
1610 1615 1620 Ser Pro Lys Val Cys Val Asp Tyr Ile Pro Asp Gly Val
Ala Asp 1625 1630 1635 Val Thr Ala Gln Lys Pro Tyr Thr Leu Thr Val
Val Gly Lys Thr 1640 1645 1650 Ile Thr Val Thr Cys Gln Gly Glu Ala
Met Ile Tyr Asp Met Asn 1655 1660 1665 Gly Arg Arg Leu Ala Ala Gly
Arg Asn Thr Val Val Tyr Thr Ala 1670 1675 1680 Gln Gly Gly Tyr Tyr
Ala Val Met Val Val Val Asp Gly Lys Ser 1685 1690 1695 Tyr Val Glu
Lys Leu Ala Ile Lys 1700 1705 621732PRTPorphyromonas gingivalis
62Met Arg Lys Leu Leu Leu Leu Ile Ala Ala Ser Leu Leu Gly Val Gly 1
5 10 15 Leu Tyr Ala Gln Ser Ala Lys Ile Lys Leu Asp Ala Pro Thr Thr
Arg 20 25 30 Thr Thr Cys Thr Asn Asn Ser Phe Lys Gln Phe Asp Ala
Ser Phe Ser 35 40 45 Phe Asn Glu Val Glu Leu Thr Lys Val Glu Thr
Lys Gly Gly Thr Phe 50 55 60 Ala Ser Val Ser Ile Pro Gly Ala Phe
Pro Thr Gly Glu Val Gly Ser 65 70 75 80 Pro Glu Val Pro Ala Val Arg
Lys Leu Ile Ala Val Pro Val Gly Ala 85 90 95 Thr Pro Val Val Arg
Val Lys Ser Phe Thr Glu Gln Val Tyr Ser Leu 100 105 110 Asn Gln Tyr
Gly Ser Glu Lys Leu Met Pro His Gln Pro Ser Met Ser 115 120 125 Lys
Ser Asp Asp Pro Glu Lys Val Pro Phe Val Tyr Asn Ala Ala Ala 130 135
140 Tyr Ala Arg Lys Gly Phe Val Gly Gln Glu Leu Thr Gln Val Glu Met
145 150 155 160 Leu Gly Thr Met Arg Gly Val Arg Ile Ala Ala Leu Thr
Ile Asn Pro 165 170 175 Val Gln Tyr Asp Val Val Ala Asn Gln Leu Lys
Val Arg Asn Asn Ile 180 185 190 Glu Ile Glu Val Ser Phe Gln Gly Ala
Asp Glu Val Ala Thr Gln Arg 195 200 205 Leu Tyr Asp Ala Ser Phe Ser
Pro Tyr Phe Glu Thr Ala Tyr Lys Gln 210 215 220 Leu Phe Asn Arg Asp
Val Tyr Thr Asp His Gly Asp Leu Tyr Asn Thr 225 230 235 240 Pro Val
Arg Met Leu Val Val Ala Gly Ala Lys Phe Lys Glu Ala Leu 245 250 255
Lys Pro Trp Leu Thr Trp Lys Ala Gln Lys Gly Phe Tyr Leu Asp Val 260
265 270 His Tyr Thr Asp Glu Ala Glu Val Gly Thr Thr Asn Ala Ser Ile
Lys 275 280 285 Ala Phe Ile His Lys Lys Tyr Asn Asp Gly Leu Ala Ala
Ser Ala Ala 290 295 300 Pro Val Phe Leu Ala Leu Val Gly Asp Thr Asp
Val Ile Ser Gly Glu 305 310 315 320 Lys Gly Lys Lys Thr Lys Lys Val
Thr Asp Leu Tyr Tyr Ser Ala Val 325 330 335 Asp Gly Asp Tyr Phe Pro
Glu Met Tyr Thr Phe Arg Met Ser Ala Ser 340 345 350 Ser Pro Glu Glu
Leu Thr Asn Ile Ile Asp Lys Val Leu Met Tyr Glu 355 360 365 Lys Ala
Thr Met Pro Asp Lys Ser Tyr Leu Glu Lys Val Leu Leu Ile 370 375 380
Ala Gly Ala Asp Tyr Ser Trp Asn Ser Gln Val Gly Gln Pro Thr Ile 385
390 395 400 Lys Tyr Gly Met Gln Tyr Tyr Tyr Asn Gln Glu His Gly Tyr
Thr Asp 405 410 415 Val Tyr Asn Tyr Leu Lys Ala Pro Tyr Thr Gly Cys
Tyr Ser His Leu 420 425 430 Asn Thr Gly Val Ser Phe Ala Asn Tyr Thr
Ala His Gly Ser Glu Thr 435 440 445 Ala Trp Ala Asp Pro Leu Leu Thr
Thr Ser Gln Leu Lys Ala Leu Thr 450 455 460 Asn Lys Asp Lys Tyr Phe
Leu Ala Ile Gly Asn Cys Cys Ile Thr Ala 465 470 475 480 Gln Phe Asp
Tyr Val Gln Pro Cys Phe Gly Glu Val Ile Thr Arg Val 485 490 495 Lys
Glu Lys Gly Ala Tyr Ala Tyr Ile Gly Ser Ser Pro Asn Ser Tyr 500 505
510 Trp Gly Glu Asp Tyr Tyr Trp Ser Val Gly Ala Asn Ala Val Phe Gly
515 520 525 Val Gln Pro Thr Phe Glu Gly Thr Ser Met Gly Ser Tyr Asp
Ala Thr 530 535 540 Phe Leu Glu Asp Ser Tyr Asn Thr Val Asn Ser Ile
Met Trp Ala Gly 545 550 555 560 Asn Leu Ala Ala Thr His Ala Gly Asn
Ile Gly Asn Ile Thr His Ile 565 570 575 Gly Ala His Tyr Tyr Trp Glu
Ala Tyr His Val Leu Gly Asp Gly Ser 580 585 590 Val Met Pro Tyr Arg
Ala Met Pro Lys Thr Asn Thr Tyr Thr Leu Pro 595 600 605 Ala Ser Leu
Pro Gln Asn Gln Ala Ser Tyr Ser Ile Gln Ala Ser Ala 610 615 620 Gly
Ser Tyr Val Ala Ile Ser Lys Asp Gly Val Leu Tyr Gly Thr Gly 625 630
635 640 Val Ala Asn Ala Ser Gly Val Ala Thr Val Ser Met Thr Lys Gln
Ile 645 650 655 Thr Glu Asn Gly Asn Tyr Asp Val Val Ile Thr Arg Ser
Asn Tyr Leu 660 665 670 Pro Val Ile Lys Gln Ile Gln Val Gly Glu Pro
Ser Pro Tyr Gln Pro 675 680 685 Val Ser Asn Leu Thr Ala Thr Thr Gln
Gly Gln Lys Val Thr Leu Lys 690 695 700 Trp Glu Ala Pro Ser Ala Lys
Lys Ala Glu Gly Ser Arg Glu Val Lys 705 710 715 720 Arg Ile Gly Asp
Gly Leu Phe Val Thr Ile Glu Pro Ala Asn Asp Val 725 730 735 Arg Ala
Asn Glu Ala Lys Val Val Leu Ala Ala Asp Asn Val Trp Gly 740 745 750
Asp Asn Thr Gly Tyr Gln Phe Leu Leu Asp Ala Asp His Asn Thr Phe 755
760 765 Gly Ser Val Ile Pro Ala Thr Gly Pro Leu Phe Thr Gly Thr Ala
Ser 770 775 780 Ser Asn Leu Tyr Ser Ala Asn Phe Glu Tyr Leu Ile Pro
Ala Asn Ala 785 790 795 800 Asp Pro Val Val Thr Thr Gln Asn Ile Ile
Val Thr Gly Gln Gly Glu 805 810 815 Val Val Ile Pro Gly Gly Val Tyr
Asp Tyr Cys Ile Thr Asn Pro Glu 820 825 830 Pro Ala Ser Gly Lys Met
Trp Ile Ala Gly Asp Gly Gly Asn Gln Pro 835 840 845 Ala Arg Tyr Asp
Asp Phe Thr Phe Glu Ala Gly Lys Lys Tyr Thr Phe 850 855 860 Thr Met
Arg Arg Ala Gly Met Gly Asp Gly Thr Asp Met Glu Val Glu 865 870 875
880 Asp Asp Ser Pro Ala Ser Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr
885 890 895 Lys Ile Lys Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp
Gly Val 900 905 910 Ala Ala Gly Asn His Glu Tyr Cys Val Glu Val Lys
Tyr Thr Ala Gly 915 920 925 Val Ser Pro Lys Val Cys Lys Asp Val Thr
Val Glu Gly Ser Asn Glu 930 935 940 Phe Ala Pro Val Gln Asn Leu Thr
Gly Ser Ser Val Gly Gln Lys Val 945 950 955 960 Thr Leu Lys Trp Asp
Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Asn 965 970 975 Pro Asn Pro
Asn Pro Gly Thr Thr Leu Ser Glu Ser Phe Glu Asn Gly 980 985 990 Ile
Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly Asp Gly His Gly 995
1000 1005 Trp Lys Pro Gly Asn Ala Pro Gly Ile Ala Gly Tyr Asn Ser
Asn 1010 1015 1020 Gly Cys Val Tyr Ser Glu Ser Phe Gly Leu Gly Gly
Ile Gly Val 1025 1030 1035 Leu Thr Pro Asp Asn Tyr Leu Ile Thr Pro
Ala Leu Asp Leu Pro 1040 1045 1050 Asn Gly Gly Lys Leu Thr Phe Trp
Val Cys Ala Gln Asp Ala Asn 1055 1060 1065 Tyr Ala Ser Glu His Tyr
Ala Val Tyr Ala Ser Ser Thr Gly Asn 1070 1075 1080 Asp Ala Ser Asn
Phe Thr Asn Ala Leu Leu Glu Glu Thr Ile Thr 1085 1090 1095 Ala Lys
Gly Val Arg Ser Pro Lys Ala Ile Arg Gly Arg Ile Gln 1100 1105 1110
Gly Thr Trp Arg Gln Lys Thr Val Asp Leu Pro Ala Gly Thr Lys 1115
1120 1125 Tyr Val Ala Phe Arg His Phe Gln Ser Thr Asp Met Phe Tyr
Ile 1130 1135 1140 Asp Leu Asp Glu Val Glu Ile Lys Ala Asn Gly Lys
Arg Ala Asp 1145 1150 1155 Phe Thr Glu Thr Phe Glu Ser Ser Thr His
Gly Glu Ala Pro Ala 1160 1165 1170 Glu Trp Thr Thr Ile Asp Ala Asp
Gly Asp Gly Gln Gly Trp Leu 1175 1180 1185 Cys Leu Ser Ser Gly Gln
Leu Asp Trp Leu Thr Ala His Gly Gly 1190 1195 1200 Ser Asn Val Val
Ser Ser Phe Ser Trp Asn Gly Met Ala Leu Asn 1205 1210 1215 Pro Asp
Asn Tyr Leu Ile Ser Lys Asp Val Thr Gly Ala Thr Lys 1220 1225 1230
Val Lys Tyr Tyr Tyr Ala Val Asn Asp Gly Phe Pro Gly Asp His 1235
1240 1245 Tyr Ala Val Met Ile Ser Lys Thr Gly Thr Asn Ala Gly Asp
Phe 1250 1255 1260 Thr Val Val Phe Glu Glu Thr Pro Asn Gly Ile Asn
Lys Gly Gly 1265 1270 1275 Ala Arg Phe Gly Leu Ser Thr Glu Ala Asn
Gly Ala Lys Pro Gln 1280 1285 1290 Ser Val Trp Ile Glu Arg Thr Val
Asp Leu Pro Ala Gly Thr Lys 1295 1300 1305 Tyr Val Ala Phe Arg His
Tyr Asn Cys Ser Asp Leu Asn Tyr Ile 1310 1315 1320 Leu Leu Asp Asp
Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro 1325 1330 1335 Thr Asp
Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys 1340 1345 1350
Glu Gly Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 1355
1360 1365 Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly
Val 1370 1375 1380 Ser Pro Lys Lys Cys Val Asn Val Thr Val Asn Ser
Thr Gln Phe 1385 1390 1395 Asn Pro Val Gln Asn Leu Thr Ala Glu Gln
Ala Pro Asn Ser Met 1400 1405 1410 Asp Ala Ile Leu Lys Trp Asn Ala
Pro Ala Ser Lys Arg Ala Glu 1415 1420 1425 Val Leu Asn Glu Asp Phe
Glu Asn Gly Ile Pro Ala Ser Trp Lys 1430 1435 1440 Thr Ile Asp Ala
Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro 1445 1450 1455 Pro Pro
Gly Gly Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys 1460 1465 1470
Val Ser Ser Ala Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro 1475
1480 1485 Asp Asn Tyr Leu Val Thr Pro Glu Leu Ser Leu Pro Gly Gly
Gly 1490 1495 1500 Thr Leu Thr Phe Trp Val Cys Ala Gln Asp Ala Asn
Tyr Ala Ser 1505 1510 1515 Glu His Tyr Ala Val Tyr Ala Ser Ser Thr
Gly Asn Asp Ala Ser 1520 1525 1530 Asn Phe Ala Asn Ala Leu Leu Glu
Glu Val Leu Thr Ala Lys Thr 1535 1540 1545 Val Val Thr Ala Pro Glu
Ala Ile Arg Gly Thr Arg Ala Gln Gly 1550 1555 1560 Thr Trp Tyr Gln
Lys Thr Val Gln Leu Pro Ala Gly Thr Lys Tyr 1565 1570 1575 Val Ala
Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn 1580 1585 1590
Leu Asp Asp Val Val Ile Thr Ser Gly Asn Ala Pro Ser Tyr Thr 1595
1600
1605 Tyr Thr Ile Tyr Arg Asn Asn Thr Gln Ile Ala Ser Gly Val Thr
1610 1615 1620 Glu Thr Thr Tyr Arg Asp Pro Asp Leu Ala Thr Gly Phe
Tyr Thr 1625 1630 1635 Tyr Gly Val Lys Val Val Tyr Pro Asn Gly Glu
Ser Ala Ile Glu 1640 1645 1650 Thr Ala Thr Leu Asn Ile Thr Ser Leu
Ala Asp Val Thr Ala Gln 1655 1660 1665 Lys Pro Tyr Thr Leu Thr Val
Val Gly Lys Thr Ile Thr Val Thr 1670 1675 1680 Cys Gln Gly Glu Ala
Met Ile Tyr Asp Met Asn Gly Arg Arg Leu 1685 1690 1695 Ala Ala Gly
Arg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly His 1700 1705 1710 Tyr
Ala Val Met Val Val Val Asp Gly Lys Ser Tyr Val Glu Lys 1715 1720
1725 Leu Ala Val Lys 1730 632164PRTPorphyromonas gingivalis 63Met
Arg Lys Leu Asn Ser Leu Phe Ser Leu Ala Val Leu Leu Ser Leu 1 5 10
15 Leu Cys Trp Gly Gln Thr Ala Ala Ala Gln Gly Gly Pro Lys Thr Ala
20 25 30 Pro Ser Val Thr His Gln Ala Val Gln Lys Gly Ile Arg Thr
Ser Lys 35 40 45 Ala Lys Asp Leu Arg Asp Pro Ile Pro Ala Gly Met
Ala Arg Ile Ile 50 55 60 Leu Glu Ala His Asp Val Trp Glu Asp Gly
Thr Gly Tyr Gln Met Leu 65 70 75 80 Trp Asp Ala Asp His Asn Gln Tyr
Gly Ala Ser Ile Pro Glu Glu Ser 85 90 95 Phe Trp Phe Ala Asn Gly
Thr Ile Pro Ala Gly Leu Tyr Asp Pro Phe 100 105 110 Glu Tyr Lys Val
Pro Val Asn Ala Asp Ala Ser Phe Ser Pro Thr Asn 115 120 125 Phe Val
Leu Asp Gly Thr Ala Ser Ala Asp Ile Pro Ala Gly Thr Tyr 130 135 140
Asp Tyr Val Ile Ile Asn Pro Asn Pro Gly Ile Ile Tyr Ile Val Gly 145
150 155 160 Glu Gly Val Ser Lys Gly Asn Asp Tyr Val Val Glu Ala Gly
Lys Thr 165 170 175 Tyr His Phe Thr Val Gln Arg Gln Gly Pro Gly Asp
Ala Ala Ser Val 180 185 190 Val Val Thr Gly Glu Gly Gly Asn Glu Phe
Ala Pro Val Gln Asn Leu 195 200 205 Gln Trp Ser Val Ser Gly Gln Thr
Val Thr Leu Thr Trp Gln Ala Pro 210 215 220 Ala Ser Asp Lys Arg Thr
Tyr Val Leu Asn Glu Ser Phe Asp Thr Gln 225 230 235 240 Thr Leu Pro
Asn Gly Trp Thr Met Ile Asp Ala Asp Gly Asp Gly His 245 250 255 Asn
Trp Leu Ser Thr Ile Asn Val Tyr Asn Thr Ala Thr His Thr Gly 260 265
270 Asp Gly Ala Met Phe Ser Lys Ser Trp Thr Ala Ser Ser Gly Ala Lys
275 280 285 Ile Asp Leu Ser Pro Asp Asn Tyr Leu Val Thr Pro Lys Phe
Thr Val 290 295 300 Pro Glu Asn Gly Lys Leu Ser Tyr Trp Val Ser Ser
Gln Glu Pro Trp 305 310 315 320 Thr Asn Glu His Tyr Gly Val Phe Leu
Ser Thr Thr Gly Asn Glu Ala 325 330 335 Ala Asn Phe Thr Ile Lys Leu
Leu Glu Glu Thr Leu Gly Ser Gly Lys 340 345 350 Pro Ala Pro Met Asn
Leu Val Lys Ser Glu Gly Val Lys Ala Pro Ala 355 360 365 Pro Tyr Gln
Glu Arg Thr Ile Asp Leu Ser Ala Tyr Ala Gly Gln Gln 370 375 380 Val
Tyr Leu Ala Phe Arg His Phe Gly Cys Thr Gly Ile Phe Arg Leu 385 390
395 400 Tyr Leu Asp Asp Val Ala Val Ser Gly Glu Gly Ser Ser Asn Asp
Tyr 405 410 415 Thr Tyr Thr Val Tyr Arg Asp Asn Val Val Ile Ala Gln
Asn Leu Thr 420 425 430 Ala Thr Thr Phe Asn Gln Glu Asn Val Ala Pro
Gly Gln Tyr Asn Tyr 435 440 445 Cys Val Glu Val Lys Tyr Thr Ala Gly
Val Ser Pro Lys Val Cys Lys 450 455 460 Asp Val Thr Val Glu Gly Ser
Asn Glu Phe Ala Pro Val Gln Asn Leu 465 470 475 480 Thr Gly Ser Ala
Val Gly Gln Lys Val Thr Leu Lys Trp Asp Ala Pro 485 490 495 Asn Gly
Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu Ser 500 505 510
Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp Gly 515
520 525 Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly Gly Ser Ser
Phe 530 535 540 Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala Ser
Tyr Ile Asn 545 550 555 560 Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr
Leu Val Thr Pro Glu Leu 565 570 575 Ser Leu Pro Asn Gly Gly Thr Leu
Thr Phe Trp Val Cys Ala Gln Asp 580 585 590 Ala Asn Tyr Ala Ser Glu
His Tyr Ala Val Tyr Ala Ser Ser Thr Gly 595 600 605 Asn Asp Ala Ser
Asn Phe Ala Asn Ala Leu Leu Glu Glu Val Leu Thr 610 615 620 Ala Lys
Thr Val Val Thr Ala Pro Glu Ala Ile Arg Gly Thr Arg Val 625 630 635
640 Gln Gly Thr Trp Tyr Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys
645 650 655 Tyr Val Ala Phe Arg His Phe Gly Cys Thr Asp Phe Phe Trp
Ile Asn 660 665 670 Leu Asp Asp Val Glu Ile Lys Ala Asn Gly Lys Arg
Ala Asp Phe Thr 675 680 685 Glu Thr Phe Glu Ser Ser Thr His Gly Glu
Ala Pro Ala Glu Trp Thr 690 695 700 Thr Ile Asp Ala Asp Gly Asp Gly
Gln Gly Trp Leu Cys Leu Ser Ser 705 710 715 720 Gly Gln Leu Gly Trp
Leu Thr Ala His Gly Gly Thr Asn Val Val Ala 725 730 735 Ser Phe Ser
Trp Asn Gly Met Ala Leu Asn Pro Asp Asn Tyr Leu Ile 740 745 750 Ser
Lys Asp Val Thr Gly Ala Thr Lys Val Lys Tyr Tyr Tyr Ala Val 755 760
765 Asn Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met Ile Ser Lys Thr
770 775 780 Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu Glu Thr
Pro Asn 785 790 795 800 Gly Ile Asn Lys Gly Gly Ala Arg Phe Gly Leu
Ser Thr Glu Ala Asn 805 810 815 Gly Ala Lys Pro Gln Ser Val Trp Ile
Glu Arg Thr Val Asp Leu Pro 820 825 830 Ala Gly Thr Lys Tyr Val Ala
Phe Arg His Tyr Asn Cys Ser Asp Leu 835 840 845 Asn Tyr Ile Leu Leu
Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro 850 855 860 Thr Pro Thr
Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile 865 870 875 880
Lys Glu Gly Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala Thr 885
890 895 Gly Asn His Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala Gly Val
Ser 900 905 910 Pro Lys Glu Cys Val Asn Val Thr Val Asp Pro Val Gln
Phe Asn Pro 915 920 925 Val Gln Asn Leu Thr Gly Ser Ala Val Gly Gln
Lys Val Thr Leu Lys 930 935 940 Trp Asp Ala Pro Asn Gly Thr Pro Asn
Pro Asn Pro Gly Thr Thr Thr 945 950 955 960 Leu Ser Glu Ser Phe Glu
Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile 965 970 975 Asp Ala Asp Gly
Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly 980 985 990 Gly Thr
Ser Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala 995 1000
1005 Ser Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu
1010 1015 1020 Val Thr Pro Glu Leu Ser Leu Pro Asn Gly Gly Thr Leu
Thr Phe 1025 1030 1035 Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser
Glu His Tyr Ala 1040 1045 1050 Val Tyr Ala Ser Ser Thr Gly Asn Asp
Ala Ser Asn Phe Ala Asn 1055 1060 1065 Ala Leu Leu Glu Glu Val Leu
Thr Ala Lys Thr Val Val Thr Ala 1070 1075 1080 Pro Glu Ala Ile Arg
Gly Thr Arg Val Gln Gly Thr Trp Tyr Gln 1085 1090 1095 Lys Thr Val
Gln Leu Pro Ala Gly Thr Lys Tyr Val Ala Phe Arg 1100 1105 1110 His
Phe Gly Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp Asp Val 1115 1120
1125 Glu Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu Thr Phe
1130 1135 1140 Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr
Thr Ile 1145 1150 1155 Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys
Leu Ser Ser Gly 1160 1165 1170 Gln Leu Asp Trp Leu Thr Ala His Gly
Gly Thr Asn Val Val Ala 1175 1180 1185 Ser Phe Ser Trp Asn Gly Met
Ala Leu Asn Pro Asp Asn Tyr Leu 1190 1195 1200 Ile Ser Lys Asp Val
Thr Gly Ala Thr Lys Val Lys Tyr Tyr Tyr 1205 1210 1215 Ala Val Asn
Asp Gly Phe Pro Gly Asp His Tyr Ala Val Met Ile 1220 1225 1230 Ser
Lys Thr Gly Thr Asn Ala Gly Asp Phe Thr Val Val Phe Glu 1235 1240
1245 Glu Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe Gly Leu
1250 1255 1260 Ser Thr Glu Ala Asn Gly Ala Lys Pro Gln Ser Val Trp
Ile Glu 1265 1270 1275 Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr
Val Ala Phe Arg 1280 1285 1290 His Tyr Asn Cys Ser Asp Leu Asn Tyr
Ile Leu Leu Asp Asp Ile 1295 1300 1305 Gln Phe Thr Met Gly Gly Ser
Pro Thr Pro Thr Asp Tyr Thr Tyr 1310 1315 1320 Thr Val Tyr Arg Asp
Gly Thr Lys Ile Lys Glu Gly Leu Thr Glu 1325 1330 1335 Thr Thr Phe
Glu Glu Asp Gly Val Ala Thr Gly Asn His Glu Tyr 1340 1345 1350 Cys
Val Glu Val Lys Tyr Thr Ala Gly Val Ser Pro Lys Glu Cys 1355 1360
1365 Val Asn Val Thr Val Asp Pro Val Gln Phe Asn Pro Val Gln Asn
1370 1375 1380 Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys
Trp Asp 1385 1390 1395 Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly
Thr Thr Thr Leu 1400 1405 1410 Ser Glu Ser Phe Glu Asn Gly Ile Pro
Ala Ser Trp Lys Thr Ile 1415 1420 1425 Asp Ala Asp Gly Asp Gly Asn
Asn Trp Thr Thr Thr Pro Pro Pro 1430 1435 1440 Gly Gly Thr Ser Phe
Ala Gly His Asn Ser Ala Ile Cys Val Ser 1445 1450 1455 Ser Ala Ser
Tyr Ile Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn 1460 1465 1470 Tyr
Leu Val Thr Pro Glu Leu Ser Leu Pro Asn Gly Gly Thr Leu 1475 1480
1485 Thr Phe Trp Val Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His
1490 1495 1500 Tyr Ala Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser
Asn Phe 1505 1510 1515 Ala Asn Ala Leu Leu Glu Glu Val Leu Thr Ala
Lys Thr Val Val 1520 1525 1530 Thr Ala Pro Glu Ala Ile Arg Gly Thr
Arg Val Gln Gly Thr Trp 1535 1540 1545 Tyr Gln Lys Thr Val Gln Leu
Pro Ala Gly Thr Lys Tyr Val Ala 1550 1555 1560 Phe Arg His Phe Gly
Cys Thr Asp Phe Phe Trp Ile Asn Leu Asp 1565 1570 1575 Asp Val Glu
Ile Lys Ala Asn Gly Lys Arg Ala Asp Phe Thr Glu 1580 1585 1590 Thr
Phe Glu Ser Ser Thr His Gly Glu Ala Pro Ala Glu Trp Thr 1595 1600
1605 Thr Ile Asp Ala Asp Gly Asp Gly Gln Gly Trp Leu Cys Leu Ser
1610 1615 1620 Ser Gly Gln Leu Gly Trp Leu Thr Ala His Gly Gly Thr
Asn Val 1625 1630 1635 Val Ala Ser Phe Ser Trp Asn Gly Met Ala Leu
Asn Pro Asp Asn 1640 1645 1650 Tyr Leu Ile Ser Lys Asp Val Thr Gly
Ala Thr Lys Val Lys Tyr 1655 1660 1665 Tyr Tyr Ala Val Asn Asp Gly
Phe Pro Gly Asp His Tyr Ala Val 1670 1675 1680 Met Ile Ser Lys Thr
Gly Thr Asn Ala Gly Asp Phe Thr Val Val 1685 1690 1695 Phe Glu Glu
Thr Pro Asn Gly Ile Asn Lys Gly Gly Ala Arg Phe 1700 1705 1710 Gly
Leu Ser Thr Glu Ala Asn Gly Ala Lys Pro Gln Ser Val Trp 1715 1720
1725 Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr Lys Tyr Val Ala
1730 1735 1740 Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile Leu
Leu Asp 1745 1750 1755 Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr
Pro Thr Asp Tyr 1760 1765 1770 Thr Tyr Thr Val Tyr Arg Asp Gly Thr
Lys Ile Lys Glu Gly Leu 1775 1780 1785 Thr Glu Thr Thr Phe Glu Glu
Asp Gly Val Ala Thr Gly Asn His 1790 1795 1800 Glu Tyr Cys Val Glu
Val Lys Tyr Thr Ala Gly Val Ser Pro Lys 1805 1810 1815 Glu Cys Val
Asn Val Thr Ile Asn Pro Thr Gln Phe Asn Pro Val 1820 1825 1830 Gln
Asn Leu Thr Ala Glu Gln Ala Pro Asn Ser Met Asp Ala Ile 1835 1840
1845 Leu Lys Trp Asn Ala Pro Ala Ser Lys Arg Ala Glu Val Leu Asn
1850 1855 1860 Glu Asp Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr
Ile Asp 1865 1870 1875 Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr
Pro Pro Pro Gly 1880 1885 1890 Gly Ser Ser Phe Ala Gly His Asn Ser
Ala Ile Cys Val Ser Ser 1895 1900 1905 Ala Ser Tyr Ile Asn Phe Glu
Gly Pro Gln Asn Pro Asp Asn Tyr 1910 1915 1920 Leu Val Thr Pro Glu
Leu Ser Leu Pro Gly Gly Gly Thr Leu Thr 1925 1930 1935 Phe Trp Val
Cys Ala Gln Asp Ala Asn Tyr Ala Ser Glu His Tyr 1940 1945 1950 Ala
Val Tyr Ala Ser Ser Thr Gly Asn Asp Ala Ser Asn Phe Ala 1955 1960
1965 Asn Ala Leu Leu Glu Glu Val Leu Thr Ala Lys Thr Val Val Thr
1970 1975 1980 Ala Pro Glu Ala Ile Arg Gly Thr Arg Val Gln Gly Thr
Trp Tyr 1985 1990 1995 Gln Lys Thr Val Gln Leu Pro Ala Gly Thr Lys
Tyr Val Ala Phe 2000 2005 2010 Arg His Phe Gly Cys Thr Asp Phe Phe
Trp Ile Asn Leu Asp Asp 2015 2020 2025 Val Val Ile Thr Ser Gly Asn
Ala Pro Ser Tyr Thr Tyr Thr Ile 2030 2035 2040 Tyr Arg Asn Asn Thr
Gln Ile Ala Ser Gly Val Thr Glu Thr Thr 2045 2050 2055 Tyr Arg Asp
Pro Asp Leu Ala Thr Gly Phe Tyr Thr Tyr Gly Val 2060 2065 2070 Lys
Val Val Tyr Pro Asn Gly Glu Ser Ala Ile Glu Thr Ala Thr 2075 2080
2085 Leu Asn Ile Thr Ser Leu Ala Asp Val Thr Ala Gln Lys Pro Tyr
2090 2095 2100 Thr Leu Thr Val Val Gly Lys Thr Ile Thr Val Thr Cys
Gln Gly 2105
2110 2115 Glu Ala Met Ile Tyr Asp Met Asn Gly Arg Arg Leu Ala Ala
Gly 2120 2125 2130 Arg Asn Thr Val Val Tyr Thr Ala Gln Gly Gly His
Tyr Ala Val 2135 2140 2145 Met Val Val Val Asp Gly Lys Ser Tyr Val
Glu Lys Leu Ala Val 2150 2155 2160 Lys 648PRTPorphyromonas
gingivalisMISC_FEATURE(2)..(2)X can be either S or
YMISC_FEATURE(3)..(3)X can be either Y or SMISC_FEATURE(6)..(6)X
can be either P or SMISC_FEATURE(7)..(7)X can be either K or
QMISC_FEATURE(8)..(8)X can be either I or V 64Asp Xaa Xaa Trp Asn
Xaa Xaa Xaa 1 5 657PRTPorphyromonas gingivalis 65Asn Ser Tyr Trp
Gly Glu Asp 1 5 6611PRTPorphyromonas
gingivalisMISC_FEATURE(4)..(4)X can be either V or I 66Ile Gly Asn
Xaa Thr His Ile Gly Ala His Tyr 1 5 10 678PRTPorphyromonas
gingivalis 67Glu Gly Gly Pro Ser Ala Asp Asn 1 5
687PRTPorphyromonas gingivalisMISC_FEATURE(1)..(1)X can be either N
or DMISC_FEATURE(3)..(3)X can be either S or YMISC_FEATURE(6)..(6)X
can be either S or P 68Xaa Gln Xaa Trp Ala Xaa Pro 1 5
6923PRTPorphyromonas gingivalis 69Pro Val Ser Asn Leu Thr Ala Thr
Thr Gln Gly Gln Lys Val Thr Leu 1 5 10 15 Lys Trp Asp Ala Pro Ser
Thr 20 7023PRTPorphyromonas gingivalis 70Pro Val Ser Asn Leu Thr
Ala Thr Thr Gln Gly Gln Lys Val Thr Leu 1 5 10 15 Lys Trp Glu Ala
Pro Ser Ala 20 7123PRTPorphyromonas gingivalis 71Pro Val Gln Asn
Leu Thr Gly Ser Ser Val Gly Gln Lys Val Thr Leu 1 5 10 15 Lys Trp
Asp Ala Pro Ser Thr 20 7223PRTPorphyromonas gingivalis 72Pro Val
Gln Asn Leu Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu 1 5 10 15
Lys Trp Asp Ala Pro Asn Gly 20 7323PRTPorphyromonas gingivalis
73Pro Val Lys Asn Leu Lys Ala Gln Pro Asp Gly Gly Asp Val Val Leu 1
5 10 15 Lys Trp Glu Ala Pro Ser Ala 20 7423PRTPorphyromonas
gingivalis 74Pro Val Gln Asn Leu Thr Ala Glu Gln Ala Pro Asn Ser
Met Asp Ala 1 5 10 15 Ile Leu Lys Trp Asn Ala Pro 20
7524PRTPorphyromonas gingivalis 75Pro Val Gln Asn Leu Thr Gln Trp
Ser Val Ser Gly Gln Thr Val Thr 1 5 10 15 Leu Thr Trp Gln Ala Pro
Ala Ser 20 7627PRTPorphyromonas gingivalis 76Tyr Thr Tyr Thr Val
Tyr Arg Asp Gly Thr Lys Ile Lys Glu Gly Leu 1 5 10 15 Thr Glu Thr
Thr Phe Glu Glu Asp Gly Val Ala 20 25 7727PRTPorphyromonas
gingivalis 77Tyr Thr Tyr Thr Val Tyr Arg Asp Asn Val Val Ile Ala
Gln Asn Leu 1 5 10 15 Thr Ala Thr Thr Phe Asn Gln Glu Asn Val Ala
20 25 7828PRTPorphyromonas gingivalis 78Tyr Thr Tyr Thr Val Tyr Arg
Asp Gly Thr Lys Ile Lys Glu Gly Leu 1 5 10 15 Thr Ala Glu Thr Thr
Phe Glu Glu Asp Gly Val Ala 20 25 7916PRTPorphyromonas
gingivalisMISC_FEATURE(6)..(7)X can be NP or NPNP or NPNPNP or
NPNPNPNP or NPNPNPNPNP or NPNPNPNPNPNP 79Pro Asn Gly Thr Pro Xaa
Xaa Gly Thr Thr Thr Leu Ser Glu Ser Phe 1 5 10 15
80325PRTPorphyromonas gingivalis 80Gly Gly Pro Lys Thr Ala Pro Ser
Val Thr His Gln Ala Val Gln Lys 1 5 10 15 Gly Ile Arg Thr Ser Lys
Ala Lys Asp Leu Arg Asp Pro Ile Pro Ala 20 25 30 Gly Met Ala Arg
Ile Ile Leu Glu Ala His Asp Val Trp Glu Asp Gly 35 40 45 Thr Gly
Tyr Gln Met Leu Trp Asp Ala Asp His Asn Gln Tyr Gly Ala 50 55 60
Ser Ile Pro Glu Glu Ser Phe Trp Phe Ala Asn Gly Thr Ile Pro Ala 65
70 75 80 Gly Leu Tyr Asp Pro Phe Glu Tyr Lys Val Pro Val Asn Ala
Asp Ala 85 90 95 Ser Phe Ser Pro Thr Asn Phe Val Leu Asp Gly Thr
Ala Ser Ala Asp 100 105 110 Ile Pro Ala Gly Thr Tyr Asp Tyr Val Ile
Ile Asn Pro Asn Pro Gly 115 120 125 Ile Ile Tyr Ile Val Gly Glu Gly
Val Ser Lys Gly Asn Asp Tyr Val 130 135 140 Val Glu Ala Gly Lys Thr
Tyr His Phe Thr Val Gln Arg Gln Gly Pro 145 150 155 160 Gly Asp Ala
Ala Ser Val Val Val Thr Gly Glu Gly Gly Asn Glu Phe 165 170 175 Ala
Pro Val Gln Asn Leu Gln Trp Ser Val Ser Gly Gln Thr Val Thr 180 185
190 Leu Thr Trp Gln Ala Pro Ala Ser Asp Lys Arg Thr Tyr Val Leu Asn
195 200 205 Glu Ser Phe Asp Thr Gln Thr Leu Pro Asn Gly Trp Thr Met
Ile Asp 210 215 220 Ala Asp Gly Asp Gly His Asn Trp Leu Ser Thr Ile
Asn Val Tyr Asn 225 230 235 240 Thr Ala Thr His Thr Gly Asp Gly Ala
Met Phe Ser Lys Ser Trp Thr 245 250 255 Ala Ser Ser Gly Ala Lys Ile
Asp Leu Ser Pro Asp Asn Tyr Leu Val 260 265 270 Thr Pro Lys Phe Thr
Val Pro Glu Asn Gly Lys Leu Ser Tyr Trp Val 275 280 285 Ser Ser Gln
Glu Pro Trp Thr Asn Glu His Tyr Gly Val Phe Leu Ser 290 295 300 Thr
Thr Gly Asn Glu Ala Ala Asn Phe Thr Ile Lys Leu Leu Glu Glu 305 310
315 320 Thr Leu Gly Ser Gly 325 81260PRTPorphyromonas gingivalis
81Ala Pro Ala Pro Tyr Gln Glu Arg Thr Ile Asp Leu Ser Ala Tyr Ala 1
5 10 15 Gly Gln Gln Val Tyr Leu Ala Phe Arg His Phe Gly Cys Thr Gly
Ile 20 25 30 Phe Arg Leu Tyr Leu Asp Asp Val Ala Val Ser Gly Glu
Gly Ser Ser 35 40 45 Asn Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Asn
Val Val Ile Ala Gln 50 55 60 Asn Leu Thr Ala Thr Thr Phe Asn Gln
Glu Asn Val Ala Pro Gly Gln 65 70 75 80 Tyr Asn Tyr Cys Val Glu Val
Lys Tyr Thr Ala Gly Val Ser Pro Lys 85 90 95 Val Cys Lys Asp Val
Thr Val Glu Gly Ser Asn Glu Phe Ala Pro Val 100 105 110 Gln Asn Leu
Thr Gly Ser Ala Val Gly Gln Lys Val Thr Leu Lys Trp 115 120 125 Asp
Ala Pro Asn Gly Thr Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu 130 135
140 Ser Glu Ser Phe Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp
145 150 155 160 Ala Asp Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro
Pro Gly Gly 165 170 175 Ser Ser Phe Ala Gly His Asn Ser Ala Ile Cys
Val Ser Ser Ala Ser 180 185 190 Tyr Ile Asn Phe Glu Gly Pro Gln Asn
Pro Asp Asn Tyr Leu Val Thr 195 200 205 Pro Glu Leu Ser Leu Pro Asn
Gly Gly Thr Leu Thr Phe Trp Val Cys 210 215 220 Ala Gln Asp Ala Asn
Tyr Ala Ser Glu His Tyr Ala Val Tyr Ala Ser 225 230 235 240 Ser Thr
Gly Asn Asp Ala Ser Asn Phe Ala Asn Ala Leu Leu Glu Glu 245 250 255
Val Leu Thr Ala 260 82258PRTPorphyromonas gingivalis 82Pro Gln Ser
Val Trp Ile Glu Arg Thr Val Asp Leu Pro Ala Gly Thr 1 5 10 15 Lys
Tyr Val Ala Phe Arg His Tyr Asn Cys Ser Asp Leu Asn Tyr Ile 20 25
30 Leu Leu Asp Asp Ile Gln Phe Thr Met Gly Gly Ser Pro Thr Pro Thr
35 40 45 Asp Tyr Thr Tyr Thr Val Tyr Arg Asp Gly Thr Lys Ile Lys
Glu Gly 50 55 60 Leu Thr Glu Thr Thr Phe Glu Glu Asp Gly Val Ala
Thr Gly Asn His 65 70 75 80 Glu Tyr Cys Val Glu Val Lys Tyr Thr Ala
Gly Val Ser Pro Lys Glu 85 90 95 Cys Val Asn Val Thr Val Asp Pro
Val Gln Phe Asn Pro Val Gln Asn 100 105 110 Leu Thr Gly Ser Ala Val
Gly Gln Lys Val Thr Leu Lys Trp Asp Ala 115 120 125 Pro Asn Gly Thr
Pro Asn Pro Asn Pro Gly Thr Thr Thr Leu Ser Glu 130 135 140 Ser Phe
Glu Asn Gly Ile Pro Ala Ser Trp Lys Thr Ile Asp Ala Asp 145 150 155
160 Gly Asp Gly Asn Asn Trp Thr Thr Thr Pro Pro Pro Gly Gly Thr Ser
165 170 175 Phe Ala Gly His Asn Ser Ala Ile Cys Val Ser Ser Ala Ser
Tyr Ile 180 185 190 Asn Phe Glu Gly Pro Gln Asn Pro Asp Asn Tyr Leu
Val Thr Pro Glu 195 200 205 Leu Ser Leu Pro Asn Gly Gly Thr Leu Thr
Phe Trp Val Cys Ala Gln 210 215 220 Asp Ala Asn Tyr Ala Ser Glu His
Tyr Ala Val Tyr Ala Ser Ser Thr 225 230 235 240 Gly Asn Asp Ala Ser
Asn Phe Ala Asn Ala Leu Leu Glu Glu Val Leu 245 250 255 Thr Ala
8315PRTPorphyromonas gingivalis 83Pro Tyr Gln Pro Val Ser Asn Leu
Thr Ala Thr Thr Gln Gly Gln 1 5 10 15 8415PRTPorphyromonas
gingivalis 84Glu Gly Leu Thr Ala Thr Thr Phe Glu Glu Asp Gly Val
Ala Ala 1 5 10 15 8517PRTPorphyromonas gingivalis 85Gly Thr Pro Asn
Pro Asn Pro Asn Pro Asn Pro Asn Pro Asn Pro Gly 1 5 10 15 Thr
* * * * *
References