U.S. patent application number 15/127537 was filed with the patent office on 2018-03-08 for mutant staphylococcal antigens.
This patent application is currently assigned to GLAXOSMITHKLINE BIOLOGICALS SA. The applicant listed for this patent is GLAXOSMITHKLINE BIOLOGICALS SA. Invention is credited to Fabio BAGNOLI, Luigi FIASCHI, Maria SCARSELLI.
Application Number | 20180064800 15/127537 |
Document ID | / |
Family ID | 52706188 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180064800 |
Kind Code |
A9 |
BAGNOLI; Fabio ; et
al. |
March 8, 2018 |
MUTANT STAPHYLOCOCCAL ANTIGENS
Abstract
Mutant protein A of Staphylococcus aureus (SpA) with decreased
affinity for the Fc.gamma. portion of human IgG is provided.
Inventors: |
BAGNOLI; Fabio; (Siena,
IT) ; FIASCHI; Luigi; (Siena, IT) ; SCARSELLI;
Maria; (Siena, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GLAXOSMITHKLINE BIOLOGICALS SA |
Rixensart |
|
BE |
|
|
Assignee: |
GLAXOSMITHKLINE BIOLOGICALS
SA
Rixensart
BE
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20170143816 A1 |
May 25, 2017 |
|
|
Family ID: |
52706188 |
Appl. No.: |
15/127537 |
Filed: |
March 24, 2015 |
PCT Filed: |
March 24, 2015 |
PCT NO: |
PCT/EP2015/056175 PCKC 00 |
371 Date: |
September 20, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/74 20130101;
A61K 2039/55511 20130101; C07K 2319/00 20130101; A61P 31/04
20180101; A61K 2039/55505 20130101; A61K 39/085 20130101; A61K
2039/70 20130101; A61K 2039/645 20130101; A61K 2039/55577 20130101;
C07K 14/31 20130101; C07K 2319/40 20130101 |
International
Class: |
A61K 39/085 20060101
A61K039/085; C07K 14/31 20060101 C07K014/31 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 26, 2014 |
EP |
14161861.1 |
Nov 12, 2014 |
EP |
14192913.3 |
Claims
1. An antigen comprising an amino acid sequence consisting of SEQ
ID NO: 50 wherein the dipeptide at positions 60 and 61 is not
QQ.
2. An antigen according to claim 1, which antigen comprises an
amino acid sequence consisting of SEQ ID NO: 49 wherein the
dipeptide at positions 60 and 61 is not QQ.
3. An antigen according to claim 1, which comprises more than one
copy of said amino acid sequence consisting of SEQ ID NO: 50
wherein the dipeptide at positions 60 and 61 is not QQ.
4. An antigen according to claim 1, wherein the antigen elicits
antibodies in a mammal that recognise SEQ ID NO: 43 or SEQ ID NO:
54.
5. An antigen according to claim 1 to which has decreased affinity,
relative to unmodified Staphylococcal protein A (SpA) having SEQ ID
NO:43 , for the Fc.gamma. portion of human IgG.
6. An antigen according to claim 5 which has decreased affinity,
relative to unmodified SpA having SEQ ID NO:43, for the Fab portion
of V.sub.H3-containing human B cell receptors.
7. An antigen according to claim 1 comprising SEQ ID NO :43,
wherein at least one amino acid selected from position 43, 44, 70,
71, 96, 97, 104, 105, 131, 132, 162, 163, 190, 191, 220, 221, 247,
248, 278, 279, 305 and 306 of SEQ ID NO: 43 is mutated.
8. An antigen according to claim 1, comprising a sequence selected
from SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 47 and SEQ ID NO:
48.
9. A fusion protein comprising an antigen according to claim 1 and
another protein.
10. An immunogenic composition comprising an antigen according to
claim 1.
11. The composition according to claim 10, wherein the composition
also comprises an adjuvant.
12. The composition according to claim 11, wherein said adjuvant is
selected from the group consisting of: aluminium salts,
particularly aluminium hydroxides and aluminium phosphates; human
TLR agonists, particularly TLR7 agonists; and a mixture
thereof.
13. The composition according to claim 11, wherein said adjuvant is
a TLR7 agonist compound of the following formula (K): ##STR00006##
wherein: R.sup.1 is H, C.sub.1-C.sub.6alkyl, --C(R.sup.5).sub.2OH,
-L.sup.1R.sup.5, -L.sup.1R.sup.6, -L.sup.2R.sup.5, -L.sup.2R.sup.6,
-OL.sup.2R.sup.5, or -OL.sup.2R.sup.6; L.sup.1 is --C(O)-- or
--O--; L.sup.2 is C.sub.1-C.sub.6alkylene,
C.sub.2-C.sub.6alkenylene, arylene, heteroarylene or
--((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p--, wherein the
C.sub.1-C.sub.6alkylene and C.sub.2-C.sub.6alkenylene of L.sup.2
are optionally substituted with 1 to 4 fluoro groups; each L.sup.3
is independently selected from C.sub.1-C.sub.6alkylene and
--((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p--, wherein the
C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1
to 4 fluoro groups; L.sup.4 is arylene or heteroarylene; R.sup.2 is
H or C.sub.1-C.sub.6alkyl; R.sup.3 is selected from
C.sub.1-C.sub.4alkyl, -L.sup.3R.sup.5, -L.sup.1R.sup.5,
-L.sup.3R.sup.7, -L.sup.3L.sup.4L.sup.3R.sup.7,
-L.sup.3L.sup.4R.sup.5, -L.sup.3L.sup.4L.sup.3R.sup.5,
-OL.sup.3R.sup.5, -OL.sup.3R.sup.7, -OL.sup.3L.sup.4R.sup.7,
-OR.sup.8, -OL.sup.3L.sup.4L.sup.3R.sup.7, -OL.sup.3L.sup.4R.sup.5,
-OL.sup.3L.sup.4L.sup.3R.sup.5 and --C(R.sup.5).sub.2OH; each
R.sup.4 is independently selected from H and fluoro; R.sup.5 is
--P(O)(OR.sup.9).sub.2, R.sup.6 is --CF.sub.2P(O)(OR.sup.9).sub.2
or --C(O)OR.sup.10; R.sup.7 is --CF.sub.2P(O)(OR.sup.9).sub.2 or
--C(O)OR.sup.10; R.sup.8 is H or C.sub.1-C.sub.4alkyl; each R.sup.9
is independently selected from H and C.sub.1-C.sub.6alkyl; R.sup.10
is H or C.sub.1-C.sub.4alkyl; each p is independently selected from
1, 2, 3, 4, 5 and 6, and q is 1, 2, 3 or 4; particularly
3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)
ethoxy)ethoxy)phenethyl) benzo[f][1,7] naphthyridin-8-yl)propanoic
acid (K1).
14. The composition according to claim 12, wherein the adjuvant
comprises a human TLR agonist adsorbed to an aluminium salt.
15. A method of treating or preventing Staphylococcus aureus
infection in a mammal, comprising administering to a mammal in need
thereof an immunologically effective amount of the immunogenic
composition according to claim 12.
16. The method according to claim 15 where said mammal is
human.
17. The antigen according to claim 1 wherein said dipeptide is
KR.
18. The antigen according to claim 2 wherein said dipeptide is
KR.
19. The antigen according to claim 2 wherein said dipeptide is
KR.
20. The fusion protein according to claim 9, comprising a protein
selected from the group consisting of EsxA, EsxB, FhuD2, Sta011 and
Hla antigens.
21. An immunogenic composition comprising a fusion protein
according to claim 9.
Description
[0001] This application claim the benefit of European patent
applications 14161861.1 (filed Mar. 26 2014) and 14192913.3 (filed
Nov. 12 2014), the complete contents of both of which are hereby
incorporated by reference for all purposes.
TECHNICAL FIELD
[0002] This invention relates to immunisation against infection by
S.aureus.
BACKGROUND ART
[0003] Staphylococcus aureus is a Gram-positive spherical
bacterium. Annual US mortality exceeds that of any other infectious
disease, including HIV/AIDS, and S.aureus is the leading cause of
bloodstream, lower respiratory tract, skin & soft tissue
infections. It is also the predominant cause of bone infections
worldwide, and these infections are painful, debilitating and
difficult to treat.
[0004] Treatment of S.aureus is becoming increasingly challenging
due to the development of antibiotic resistance by many strains of
S.aureus . Methicillin-resistant S.aureus (MRSA) is found in over
half of all community and hospital infections. Recent years have
seen the emergence of MRSA strains which are also resistant to
vancomycin, the antibiotic of last resort, and which are
essentially untreatable.
[0005] There is currently no authorised vaccine. A vaccine based on
a mixture of surface polysaccharides from bacterial types 5 and 8,
StaphVAX.TM., failed to reduce infections when compared to the
placebo group in a phase III clinical trial. Similarly, the V710
vaccine [1], based on the IsdB antigen [2], failed to reduce the
rate of post-operative S.aureus infections [3].
[0006] The need for a vaccine is particularly acute due to problem
of antibiotic resistance and the fact that S.aureus infection does
not provide immunity from future infection thanks to its
well-developed immune evasion capabilities. The immune evasion
properties of S.aureus in turn render the development of effective
vaccines more difficult. The mechanisms of immune evasion are not
fully understood, but are at least in part due to staphylococcal
protein A (SpA), an S.aureus surface molecule that binds to Fc of
immunoglobulin (Ig) and to the Fab portion of VH3-type B cell
receptors. Interaction of SpA with B cell receptors leads to clonal
explansion and subsequent cell death of B cell populations, leading
to ablation of the adaptive immune response. SpA binding to Ig Fc
interferes with opsonophagocytic clearance of staphylococci by
polymorphonuclear leukocytes.
[0007] Mutant forms of SpA with reduced affinity to immunoglobulins
have been developed. WO2011/005341 describes a SpA with point
mutations in each of the five Ig-binding domains which reduce the
ability of the protein to bind to IgG.
[0008] Reference 4 discloses various approaches to providing
improved S.aureus vaccines, including two preferred immunogen
combinations referred to as "Combo-1" and "Combo-2". "Combo-1"
included five antigens EsxA, EsxB, a mutant Hla, FhuD2, and Sta011,
whereas "Combo-2" used a fragment of IsdA in place of the mutant
Hla. Both of these combinations were tested with aluminium
hydroxide adjuvants and they increased median survival time in a
mouse model of infection when compared to buffer alone or to the
IsdB antigen. "Combo-1" has also been tested with an aluminium
hydroxide adjuvant and a TLR7 agonist, and the addition of the TLR7
agonist improved responses [5].
[0009] Despite the positive results with "Combo-1" and "Combo-2",
there remains a need for further and improved compositions for
immunising against S.aureus.
DISCLOSURE OF THE INVENTION
[0010] Protein A (SpA) (SEQ ID NO: 43), a cell wall anchored
surface protein of Staphylococcus aureus, provides for bacterial
evasion from innate and adaptive immune responses. Protein A binds
immunoglobulins at their Fc portion, interacts with the VH3 domain
of B cell receptors inappropriately stimulating B cell
proliferation and apotosis, binds to von Willebrand factor Al
domains to activate intracellular clotting, and also binds to the
TNF Receptor-1 to contribute to the pathogenesis of staphylococcal
pneumonia. Due to the fact that Protein A captures immunoglobulin
and displays toxic attributes, the possibility that this surface
molecule may function as a vaccine in humans has not been
rigorously pursued. Here the inventors demonstrate that Protein A
variants no longer able to bind to immunoglobulins, which are
thereby removed of their toxigenic potential, i.e, are
non-toxigenic, stimulate humoral immune responses that protect
against staphylococcal disease.
[0011] The invention therefore provides mutant SpA antigens as
described below. Said mutants preferably have decreased affinity
for the Fc.gamma. portion of human IgG relative to unmodified SpA.
The mutants may also have decreased affinity, relative to
unmodified SpA, for the Fab portion of V.sub.H3-containing human B
cell receptors.
[0012] The inventors have found that the known "Combo-1" vaccine
can be improved by adding a mutant staphylococcal protein A (SpA)
which has been modified to decrease its affinity for the Fc.gamma.
portion of human IgG and for the Fab portion of V.sub.H3-containing
B cell receptors. This extra antigen increases the combination's
protective efficacy and provides striking results in a renal
abscess model. None of the antigen combinations tested in reference
4 included a SpA antigen.
[0013] The invention therefore further provides an immunogenic
composition comprising EsxA, EsxB, FhuD2, Sta011, and Hla antigens,
characterised in that the composition additionally includes a
mutant SpA antigen, wherein the mutant has decreased affinity,
relative to unmodified SpA, for the Fc.gamma. portion of human IgG
and for the Fab portion of V.sub.H3-containing human B cell
receptors.
[0014] The invention also provides an immunogenic composition
comprising: (i) at least one antigen selected from the group
consisting of EsxA, EsxB, FhuD2, Sta011, and Hla antigens; and (ii)
a mutant SpA antigen which has decreased affinity, relative to
unmodified SpA, for the Fc.gamma. portion of human IgG and for the
Fab portion of V.sub.H3-containing human B cell receptors. Thus 1,
2, 3, 4 or preferably all 5 of EsxA, EsxB, FhuD2, Sta011, and Hla
can be used in combination with the mutant SpA. Unless defined
otherwise, all technical and scientific terms used herein have the
same meaning as commonly understood to one skilled in the relevant
art.
[0015] For convenience, the meaning of certain terms and phrases
employed in the specification, examples and claims are
provided.
[0016] S.aureus Antigens
[0017] The invention relates inter alia to a mutant SpA antigen.
The wild-type SpA (staphylococcal protein A) is a cell
wall-anchored surface protein which is a crucial virulence factor
for lung infections, septicemia, and abscess development and is
expressed by most clinical S. aureus isolates. Wild-type SpA binds
to the Fc portion of human IgG, to V.sub.H3-containing B cell
receptors, to von Willebrand factor at its Al domain, and to the
TNF-.alpha. receptor 1. Interaction of SpA with B cell receptors
leads to clonal expansion and subsequent cell death of B cell
populations with effects on adaptive and innate immune responses,
whereas its binding to the Fc.gamma. of IgG interferes with
opsonophagocytic clearance of staphylococci by polymorphonuclear
leukocytes. The N-terminal part of mature SpA is comprised of four
or five 56-61-residue Ig-binding domains, which fold into triple
helical bundles connected by short linkers, and are designated in
order E, D, A, B, and C [6]. These domains display .about.80%
identity at the amino acid level, are 56 to 61 residues in length,
and are organized as tandem repeats [7]. The C-terminal region is
comprised of "Xr", a highly repetitive yet variable octapeptide,
and "Xc", a domain which abuts the cell wall anchor structure of
SpA.
[0018] In the NCTC 8325 strain spa is SAOUHSC_00069 and has amino
acid sequence SEQ ID NO: 43 (GI:88193885). In the Newman strain it
is nwmn_0055 (GI:151220267). SpA antigens used with the invention
can elicit an antibody (e.g. when administered to a human) that
recognises SEQ ID NO: 43 and/or may comprise an amino acid
sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%, 75%,
80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%
or more) to SEQ ID NO: 43; and/or (b) comprising a fragment of at
least `n` consecutive amino acids of SEQ ID NO: 43, wherein `n` is
7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60,
70, 80, 90, 100, 150, 200, 250 or more). These SpA antigens include
variants of SEQ ID NO: 43. Preferred fragments of (b) comprise an
epitope from SEQ ID NO: 43. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus
of SEQ ID NO: 43 while retaining at least one epitope of SEQ ID NO:
43. The final 35 C-terminal amino acids of SEQ ID NO: 43 can
usefully be omitted. The first 36 N-terminal amino acids of SEQ ID
NO: 43 can usefully be omitted. Reference 8 suggests that
individual IgG-binding domains might be useful immunogens, alone or
in combination.
[0019] A useful fragment of SEQ ID NO: 43 is amino acids 37 to 325.
This fragment contains all the five SpA Ig-binding domains (which
are naturally arranged from N- to C-terminus in the order E, D, A,
B, C) and includes the most exposed domain of SpA. It also reduces
the antigen's similarity with human proteins. Other useful
fragments may omit 1, 2, 3 or 4 of the natural A, B, C, D and/or E
domains to prevent the excessive B cell expansion and then
apoptosis which might occur if spa functions as a B cell
superantigen. As reported in reference 18, other useful fragments
may include only 1, 2, 3 or 4 of the natural A, B, C, D and/or E
domains e.g. comprise only the SpA(A) domain but not B to E, or
comprise only the SpA(D) domain but not A, B, C or E, etc. Thus a
spa antigen useful with the invention may include 1, 2, 3, 4 or 5
IgG-binding domains, but ideally has 4 or fewer
[0020] Thus, another useful fragment of SpA comprises or consists
of the amino acid sequence of SEQ ID NO: 50 wherein the amino acid
doublet at positions 60 and 61 is not Gln-Gln. Said doublet may be
mutated as described below, for example to Lys-Arg. Thus, said
fragment may comprise or consist of SEQ ID NO: 51 or 52.
[0021] If an antigen includes only one type of spa domain (e.g.
only the Spa(A), SpA(D) or Spa(E) domain), it may include more than
one copy of this domain e.g. multiple SpA(E) domains in a single
polypeptide chain. It may also include one type of SpA domain and
another protein or polypeptide. Thus, an antigen of the invention
may be a fusion protein comprising only one type of SpA domain,
such as the SpA(E) domain, and another protein antigen, such as
EsxA; EsxB; FhuD2; Sta011; and Hla.
[0022] SpA antigens of the invention are mutated relative to SEQ ID
NO: 43, such that they have decreased affinity for the Fc.gamma.
portion of human IgG. For instance, the QQ dipeptide at residues
60-61 of SEQ ID NO: 43 can be mutated to reduce affinity for
immunoglobulins. Useful dipeptide substitutions for a QQ dipeptide
are discussed below, and a preferred substitution is KR dipeptide.
Thus a useful SpA antigen can comprise SEQ ID NO: 49, in which one
or more (preferably all) of the 11 XX dipeptides differ from the
corresponding dipeptides within SEQ ID NO: 43. For instance, the
SpA antigen can comprise SEQ ID NO: 46, and a preferred example of
SEQ ID NO: 46 is SEQ ID NO: 47. When expressed with a N-terminal
methionine, the SpA antigen comprising SEQ ID NO: 47 can consist of
SEQ ID NO: 48.
[0023] SpA antigens used with the invention may be further mutated
relative to SEQ ID NO: 43, such that they have decreased affinity
for the Fc.gamma. portion of human IgG and for the Fab portion of
V.sub.H3-containing human B cell receptors. This can be achieved
and assessed by, for instance, following the guidance in reference
9. Thus at least one Gln-Gln dipeptide in wild-type SpA can be
mutated (e.g. to Lys-Lys; other possible mutations include Arg-Arg,
Arg-Lys, Lys-Arg, Ala-Ala, Ser-Ser, Ser-Thr, Thr-Thr, etc.) and/or
at least one Asp-Asp dipeptide in wild-type SpA can be mutated
(e.g. to Ala-Ala; other possible mutations include Lys-Lys,
Arg-Arg, Lys-Arg, Arg-Lys, His-His, Val-Val, etc.). These target
sequences for mutation are underlined below, where dashes separate
the five Ig-binding domains:
TABLE-US-00001 (SEQ ID NO: 43)
MKKKNIYSIRKLGVGIASVTLGTLLISGGVTP-AANAAQHDEAQQNAFYQ
VLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPK-ADAQQN
NFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLNE
SQAPK-ADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANL
LSEAKKLNESQAPK-ADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLK
DDPSQSANLLAEAKKLNDAQAPK-ADNKFNKEQQNAFYEILHLPNLTEEQ
RNGFIQSLKDDPSVSKEILAEAKKLNDAQAPK-EEDNNKPGKEDNNKPGK
EDNNKPGKEDNNKPGKEDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKED
NKKPGKEDGNKPGKEDGNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADK
IAADNKLADKNMIKPGQELVVDKKQPANHADANKAQALPETGEENPFIGT
TVFGGLSLALGAALLAGRRREL
[0024] An individual domain within the antigen may be mutated at 1,
2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids relative to SEQ ID
NO: 43 (e.g. see above in relation to Gln-Gln and Asp-Asp
sequences, but also see reference Error! Bookmark not defined.
which discloses mutations at residues 3 and/or 24 of domain D, at
residue 46 and/or 53 of domain A, etc.). Such mutations should not
remove the antigen's ability to elicit an antibody that recognises
SEQ ID NO: 43, but will remove the antigen's binding to IgG and/or
other human proteins (such as human blood proteins) as noted above.
Particularly, the mutant SpA antigen is of sequence comprising or
consisting of SEQ ID NO :43 mutated in at least 1, more
particularly at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19 and even more particularly 20 amino acids at
position 43, 44, 70, 71, 104, 105, 131, 132, 162, 163, 190, 191,
220, 221, 247, 248, 278, 279, 305 and/or 306 of SEQ ID NO: 43.
Useful substitutions for these positions are mentioned above.
[0025] Furthermore, the native N-terminus can be removed, and the
first 36 amino acids of SEQ ID NO: 43 can usefully be omitted.
Similarly, the native C-terminal can be removed, and the sequence
downstream of the fifth Ig-binding domain can usefully be omitted
(i.e. downstream of Lys-327 in SEQ ID NO: 43). Thus a useful SpA
antigen comprises SEQ ID NO: 44:
TABLE-US-00002 AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLN
DSQAPKADAQQNNFNKDXXSAFYEILNMPNLNEAQRNGFIQSLKXXPSQS
TNVLGEAKKLNESQAPKADNNFNKEXXNAFYEILNMPNLNEEQRNGFIQS
LKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLPNLNEE
QRNGFIQSLKXXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEIL
HLPNLTEEQRNGFIQSLKXXPSVSKEILAEAKKLNDAQAPK
wherein the 10 underlined XX dipeptides differ from the
corresponding dipeptides within SEQ ID NO: 43. Thus a QQ at these
positions in SEQ ID NO: 43 will not be QQ in SEQ ID NO: 44, and
ideally includes no glutamine residue e.g. it is KK. Similarly, a
DD at these positions in SEQ ID NO: 43 will not be DD in SEQ ID NO:
44, and ideally includes no aspartate residue e.g. it is AA. The
preferred form of SpA for use with the invention thus comprises SEQ
ID NO: 45.
[0026] In addition to substitutions at the XX dipeptides in SEQ ID
NO: 44 it is possible to modify the amino acid sequence with up to
5 single amino changes provided that the modified sequence can
elicit antibodies which still bind to a polypeptide consisting of
SEQ ID NO: 44. Thus, SEQ ID NO: 45 can be modified by 1, 2 or 3
substitutions at positions outside the XX dipeptides within SEQ ID
NO: 44. For instance, the QQ dipeptide at residues 60-61 of SEQ ID
NO: 44 can be mutated to further reduce affinity for
immunoglobulins. Useful dipeptide substitutions for a QQ dipeptide
are discussed above, and a preferred substitution is KR dipeptide.
Thus a useful SpA antigen can comprise SEQ ID NO: 49, in which one
or more (preferably all) of the 11 XX dipeptides differ from the
corresponding dipeptides within SEQ ID NO: 43. For instance, the
SpA antigen can comprise SEQ ID NO: 46, and a preferred example of
SEQ ID NO: 46 is SEQ ID NO: 47. When expressed with a N-terminal
methionine, the SpA antigen comprising SEQ ID NO: 47 can consist of
SEQ ID NO: 48.
[0027] As discussed above, a useful fragment of SpA may include
only 1, 2, 3 or 4 of the natural A, B, C, D and/or E domains e.g.
comprise only the SpA(E) domain but not D, A B or C. Thus a SpA
antigen useful with the invention may comprise only the SpA(E)
domain mutated as described above, i.e. amino acid sequence
comprising or consisting of SEQ ID NO: 54 mutated in at least 1
amino acid at positions 60 and 61 of SEQ ID NO: 54 amino acids 1 to
67 of SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 or
SEQ ID NO: 49, or amino acids 1 to 68 of SEQ ID NO: 48. For
example, said antigen may comprise SEQ ID NO. 50, SEQ ID NO. 51,
SEQ ID NO. 52 or SEQ ID NO. 53. Said antigen will preferably not
include other sequence from SpA. It may include more than one copy
of the SpA(E) domain, and/or additionally comprise another protein
antigen such as an EsxA, EsxB, FhuD2, Sta011, or Hla antigen as
described herein.
[0028] The antigen combinations of the invention use 1, 2, 3, 4, or
all 5 of the following antigens: EsxA; EsxB; FhuD2; Sta011; and
Hla. These five antigens are already known in the art (e.g. see
references 4-14) and further details are given below. A
particularly useful composition includes all five of these antigens
(preferably where the Hla is a non-toxic (i.e. detoxified) mutant
form).
[0029] The `EsxA` antigen in the NCTC 8325 strain has amino acid
sequence SEQ ID NO: 1 (GI:88194063). EsxA antigens used with the
present invention can elicit an antibody (e.g. when administered to
a human) that recognises SEQ ID NO: 1 and/or may comprise an amino
acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 1; and/or (b) comprising a fragment of
at least `n` consecutive amino acids of SEQ ID NO: 1, wherein `n`
is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90 or more). These EsxA polypeptides include variants
of SEQ ID NO: 1. Preferred fragments of (b) comprise an epitope
from SEQ ID NO: 1. Other preferred fragments lack one or more amino
acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from
the C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID
NO: 1 while retaining at least one epitope of SEQ ID NO: 1.
[0030] The `EsxB` antigen in the NCTC 8325 strain has amino acid
sequence SEQ ID NO: 2 (GI:88194070). EsxB used with the present
invention can elicit an antibody (e.g. when administered to a
human) that recognises SEQ ID NO: 2 and/or may comprise an amino
acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 2; and/or (b) comprising a fragment of
at least `n` consecutive amino acids of SEQ ID NO: 2, wherein `n`
is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90, 100 or more). These EsxB polypeptides include
variants of SEQ ID NO: 2. Preferred fragments of (b) comprise an
epitope from SEQ ID NO: 2. Other preferred fragments lack one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the C-terminus and/or one or more amino acids (e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus
of SEQ ID NO: 2 while retaining at least one epitope of SEQ ID NO:
2. A useful EsxB antigen lacks the internal cysteine residue of SEQ
ID NO: 2 e.g. it comprises SEQ ID NO: 35, wherein residue X at
position 30 is either absent or is an amino acid residue without a
free thiol group (under reducing conditions) e.g. is any natural
amino acid except cysteine.
[0031] The `FhuD2` antigen is annotated as `ferrichrome-binding
protein`, and has also been studied in the literature [15]. It has
also been known as `Sta006` (e.g. in references 4-14). In the NCTC
8325 strain FhuD2 has amino acid sequence SEQ ID NO: 3
(GI:88196199). FhuD2 used with the present invention can elicit an
antibody (e.g. when administered to a human) that recognises SEQ ID
NO: 3 and/or may comprise an amino acid sequence: (a) having 50% or
more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 3;
and/or (b) comprising a fragment of at least `n` consecutive amino
acids of SEQ ID NO: 3, wherein `n` is 7 or more (e.g. 8, 10, 12,
14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200,
250 or more). These FhuD2 polypeptides include variants of SEQ ID
NO: 3. Preferred fragments of (b) comprise an epitope from SEQ ID
NO: 3. Other preferred fragments lack one or more amino acids (e.g.
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the
C-terminus and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 15, 20, 25 or more) from the N-terminus of SEQ ID NO:
3 while retaining at least one epitope of SEQ ID NO: 3. The first
17 N-terminal amino acids of SEQ ID NO: 3 can usefully be omitted
(to provide SEQ ID NO: 6). Mutant forms of FhuD2 are reported in
reference 16. A useful FhuD2 antigen lacks the cysteine residue of
SEQ ID NO: 3 e.g. it comprises SEQ ID NO: 34 and does not include
any amino acid residue with a free thiol group (under reducing
conditions) e.g. it is cysteine-free. A FhuD2 antigen may be
lipidated e.g. with an acylated N-terminus cysteine. One useful
FhuD2 sequence is SEQ ID NO: 7, which has a Met-Ala-Ser- sequence
at the N-terminus; SEQ ID NO: 37 is another such sequence, but it
lacks the cysteine present in SEQ ID NO: 7.
[0032] The `Sta011` antigen has amino acid sequence SEQ ID NO: 4
(GI:88193872) in the NCTC 8325 strain. Sta011 antigens used with
the invention can elicit an antibody (e.g. when administered to a
human) that recognises SEQ ID NO: 4 and/or may comprise an amino
acid sequence: (a) having 50% or more identity (e.g. 60%, 65%, 70%,
75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,
99.5% or more) to SEQ ID NO: 4; and/or (b) comprising a fragment of
at least `n` consecutive amino acids of SEQ ID NO: 4, wherein `n`
is 7 or more (e.g. 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50,
60, 70, 80, 90, 100, 150, 200, 250 or more). These Sta011
polypeptides include variants of SEQ ID NO: 4. Preferred fragments
of (b) comprise an epitope from SEQ ID NO: 4. Other preferred
fragments lack one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 15, 20, 25 or more) from the C-terminus and/or one or
more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or
more) from the N-terminus of SEQ ID NO: 4 while retaining at least
one epitope of SEQ ID NO: 4. The first 23 N-terminal amino acids of
SEQ ID NO: 4 can usefully be omitted (to provide SEQ ID NO: 33). A
useful Sta011 antigen lacks the cysteine residue of SEQ ID NO: 4
e.g. it comprises SEQ ID NO: 36 and does not include any amino acid
residue with a free thiol group (under reducing conditions) e.g. it
is cysteine-free. A Sta011 antigen may be lipidated e.g. with an
acylated N-terminus cysteine. One useful Sta011 sequence is SEQ ID
NO: 8, which has a N-terminus methionine; SEQ ID NO: 39 is another
such sequence, but it lacks the cysteine present in SEQ ID NO: 8.
Variant forms of SEQ ID NO: 4 which may be used as or for preparing
Sta011 antigens include, but are not limited to, SEQ ID NOs: 9, 10
and 11 with various Ile/Val/Leu substitutions (and
[0033] Cys-free variants of these sequences can also be used with
the invention). Sta011 can exist as a monomer or an oligomer, with
Ca.sup.++ ions favouring oligomerisation. The invention can use
monomers and/or oligomers of Sta011.
[0034] The `Hla` antigen is the `alpha-hemolysin precursor` also
known as `alpha toxin` or simply `hemolysin`. In the NCTC 8325
strain Hla has amino acid sequence SEQ ID NO: 5 (GI:88194865). Hla
is an important virulence determinant produced by most strains of
S.aureus, having pore-forming and haemolytic activity. Anti-Hla
antibodies can neutralise the detrimental effects of the toxin in
animal models, and Hla is particularly useful for protecting
against pneumonia.
[0035] Useful Hla antigens can elicit an antibody (e.g. when
administered to a human) that recognises SEQ ID NO: 5 and/or may
comprise an amino acid sequence: (a) having 50% or more identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 5; and/or (b)
comprising a fragment of at least `n` consecutive amino acids of
SEQ ID NO: 5, wherein `n` is 7 or more (e.g. 8, 10, 12, 14, 16, 18,
20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more). These Hla antigens include variants of SEQ ID NO: 5.
Preferred fragments of (b) comprise an epitope from SEQ ID NO: 5.
Other preferred fragments lack one or more amino acids (e.g. 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more) from the C-terminus
and/or one or more amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
15, 20, 25 or more) from the N-terminus of SEQ ID NO: 5 while
retaining at least one epitope of SEQ ID NO: 5. The first 26
N-terminal amino acids of SEQ ID NO: 5 can usefully be omitted
(e.g. to give SEQ ID NO: 12). Truncation at the C-terminus can also
be used e.g. leaving only 50 amino acids (residues 27-76 of SEQ ID
NO: 5) [17].
[0036] Particularly, the Hla antigen is a detoxified Hla antigen,
i.e. a mutant form of Hla, wherein Hla's natural toxicity has been
removed. Hla's toxicity can be avoided by chemical inactivation
(e.g. using formaldehyde, glutaraldehyde or other cross-linking
reagents). Instead, however, it is preferred to use mutant forms of
Hla which remove its toxic activity while retaining its
immunogenicity. More particularly, the detoxified Hla antigen is a
mutant form of Hla, wherein Hla's toxicity has been removed while
Hla's immunogenicity has been retained. Such detoxified mutants are
already known in the art. A preferred Hla antigen is a mutant
S.aureus hemolysin having a mutation at residue 61 of SEQ ID NO: 5,
which is residue 35 of the mature antigen (i.e. after omitting the
first 26 N-terminal amino acids=residue 35 of SEQ ID NO: 12). Thus
residue 61 may not be histidine, and may instead be e.g. Ile, Val
or preferably Leu. A His-Arg mutation at this position can also be
used. For example, SEQ ID NO: 13 is the sequence of the mature H35L
mutant form of Hla (i.e. SEQ ID NO: 12 with a H35L mutation) and a
useful detoxified Hla antigen is of sequence comprising or
consisting of SEQ ID NO: 13. Another useful mutation replaces a
long loop with a short sequence e.g. to replace the 39 mer at
residues 136-174 of SEQ ID NO: 5 with a tetramer such as PSGS (SEQ
ID NO: 14), as in SEQ ID NO: 15 (which also includes the H35L
mutation) and SEQ ID NO: 16 (which does not include the H35L
mutation). Another useful mutation replaces residue Y101 e.g. with
a leucine (SEQ ID NO: 17). Another useful mutation replaces residue
D152 e.g. with a leucine (SEQ ID NO: 18). Another useful mutant
replaces residues H35 and Y101 e.g. with a leucine (SEQ ID NO: 19).
Another useful mutant replaces residues H35 and D152 e.g. with a
leucine (SEQ ID NO: 20).
[0037] Further useful Hla antigens are disclosed in references 18
and 19.
[0038] SEQ ID NOs: 21, 22 & 23 are three useful fragments of
SEQ ID NO: 5 (`Hla.sub.27-76`, `Hla.sub.27-89` and `Hla.sub.27-79`,
respectively). SEQ ID NOs: 24, 25 & 26 are the corresponding
fragments from SEQ ID NO: 13.
[0039] One useful Hla sequence is SEQ ID NO: 27. It has a
N-terminal Met, then an Ala-Ser dipeptide from the expression
vector, then SEQ ID NO: 13 (from NCTC8325 strain) including the
H35L mutation.
[0040] Where a composition includes both EsxA and EsxB antigens,
these may be present as a single polypeptide (i.e. as a fusion
polypeptide comprising or consisting of both EsxA and EsxB). Thus a
single polypeptide can elicit antibodies (e.g. when administered to
a human) that recognise both SEQ ID NO: 1 and SEQ ID NO: 2. The
single polypeptide can include: (i) a first polypeptide sequence
having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to
SEQ ID NO: 1 and/or comprising a fragment of at least `n`
consecutive amino acids of SEQ ID NO: 1, as defined above for EsxA;
and (ii) a second polypeptide sequence having 50% or more identity
(e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 2 and/or
comprising a fragment of at least `n` consecutive amino acids of
SEQ ID NO: 2, as defined above for EsxB. The first and second
polypeptide sequences can be in either order, N- to C- terminus.
SEQ ID NOs: 28 (`EsxAB`) and 29 (`EsxBA`) are examples of such
polypeptides, both having hexapeptide linkers ASGGGS (SEQ ID NO:
30). Another `EsxAB` hybrid comprises SEQ ID NO: 31, which may be
provided with a N-terminus methionine (e.g. SEQ ID NO: 32). A
useful variant of EsxAB lacks the internal cysteine residue of EsxB
e.g. it comprises SEQ ID NO: 40 wherein residue X at position 132
is either absent or is an amino acid residue without a free thiol
group (under reducing conditions) e.g. is any natural amino acid
except cysteine. Thus a preferred EsxAB antigen for use with the
invention has amino acid sequence SEQ ID NO: 38.
[0041] Thus a useful polypeptide comprises an amino acid sequence
(a) having 80% or more identity (e.g. 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQ ID NO: 31;
and/or (b) comprising both a fragment of at least `n` consecutive
amino acids from amino acids 1-96 of SEQ ID NO: 31 and a fragment
of at least `n` consecutive amino acids from amino acids 103-205 of
SEQ ID NO: 31, wherein `n` is 7 or more (e.g. 8, 10, 12, 14, 16,
18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 or
more). These polypeptides (e.g. SEQ ID NO: 32) can elicit
antibodies (e.g. when administered to a human) which recognise both
the wild-type staphylococcal protein comprising SEQ ID NO: 1 and
the wild-type staphylococcal protein comprising SEQ ID NO: 2. Thus
the immune response will recognise both of antigens EsxA and EsxB.
Preferred fragments of (b) provide an epitope from SEQ ID NO: 1 and
an epitope from SEQ ID NO: 2.
[0042] The invention uses 1, 2, 3, 4, or all 5 of EsxA, EsxB,
FhuD2, Sta011, and Hla (preferably a non-toxic mutant Hla). As
mentioned above a particularly useful composition includes all five
of these antigens, but in some embodiments the invention includes
only 1, 2, 3 or 4 of these five antigens i.e. 1, 2, 3 or 4 of EsxA,
EsxB, FhuD2, Sta011, and Hla is absent from the composition.
[0043] A preferred composition includes all four of: (i) a single
polypeptide including both an EsxA antigen and an EsxB antigen e.g.
comprising SEQ ID NO: 31; (ii) a FhuD2 antigen e.g. comprising SEQ
ID NO: 6; (iii) a Sta011 antigen e.g. comprising SEQ ID NO: 33; and
(iv) a H35L mutant form of Hla e.g. comprising SEQ ID NO: 13.
[0044] Although SEQ ID NOs: 31, 6, 33 and 13 are useful amino acid
sequences in a combination, the invention is not limited to these
precise sequences. Thus 1, 2, 3 or all 4 of these sequences can
independently be modified by up to 5 single amino changes (i.e. 1,
2, 3, 4 or 5 single amino acid substitutions, deletions and/or
insertions) provided that the modified sequence can elicit
antibodies which still bind to a polypeptide consisting of the
unmodified sequence.
[0045] Another useful composition includes all four of: (i) a first
polypeptide having amino acid sequence SEQ ID NO: 32; (ii) a second
polypeptide having amino acid sequence SEQ ID NO: 7; (iii) a third
polypeptide having amino acid sequence SEQ ID NO: 8; and (iv) a
fourth polypeptide having amino acid sequence SEQ ID NO: 27.
[0046] Although SEQ ID NOs: 32, 7, 8 and 27 are useful amino acid
sequences in a combination, the invention is not limited to these
precise sequences. Thus 1, 2, 3 or all 4 of these four sequences
can independently be modified by 1, 2, 3, 4 or 5 single amino
changes (i.e. 1, 2, 3, 4 or 5 single amino acid substitutions,
deletions and/or insertions) provided that the modified sequence
can elicit antibodies which still bind to a polypeptide consisting
of the unmodified sequence. In a preferred embodiment, a
composition thus includes these four specified polypeptides with 1,
2, 3 or all 4 of SEQ ID NO: 32, 7, 8 and 27 independently modified
by 1 single amino acid substitution, deletion and/or insertion.
[0047] For instance, wild-type FhuD2, Sta011 and EsxAB polypeptide
sequences (e.g. SEQ ID NOs: 6, 31 and 33) each include a single
cysteine residue which can lead to inter-polypeptide disulfide
bridges, forming both homodimers and heterodimers. Such
inter-linked polypeptides are undesirable and so Sta006, Sta011 and
EsxB sequences can be modified to remove their natural cysteine
residues, such that they do not contain free thiol groups (under
reducing conditions). The wild-type cysteine can be deleted or can
be substituted with a different amino acid.
[0048] Thus: a FhuD2antigen can comprise SEQ ID NO: 34; a Sta011
antigen can comprise SEQ ID NO: 36; and a EsxB antigen can comprise
SEQ ID NO: 35 (e.g. as an EsxAB hybrid comprising SEQ ID NO: 40).
Examples of such sequences include, but are not limited to, SEQ ID
NOs: 37, 39, and 38. These sequences can be used singly as
substitutes for the corresponding wild-type sequences, or in
combination. Thus a particularly useful composition includes all
four of: (i) a first polypeptide having amino acid sequence SEQ ID
NO: 38; (ii) a second polypeptide having amino acid sequence SEQ ID
NO: 37; (iii) a third polypeptide having amino acid sequence SEQ ID
NO: 39; and (iv) a fourth polypeptide having amino acid sequence
SEQ ID NO: 27.
[0049] Thus a preferred composition of the invention comprises all
five of: (i) a single polypeptide including both an EsxA antigen
and an EsxB antigen e.g. comprising SEQ ID NO: 31; (ii) a FhuD2
antigen e.g. comprising SEQ ID NO: 6; (iii) a Sta011 antigen e.g.
comprising SEQ ID NO: 33; (iv) a H35L mutant form of Hla e.g.
comprising SEQ ID NO: 13; and (v) a mutant SpA e.g. comprising SEQ
ID NO: 45 or 47, or an antigen comprising a single domain thereof
e.g. SEQ ID NO. 50, 51, 52 or 53. So, in particular the composition
according to the invention comprises: [0050] (i) a single
polypeptide including both an EsxA antigen and an EsxB antigen,
particularly of sequence comprising or consisting of SEQ ID NO: 31;
[0051] (ii) a FhuD2 antigen, particularly of sequence comprising or
consisting of SEQ ID NO: 6; [0052] (iii) a Sta011 antigen,
particularly of sequence comprising or consisting of SEQ ID NO: 33;
[0053] (iv) a H35L mutant form of Hla, particularly of sequence
comprising or consisting of SEQ ID NO: 13; and [0054] (v) a mutant
SpA antigen, particularly of sequence comprising or consisting of
SEQ ID NO: 45,SEQ ID NO: 47, or SEQ ID NO. 52.
[0055] Another preferred composition according to the invention
comprises: [0056] (i) a first polypeptide of sequence comprising or
consisting of SEQ ID NO: 32, more particularly consisting of SEQ ID
NO: 32; [0057] (ii) a second polypeptide of sequence comprising or
consisting of SEQ ID NO: 7, more particularly consisting of SEQ ID
NO: 7; [0058] (iii) a third polypeptide of sequence comprising or
consisting of SEQ ID NO: 8, more particularly consisting of SEQ ID
NO: 8; [0059] (iv) a fourth polypeptide of sequence comprising or
consisting of SEQ ID NO: 27, more particularly consisting of SEQ ID
NO: 27; and [0060] (v) a fifth polypeptide of sequence comprising
or consisting of SEQ ID NO. 52, more particularly comprising or
consisting of comprising or consisting of SEQ ID NO: 45 modified by
up to 3 amino acid substitutions (e.g. comprising SEQ ID NO: 47 or
consisting of SEQ ID NO: 48).
[0061] Another preferred composition according to the invention
comprises: [0062] (i) a first polypeptide of sequence comprising or
consisting of SEQ ID NO: 38, more particularly consisting of SEQ ID
NO: 38; [0063] (ii) a second polypeptide of sequence comprising or
consisting of SEQ ID NO: 37, more particularly consisting of SEQ ID
NO: 37; [0064] (iii) a third polypeptide of sequence comprising or
consisting of SEQ ID NO: 39, more particularly consisting of SEQ ID
NO: 39; [0065] (iv) a fourth polypeptide of sequence comprising or
consisting of SEQ ID NO: 27, more particularly consisting of SEQ ID
NO: 27; and [0066] (v) a fifth polypeptide of sequence comprising
or consisting of SEQ ID NO. 52, more particularly SEQ ID NO: 45
modified by up to 3 amino acid substitutions (e.g. comprising SEQ
ID NO: 47 or consisting of SEQ ID NO: 48).
[0067] Proteins (i) to (v) in these combinations can, as explained
above, independently be modified by up to 5 single amino changes
provided that the modified sequence can elicit antibodies which
still bind to a polypeptide consisting of the unmodified sequence.
In some embodiments, a composition may include one or more further
polypeptides; in other embodiments the only polypeptides in a
composition are these five specified polypeptides, and these
polypeptides can even be the only immunogenic components in a
composition.
[0068] When more than one polypeptide is present, they may be
present at substantially equal masses i.e. the mass of each of them
is within .+-.5% of the mean mass of all the polypeptides. Thus,
when five polypeptides are present, they may be present at a mass
ratio of a:b:c:d:e, where each of a-e is between 0.95 and 1.05.
[0069] Aside from EsxA, EsxB, Hla, FhuD2, Sta011, and SpA, other
S.aureus antigens exist, and a composition can optionally include
one or more further S.aureus antigens. For instance, both
saccharide and polypeptide antigens are known for S.aureus. Thus a
composition might include a S.aureus saccharide antigen e.g. known
saccharide antigens include the exopolysaccharide of S.aureus,
which is a poly-N-acetylglucosamine (PNAG), and the capsular
saccharides of S.aureus, which can be e.g. from type 5, type 8 or
type 336. A composition might also include a ClfA antigen, an IsdA
antigen, an IsdB antigen, an IsdC antigen, and/or an IsdH antigen
(each as defined on pages 15-17 of reference 5).
[0070] In some embodiments, a composition includes a S.aureus
antigen as defined above, and also an antigen from a different
organism (e.g. from a virus or from another bacterium).
Immunogenic Compositions and Medicaments
[0071] Immunogenic compositions according to the invention may be
useful as vaccines. Vaccines according to the invention may either
be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to
treat infection), but will typically be prophylactic.
[0072] Compositions may thus be pharmaceutically acceptable. They
will usually include components in addition to the antigens e.g.
they typically include one or more pharmaceutical carrier(s) and/or
excipient(s). A thorough discussion of such components is available
in reference 121.
[0073] Compositions will generally be administered to a mammal in
aqueous form. Prior to administration, however, the composition may
have been in a non-aqueous form. For instance, although some
vaccines are manufactured in aqueous form, then filled and
distributed and administered also in aqueous form, other vaccines
are lyophilised during manufacture and are reconstituted into an
aqueous form at the time of use. Thus a composition may be dried,
such as a lyophilised formulation. Reference 10 discloses the use
of lyophilisation with S.aureus immunogenic compositions.
[0074] A composition may include preservatives such as thiomersal
or 2-phenoxyethanol. It is preferred, however, that the vaccine
should be substantially free from (i.e. less than 5 .mu.g/ml)
mercurial material e.g. thiomersal-free. Vaccines containing no
mercury are more preferred. Preservative-free vaccines are
particularly preferred.
[0075] To improve thermal stability, a composition may include a
temperature protective agent (see below).
[0076] To control tonicity, it is preferred to include a
physiological salt, such as a sodium salt. Sodium chloride (NaCl)
is preferred, which may be present at between 1 and 20 mg/ml e.g.
about 10.+-.2 mg/ml NaCl. Other salts that may be present include
potassium chloride, potassium dihydrogen phosphate, disodium
phosphate dehydrate, magnesium chloride, calcium chloride, etc.
[0077] Compositions will generally have an osmolality of between
200 mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg,
and will more preferably fall within the range of 290-310
mOsm/kg.
[0078] Compositions may include one or more buffers. Typical
buffers include: a phosphate buffer; a Tris buffer; a borate
buffer; a succinate buffer; a histidine buffer (particularly with
an aluminum hydroxide adjuvant); or a citrate buffer. Buffers will
typically be included in the 5-20 mM range.
[0079] Compositions may include a metal ion chelator, in particular
a divalent metal ion chelator such as EDTA. Reference 10 discloses
that inclusion of EDTA can improve stability of the compositions
disclosed herein. The final concentration of EDTA in an immunogenic
composition can be about 1-50 mM, about 1-10 mM or about 1-5 mM,
preferably about 2.5 mM.
[0080] The pH of a composition will generally be between 5.0 and
8.1, and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or
between 7.0 and 7.8.
[0081] The composition is preferably sterile. The composition is
preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit,
a standard measure) per dose, and preferably <0.1 EU per dose.
The composition is preferably gluten-free.
[0082] The composition may include material for a single
immunisation, or may include material for multiple immunisations
(i.e. a `multidose` kit). The inclusion of a preservative is
preferred in multidose arrangements. As an alternative (or in
addition) to including a preservative in multidose compositions,
the compositions may be contained in a container having an aseptic
adaptor for removal of material.
[0083] S.aureus infections can affect various areas of the body and
so a composition may be prepared in various forms. For example, a
composition may be prepared as injectables, either as liquid
solutions or suspensions. Solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection can also be
prepared (e.g. a lyophilised composition or a spray-freeze dried
composition). A composition may be prepared for topical
administration e.g. as an ointment, cream or powder. A composition
may be prepared for oral administration e.g. as a tablet or
capsule, as a spray, or as a syrup (optionally flavoured). A
composition may be prepared for pulmonary administration e.g. as an
inhaler, using a fine powder or a spray. A composition may be
prepared as a suppository or pessary. A composition may be prepared
for nasal, aural or ocular administration e.g. as drops. A
composition may be in kit form, designed such that a combined
composition is reconstituted just prior to administration to a
patient. Such kits may comprise one or more antigens in liquid form
and one or more lyophilised antigens.
[0084] Where a composition is to be prepared extemporaneously prior
to use (e.g. where a component is presented in lyophilised form)
and is presented as a kit, the kit may comprise two vials, or it
may comprise one ready-filled syringe and one vial, with the
contents of the syringe being used to reactivate the contents of
the vial prior to injection.
[0085] Human vaccines are typically administered in a dosage volume
of about 0.5 ml, although a half volume (i.e. about 0.25 ml) may
also be useful e.g. for children.
[0086] Immunogenic compositions administered according to the
invention may also comprise one or more immunoregulatory agents.
Preferably, one or more of the immunoregulatory agents include one
or more adjuvants (see below).
[0087] The compositions may elicit both a cell mediated immune
response as well as a humoral immune response. This immune response
will preferably induce long lasting (e.g. neutralising) antibodies
and a cell mediated immunity that can quickly respond upon exposure
to S.aureus.
[0088] Immunogenic compositions used as vaccines comprise an
immunologically effective amount of antigen(s), as well as any
other components, as needed. By `immunologically effective amount`,
it is meant that the administration of that amount to an
individual, either in a single dose or as part of a series, is
effective for treatment or prevention. This amount varies depending
upon the health and physical condition of the individual to be
treated, age, the taxonomic group of individual to be treated (e.g.
non-human primate, primate, etc.), the capacity of the individual's
immune system to synthesise antibodies, the degree of protection
desired, the formulation of the vaccine, the treating doctor's
assessment of the medical situation, and other relevant factors. It
is expected that the amount will fall in a relatively broad range
that can be determined through routine trials. Where more than one
antigen is included in a composition then two antigens may be
present at the same dose as each other or at different doses.
[0089] As mentioned above, a composition may include a temperature
protective agent, and this component may be particularly useful in
adjuvanted compositions (particularly those containing a mineral
adjuvant, such as an aluminium salt). As described in reference 20,
a liquid temperature protective agent may be added to an aqueous
vaccine composition to lower its freezing point e.g. to reduce the
freezing point to below 0.degree. C. Thus the composition can be
stored below 0.degree. C., but above its freezing point, to inhibit
thermal breakdown. The temperature protective agent also permits
freezing of the composition while protecting mineral salt adjuvants
against agglomeration or sedimentation after freezing and thawing,
and may also protect the composition at elevated temperatures e.g.
above 40.degree. C. A starting aqueous vaccine and the liquid
temperature protective agent may be mixed such that the liquid
temperature protective agent forms from 1-80% by volume of the
final mixture. Suitable temperature protective agents should be
safe for human administration, readily miscible/soluble in water,
and should not damage other components (e.g. antigen and adjuvant)
in the composition. Examples include glycerin, propylene glycol,
and/or polyethylene glycol (PEG). Suitable PEGs may have an average
molecular weight ranging from 200-20,000 Da. In a preferred
embodiment, the polyethylene glycol can have an average molecular
weight of about 300 Da (TEG-300').
Methods of Treatment, and Administration of an Immunogenic
Composition
[0090] The invention relates to the immunogenic composition
according to the invention for use as a medicament.
[0091] The invention also relates to the immunogenic composition
according to the invention for use as a medicament in the
prevention and/or treatment of a S. aureus infection.
[0092] The invention also provides a method for the prevention
and/or treatment of a S. aureus infection in a mammal comprising
the step of administering to a mammal in need thereof an
immunologically effective amount of an immunogenic composition
according to the invention as defined above. The advantageous
embodiments are as defined above.
[0093] The invention also provides the use of (i) at least one
antigen selected from the group consisting of EsxA, EsxB, FhuD2,
Sta011, and Hla antigens, and (ii) a mutant SpA antigen which has
decreased affinity, relative to unmodified SpA, for the Fc.gamma.
portion of human IgG and for the Fab portion of V.sub.H3-containing
human B cell receptors, in the manufacture of a medicament for
preventing or treating S.aureus infection in a mammal. The
advantageous embodiments are as defined above
[0094] The invention also provides (i) at least one antigen
selected from the group consisting of EsxA, EsxB, FhuD2, Sta011,
and Hla antigens, and (ii) a mutant SpA antigen which has decreased
affinity, relative to unmodified SpA, for the Fc.gamma. portion of
human IgG and for the Fab portion of V.sub.H3-containing human B
cell receptors, for use in immunising a mammal to prevent or treat
S.aureus infection. The advantageous embodiments are as defined
above.
[0095] The invention also provides (i) at least one antigen
selected from the group consisting of EsxA, EsxB, FhuD2, Sta011,
and Hla antigens, and (ii) a mutant SpA antigen which has decreased
affinity, relative to unmodified SpA, for the Fc.gamma. portion of
human IgG and for the Fab portion of V.sub.H3-containing human B
cell receptors, for use in a method of immunising a mammal to
prevent or treat S.aureus infection by administering a
therapeutically effective amount of the antigens to the mammal. The
advantageous embodiments are as defined above.
[0096] As noted above, 1, 2, 3, 4 or preferably all 5 of EsxA,
EsxB, FhuD2, Sta011, and Hla can be used in combination with the
mutant SpA. In this manner the methods, uses, compositions and
antigen combinations of the invention elicit an immune response
which is effective for preventing or treating S.aureus infections.
The immune response can involve antibodies and/or cell-mediated
immunity. By raising an immune response in the mammal by these uses
and methods, the mammal can be protected against S.aureus
infection, including a nosocomial infection. More particularly, the
mammal may be protected against a skin infection, pneumonia,
meningitis, osteomyelitis endocarditis, toxic shock syndrome,
and/or septicaemia. The invention is also useful for protecting
against S.aureus infection of a mammal's bones and joints (and thus
for preventing disorders including, but not limited to,
osteomyelitis, septic arthritis, and prosthetic joint infection).
In many cases these disorders may be associated with the formation
of a S.aureus biofilm.
[0097] S.aureus infects various mammals (including cows, dogs,
horses, and pigs), but the preferred mammal for use with the
invention is a human. The human can be a child (e.g. a toddler or
infant), a teenager, or an adult. In some embodiments the human may
have a prosthetic bone or joint, or may be an intended recipient of
such prostheses (e.g. a pre-operative orthopedic surgery patient).
A vaccine intended for children may also be administered to adults
e.g. to assess safety, dosage, immunogenicity, etc. The vaccines
are not suitable solely for these groups, however, and may be used
more generally in a human population.
[0098] One way of checking efficacy of therapeutic treatment
involves monitoring S.aureus infection after administration of the
compositions or antigens according to the invention. One way of
checking efficacy of prophylactic treatment involves monitoring
immune responses, systemically (such as monitoring the level of
IgG1 and IgG2a production) and/or mucosally (such as monitoring the
level of IgA production), against the antigens in the administered
composition after its administration. Another way of assessing the
immunogenicity of the compositions is to express the antigens
recombinantly for screening patient sera or mucosal secretions by
immunoblot and/or microarrays. A positive reaction between the
protein and the patient sample indicates that the patient has
mounted an immune response to the protein in question.
[0099] The efficacy of vaccine compositions can also be determined
in vivo by challenging animal models of S.aureus infection, e.g.,
guinea pigs or mice, with the vaccine compositions. There are three
generally useful animal models for the study of S.aureus infectious
disease, namely: (i) the murine abscess model [21], (ii) the murine
lethal infection model [21], and (iii) the murine pneumonia model
[22]. The abscess model looks at abscesses in mouse kidneys after
intravenous challenge. The lethal infection model looks at the
number of mice which survive after being infected by a
normally-lethal dose of S.aureus by the intravenous or
intraperitoneal route. The pneumonia model also looks at the
survival rate, but uses intranasal infection. Further useful models
are disclosed in reference 23 for studying both S.aureus disease in
relation to biofilm-mediated implant infection, skin and soft
tissue infection (SSTI), and sepsis. A useful vaccine may be
effective in one or more of these models. For instance, for some
clinical situations it may be desirable to protect against
pneumonia, without needing to prevent hematic spread or to promote
opsonisation; in other situations the main desire may be to prevent
hematic spread or sepsis. Different antigens, and different antigen
combinations, may contribute to different aspects of an effective
vaccine.
[0100] Compositions will generally be administered directly to a
patient. Direct delivery may be accomplished by parenteral
injection (e.g. subcutaneously, intraperitoneally, intravenously,
intramuscularly, or to the interstitial space of a tissue), or
mucosally, such as by rectal, oral (e.g. tablet, spray), vaginal,
topical, transdermal or transcutaneous, intranasal, ocular, aural,
pulmonary or other mucosal administration. Intramuscular injection
is the most typical route for administering compositions according
to the invention.
[0101] The invention may be used to elicit systemic and/or mucosal
immunity, preferably to elicit an enhanced systemic and/or mucosal
immunity. Preferably the enhanced systemic and/or mucosal immunity
is reflected in an enhanced TH1 and/or TH2 immune response.
Preferably, the enhanced immune response includes an increase in
the production of IgG1 and/or IgG2a and/or IgA.
[0102] Dosage can be by a single dose schedule or a multiple dose
schedule. Multiple doses may be used in a primary immunisation
schedule and/or in a booster immunisation schedule. In a multiple
dose schedule the various doses may be given by the same or
different routes e.g. a parenteral prime and mucosal boost, a
mucosal prime and parenteral boost, etc. Multiple doses will
typically be administered at least 1 week apart (e.g. about 2
weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks,
about 10 weeks, about 12 weeks, about 16 weeks, etc.).
[0103] Immunogenic compositions may be administered to patients at
substantially the same time as (e.g. during the same medical
consultation or visit to a healthcare professional or vaccination
centre) other vaccines.
[0104] Immunogenic compositions may be administered to patients in
combination with an antibiotic. For instance, they may be
administered at substantially the same time as an antibiotic.
Similarly, they may be administered to a subject who is receiving
antibiotic therapy. Similarly, they may be administered as part of
a co-therapy which involves administration of both a composition as
discussed herein and an antibiotic. The antibiotic will be
effective against a S.aureus bacterium, for instance a
beta-lactam.
Strains and Variants
[0105] Antigens are discussed above by reference to existing
nomenclature (e.g. "EsxA") and exemplary sequences given as GI
numbers and also in the sequence listing. The invention is not
limited to these precise sequences. Genome sequences of several
strains of S.aureus are available, including those of MRSA strains
N315 and Mu50 [24], MW2, N315, COL, MRSA252, MSSA476, RF122, USA300
(very virulent), JH1, JH9, NCTC 8325, and Newman. Standard search
and alignment techniques can be used to identify in any of these
(or other) further genome sequences the homolog of any particular
sequence mentioned herein Moreover, the specific sequences
disclosed herein can be used to design primers for amplification of
homologous sequences from other strains. Thus the invention
encompasses such variants and homologs from any strain of S.aureus
, as well as non-natural variants. In general, suitable variants of
a particular SEQ ID NO include its allelic variants, its
polymorphic forms, its homologs, its orthologs, its paralogs, its
mutants, etc.
[0106] Thus, for instance, polypeptides used with the invention
may, compared to the SEQ ID NO herein, include one or more (e.g. 1,
2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid substitutions, such as
conservative substitutions (i.e. substitutions of one amino acid
with another which has a related side chain). Genetically-encoded
amino acids are generally divided into four families: (1) acidic
i.e. aspartate, glutamate; (2) basic i.e. lysine, arginine,
histidine; (3) non-polar i.e. alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan; and (4) uncharged
polar i.e. glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are
sometimes classified jointly as aromatic amino acids. In general,
substitution of single amino acids within these families does not
have a major effect on the biological activity. The polypeptides
may also include one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.)
single amino acid deletions relative to the SEQ ID NO sequences.
The polypeptides may also include one or more (e.g. 1, 2, 3, 4, 5,
6, 7, 8, 9, etc.) insertions (e.g. each of 1, 2, 3, 4 or 5 amino
acids) relative to the SEQ ID NO sequences.
[0107] Similarly, a polypeptide used with the invention may
comprise an amino acid sequence that: [0108] (a) is identical (i.e.
100% identical) to a sequence disclosed in the sequence listing;
[0109] (b) shares sequence identity (e.g. 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) with a sequence
disclosed in the sequence listing (ideally over the entire length
of said sequence); [0110] (c) has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
(or more) single amino acid alterations (deletions, insertions,
substitutions), which may be at separate locations or may be
contiguous, as compared to the sequences of (a) or (b); or [0111]
(d) when aligned with a particular sequence from the sequence
listing using a pairwise alignment algorithm, each moving window of
x amino acids from N-terminus to C-terminus (such that for an
alignment that extends to p amino acids, where p>x, there are
p-x+1 such windows) has at least xy identical aligned amino acids,
where: x is selected from 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,
90, 100, 150, 200; y is selected from 0.50, 0.60, 0.70, 0.75, 0.80,
0.85, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99;
and if xy is not an integer then it is rounded up to the nearest
integer. The preferred pairwise alignment algorithm is the
Needleman-Wunsch global alignment algorithm [25], using default
parameters (e.g. with Gap opening penalty=10.0, and with Gap
extension penalty=0.5, using the EBLOSUM62 scoring matrix). This
algorithm is conveniently implemented in the needle tool in the
EMBOSS package [26].
[0112] Where hybrid polypeptides are used, the individual antigens
within the hybrid (i.e. individual --X-moieties) may be from one or
more strains. Where n=2, for instance, X.sub.2 may be from the same
strain as X.sub.1 or from a different strain. Where n=3, the
strains might be (i) X.sub.1=X.sub.2=X.sub.3 (ii)
X.sub.1=X.sub.2.noteq.X.sub.3 (iii) X.sub.1.noteq.X.sub.2=X.sub.3
(iv) X.sub.1.noteq.X.sub.2.noteq.X.sub.3 or (v)
X.sub.1=X.sub.3.noteq.X.sub.2, etc.
[0113] Within group (c), deletions or substitutions may be at the
N-terminus and/or C-terminus, or may be between the two termini.
Thus a truncation is an example of a deletion. Truncations may
involve deletion of up to 40 (or more) amino acids at the
N-terminus and/or C-terminus. N-terminus truncation can remove
leader peptides e.g. to facilitate recombinant expression in a
heterologous host. C-terminus truncation can remove anchor
sequences e.g. to facilitate recombinant expression in a
heterologous host.
[0114] In general, when an antigen comprises a sequence that is not
identical to a complete S.aureus sequence from the sequence listing
(e.g. when it comprises a sequence listing with <100% sequence
identity thereto, or when it comprises a fragment thereof) it is
preferred in each individual instance that the antigen can elicit
an antibody which recognises the respective complete S.aureus
sequence.
Polypeptides Used With the Invention
[0115] Polypeptides used with the invention can take various forms
(e.g. native, fusions, glycosylated, non-glycosylated, lipidated,
non-lipidated, phosphorylated, non-phosphorylated, myristoylated,
non-myristoylated, monomeric, multimeric, etc.).
[0116] Polypeptides used with the invention can be prepared by
various means (e.g. recombinant expression, purification from cell
culture, chemical synthesis, etc.). Recombinantly-expressed
proteins are preferred, particularly for hybrid polypeptides.
[0117] Polypeptides used with the invention are preferably provided
in purified or substantially purified form i.e. substantially free
from other polypeptides (e.g. free from naturally-occurring
polypeptides), particularly from other staphylococcal or host cell
polypeptides, and are generally at least about 50% pure (by
weight), and usually at least about 90% pure i.e. less than about
50%, and more preferably less than about 10% (e.g. 5%) of a
composition is made up of other expressed polypeptides. Thus the
antigens in the compositions are separated from the whole organism
with which the molecule is expressed.
[0118] The term "polypeptide" refers to amino acid polymers of any
length. The polymer may be linear or branched, it may comprise
modified amino acids, and it may be interrupted by non-amino acids.
The terms also encompass an amino acid polymer that has been
modified naturally or by intervention; for example, disulfide bond
formation, glycosylation, lipidation, acetylation, phosphorylation,
or any other manipulation or modification, such as conjugation with
a labeling component. Also included are, for example, polypeptides
containing one or more analogs of an amino acid (including, for
example, unnatural amino acids, etc.), as well as other
modifications known in the art. Polypeptides can occur as single
chains or associated chains.
[0119] Although expression of the polypeptides of the invention may
take place in a Staphylococcus, the invention will usually use a
heterologous host for expression (recombinant expression). The
heterologous host may be prokaryotic (e.g. a bacterium) or
eukaryotic. It may be E.coli, but other suitable hosts include
Bacillus subtilis, Vibrio cholerae, Salmonella typhi, Salmonella
typhimurium, Neisseria lactamica, Neisseria cinerea, Mycobacteria
(e.g. M.tuberculosis), yeasts, etc. Compared to the wild-type
S.aureus genes encoding polypeptides of the invention, it is
helpful to change codons to optimise expression efficiency in such
hosts without affecting the encoded amino acids.
Adjuvants
[0120] As mentioned above, immunogenic compositions used according
to the invention may include one or more adjuvants. Adjuvants which
may be used with the invention include, but are not limited to:
[0121] A. Mineral-Containing Compositions
[0122] Mineral containing compositions suitable for use as
adjuvants in the invention include mineral salts, such as aluminium
salts and calcium salts (or mixtures thereof). Calcium salts
include calcium phosphate (e.g. the "CAP" particles disclosed in
ref. 27). Aluminum salts include hydroxides and phosphates etc.,
with the salts taking any suitable form (e.g. gel, crystalline,
amorphous, etc.). Adsorption to these salts is preferred (e.g. all
antigens may be adsorbed). The mineral containing compositions may
also be formulated as a particle of metal salt [28].
[0123] The adjuvants known as aluminum hydroxide and aluminum
phosphate may be used. These names are conventional, but are used
for convenience only, as neither is a precise description of the
actual chemical compound which is present (e.g. see chapter 9 of
reference 29). The invention can use any of the "hydroxide" or
"phosphate" adjuvants that are in general use as adjuvants. The
adjuvants known as "aluminium hydroxide" are typically aluminium
oxyhydroxide salts, which are usually at least partially
crystalline. The adjuvants known as "aluminium phosphate" are
typically aluminium hydroxyphosphates, often also containing a
small amount of sulfate (i. e. aluminium hydroxyphosphate sulfate).
They may be obtained by precipitation, and the reaction conditions
and concentrations during precipitation influence the degree of
substitution of phosphate for hydroxyl in the salt.
[0124] A fibrous morphology (e.g. as seen in transmission electron
micrographs) is typical for aluminium hydroxide adjuvants. The pI
of aluminium hydroxide adjuvants is typically about 11 i.e. the
adjuvant itself has a positive surface charge at physiological pH.
Adsorptive capacities of between 1.8-2.6 mg protein per mg
Al.sup.+++ at pH 7.4 have been reported for aluminium hydroxide
adjuvants.
[0125] Aluminium phosphate adjuvants generally have a PO.sub.4/Al
molar ratio between 0.3 and 1.2, preferably between 0.8 and 1.2,
and more preferably 0.95.+-.0.1. The aluminium phosphate will
generally be amorphous, particularly for hydroxyphosphate salts. A
typical adjuvant is amorphous aluminium hydroxyphosphate with
PO.sub.4/Al molar ratio between 0.84 and 0.92, included at 0.6 mg
A.sup.3+/ml. The aluminium phosphate will generally be particulate
(e.g. plate-like morphology as seen in transmission electron
micrographs). Typical diameters of the particles are in the range
0.5-20 .mu.m (e.g. about 5-10 .mu.m) after any antigen adsorption.
Adsorptive capacities of between 0.7-1.5 mg protein per mg
Al.sup.+++ at pH 7.4 have been reported for aluminium phosphate
adjuvants.
[0126] The point of zero charge (PZC) of aluminium phosphate is
inversely related to the degree of substitution of phosphate for
hydroxyl, and this degree of substitution can vary depending on
reaction conditions and concentration of reactants used for
preparing the salt by precipitation. PZC is also altered by
changing the concentration of free phosphate ions in solution (more
phosphate=more acidic PZC) or by adding a buffer such as a
histidine buffer (makes PZC more basic). Aluminium phosphates used
according to the invention will generally have a PZC of between 4.0
and 7.0, more preferably between 5.0 and 6.5 e.g. about 5.7.
[0127] Suspensions of aluminium salts used to prepare compositions
of the invention may contain a buffer (e.g. a phosphate or a
histidine or a Tris buffer), but this is not always necessary. The
suspensions are preferably sterile and pyrogen-free. A suspension
may include free aqueous phosphate ions e.g. present at a
concentration between 1.0 and 20 mM, preferably between 5 and 15
mM, and more preferably about 10 mM. The suspensions may also
comprise sodium chloride.
[0128] The invention can use a mixture of both an aluminium
hydroxide and an aluminium phosphate. In this case there may be
more aluminium phosphate than hydroxide e.g. a weight ratio of at
least 2:1 e.g. .gtoreq.5:1, .gtoreq.6:1, .gtoreq.7:1, .gtoreq.8:1,
.gtoreq.9:1, etc.
[0129] The concentration of Al.sup.+++ in a composition for
administration to a patient is preferably less than 10mg/ml e.g.
.ltoreq.5 mg/ml, .ltoreq.4 mg/ml, .ltoreq.3 mg/ml, .ltoreq.2 mg/ml,
.ltoreq.1 mg/ml, etc. A preferred range is between 0.3 and 1 mg/ml.
A maximum of 0.85 mg/dose is preferred.
[0130] B. Oil-in-Water Emulsions
[0131] Oil-in-water emulsion compositions suitable for use as
adjuvants in the invention include squalene-in-water emulsions,
such as MF59 (see Chapter 10 of ref. 29; see also ref. 30) and AS03
[31].
[0132] Various oil-in-water emulsion adjuvants are known, and they
typically include at least one oil and at least one surfactant,
with the oil(s) and surfactant(s) being biodegradable
(metabolisable) and biocompatible. The emulsion will include
submicron oil droplets, and emulsions with droplets having a
diameter less than 220 nm are preferred as they can be subjected to
filter sterilization.
[0133] The emulsion comprises one or more oils. Suitable oil(s)
include those from, for example, an animal (such as fish) or a
vegetable source. The oil is ideally biodegradable (metabolisable)
and biocompatible. Sources for vegetable oils include nuts, seeds
and grains. Peanut oil, soybean oil, coconut oil, and olive oil,
the most commonly available, exemplify the nut oils. Jojoba oil can
be used e.g. obtained from the jojoba bean. Seed oils include
safflower oil, cottonseed oil, sunflower seed oil, sesame seed oil
and the like. In the grain group, corn oil is the most readily
available, but the oil of other cereal grains such as wheat, oats,
rye, rice, teff, triticale and the like may also be used. 6-10
carbon fatty acid esters of glycerol and 1,2-propanediol, while not
occurring naturally in seed oils, may be prepared by hydrolysis,
separation and esterification of the appropriate materials starting
from the nut and seed oils. Fats and oils from mammalian milk are
metabolisable and so may be used. The procedures for separation,
purification, saponification and other means necessary for
obtaining pure oils from animal sources are well known in the
art.
[0134] Most fish contain metabolisable oils which may be readily
recovered. For example, cod liver oil, shark liver oils, and whale
oil such as spermaceti exemplify several of the fish oils which may
be used herein. A number of branched chain oils are synthesized
biochemically in 5-carbon isoprene units and are generally referred
to as terpenoids. Preferred emulsions comprise squalene, a shark
liver oil which is a branched, unsaturated terpenoid. Squalane, the
saturated analog to squalene, can also be used. Fish oils,
including squalene and squalane, are readily available from
commercial sources or may be obtained by methods known in the
art.
[0135] Other useful oils are the tocopherols, particularly in
combination with squalene. Where the oil phase of an emulsion
includes a tocopherol, any of the .alpha., .beta., .gamma.,
.delta., .epsilon. or .xi. tocopherols can be used, but
.alpha.-tocopherols are preferred. D-.alpha.-tocopherol and
DL-a-tocopherol can both be used. A preferred .alpha.-tocopherol is
DL-.alpha.-tocopherol. An oil combination comprising squalene and a
tocopherol (e.g. DL-.alpha.-tocopherol) can be used.
[0136] The oil in the emulsion may comprise a combination of oils
e.g. squalene and at least one other oil.
[0137] The aqueous component of the emulsion can be plain water
(e.g. w.f.i.) or can include further components e.g. solutes. For
instance, it may include salts to form a buffer e.g. citrate or
phosphate salts, such as sodium salts. Typical buffers include: a
phosphate buffer; a Tris buffer; a borate buffer; a succinate
buffer; a histidine buffer; or a citrate buffer. A buffered aqueous
phase is preferred, and buffers will typically be included in the
5-20 mM range.
[0138] In addition to the oil and cationic lipid, an emulsion can
include a non-ionic surfactant and/or a zwitterionic surfactant.
Such surfactants include, but are not limited to: the
polyoxyethylene sorbitan esters surfactants (commonly referred to
as the Tweens), especially polysorbate 20 and polysorbate 80;
copolymers of ethylene oxide (EO), propylene oxide (PO), and/or
butylene oxide (BO), sold under the DOWFAX.TM. tradename, such as
linear EO/PO block copolymers; octoxynols, which can vary in the
number of repeating ethoxy (oxy-1,2-ethanediyl) groups, with
octoxynol-9 (Triton X-100, or t-octylphenoxypolyethoxyethanol)
being of particular interest; (octylphenoxy)polyethoxyethanol
(IGEPAL CA-630/NP-40); phospholipids such as phosphatidylcholine
(lecithin); polyoxyethylene fatty ethers derived from lauryl,
cetyl, stearyl and oleyl alcohols (known as Brij surfactants), such
as triethyleneglycol monolauryl ether (Brij 30);
polyoxyethylene-9-lauryl ether; and sorbitan esters (commonly known
as the Spans), such as sorbitan trioleate (Span 85) and sorbitan
monolaurate. Preferred surfactants for including in the emulsion
are polysorbate 80 (Tween 80; polyoxyethylene sorbitan monooleate),
Span 85 (sorbitan trioleate), lecithin and Triton X-100.
[0139] Mixtures of surfactants can be used e.g. Tween 80/Span 85
mixtures, or Tween 80/Triton-X100 mixtures. A combination of a
polyoxyethylene sorbitan ester such as polyoxyethylene sorbitan
monooleate (Tween 80) and an octoxynol such as
t-octylphenoxy-polyethoxyethanol (Triton X-100) is also suitable.
Another useful combination comprises laureth 9 plus a
polyoxyethylene sorbitan ester and/or an octoxynol. Useful mixtures
can comprise a surfactant with a HLB value in the range of 10-20
(e.g. polysorbate 80, with a HLB of 15.0) and a surfactant with a
HLB value in the range of 1-10 (e.g. sorbitan trioleate, with a HLB
of 1.8).
[0140] Preferred amounts of oil (% by volume) in the final emulsion
are between 2-20% e.g. 5-15%, 6-14%, 7-13%, 8-12%. A squalene
content of about 4-6% or about 9-11% is particularly useful.
[0141] Preferred amounts of surfactants (% by weight) in the final
emulsion are between 0.001% and 8%. For example: polyoxyethylene
sorbitan esters (such as polysorbate 80) 0.2 to 4%, in particular
between 0.4-0.6%, between 0.45-0.55%, about 0.5% or between 1.5-2%,
between 1.8-2.2%, between 1.9-2.1%, about 2%, or 0.85-0.95%, or
about 1%; sorbitan esters (such as sorbitan trioleate) 0.02 to 2%,
in particular about 0.5% or about 1%; octyl- or nonylphenoxy
polyoxyethanols (such as Triton X-100) 0.001 to 0.1%, in particular
0.005 to 0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to
8%, preferably 0.1 to 10% and in particular 0.1 to 1% or about
0.5%.
[0142] The absolute amounts of oil and surfactant, and their ratio,
can be varied within wide limits while still forming an emulsion. A
skilled person can easily vary the relative proportions of the
components to obtain a desired emulsion, but a weight ratio of
between 4:1 and 5:1 for oil and surfactant is typical (excess
oil).
[0143] An important parameter for ensuring immunostimulatory
activity of an emulsion, particularly in large animals, is the oil
droplet size (diameter). The most effective emulsions have a
droplet size in the submicron range. Suitably the droplet sizes
will be in the range 50-750 nm. Most usefully the average droplet
size is less than 250 nm e.g. less than 200 nm, less than 150 nm.
The average droplet size is usefully in the range of 80-180 nm.
Ideally, at least 80% (by number) of the emulsion's oil droplets
are less than 250 nm in diameter, and preferably at least 90%.
These droplet sizes can conveniently be achieved by techniques such
as microfluidisation. Apparatuses for determining the average
droplet size in an emulsion, and the size distribution, are
commercially available. These typically use the techniques of
dynamic light scattering and/or single-particle optical sensing
e.g. the Accusizer.TM. and Nicomp.TM. series of instruments
available from Particle Sizing Systems (Santa Barbara, USA), or the
Zetasizer.TM. instruments from Malvern Instruments (UK), or the
Particle Size Distribution Analyzer instruments from Horiba (Kyoto,
Japan). Ideally, the distribution of droplet sizes (by number) has
only one maximum i.e. there is a single population of droplets
distributed around an average (mode), rather than having two
maxima. Preferred emulsions have a polydispersity of <0.4 e.g.
0.3, 0.2, or less.
[0144] Specific oil-in-water emulsion adjuvants useful with the
invention include, but are not limited to: [0145] A submicron
emulsion of squalene, Tween 80, and Span 85. The composition of the
emulsion by volume can be about 5% squalene, about 0.5% polysorbate
80 and about 0.5% Span 85. In weight terms, these ratios become
4.3% squalene, 0.5% polysorbate 80 and 0.48% Span 85. This adjuvant
is known as `MF59` [32-34], as described in more detail in Chapter
10 of ref. 35 and chapter 12 of ref. 36. The MF59 emulsion
advantageously includes citrate ions e.g. 10 mM sodium citrate
buffer. [0146] An emulsion comprising squalene, a tocopherol, and
polysorbate 80. The emulsion may include phosphate buffered saline.
These emulsions may have by volume from 2 to 10% squalene, from 2
to 10% tocopherol and from 0.3 to 3% polysorbate 80, and the weight
ratio of squalene:tocopherol is preferably <1 (e.g. 0.90) as
this can provide a more stable emulsion. Squalene and polysorbate
80 may be present volume ratio of about 5:2 or at a weight ratio of
about 11:5. Thus the three components (squalene, tocopherol,
polysorbate 80) may be present at a weight ratio of 1068:1186:485
or around 55:61:25. One such emulsion (`AS03`) can be made by
dissolving Tween 80 in PBS to give a 2% solution, then mixing 90 ml
of this solution with a mixture of (5 g of DL a tocopherol and 5 ml
squalene), then microfluidising the mixture. The resulting emulsion
may have submicron oil droplets e.g. with an average diameter of
between 100 and 250 nm, preferably about 180 nm. The emulsion may
also include a 3-de-O-acylated monophosphoryl lipid A (3d MPL).
Another useful emulsion of this type may comprise, per human dose,
0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate
80 [37] e.g. in the ratios discussed above. [0147] An emulsion of
squalene, a tocopherol, and a Triton detergent (e.g. Triton X-100).
The emulsion may also include a 3d-MPL (see below). The emulsion
may contain a phosphate buffer. [0148] An emulsion comprising a
polysorbate (e.g. polysorbate 80), a Triton detergent (e.g.
Triton
[0149] X-100) and a tocopherol (e.g. an .alpha.-tocopherol
succinate). The emulsion may include these three components at a
mass ratio of about 75:11:10 (e.g. 750 .mu.g/ml polysorbate 80, 110
.mu.g/ml Triton X-100 and 100 .mu.g/ml .alpha.-tocopherol
succinate), and these concentrations should include any
contribution of these components from antigens. The emulsion may
also include squalene. The emulsion may also include a 3d-MPL (see
below). The aqueous phase may contain a phosphate buffer. [0150] An
emulsion of squalane, polysorbate 80 and poloxamer 401
("Pluronic.TM. L121"). The emulsion can be formulated in phosphate
buffered saline, pH 7.4. This emulsion is a useful delivery vehicle
for muramyl dipeptides, and has been used with threonyl-MDP in the
"SAF-1" adjuvant [38] (0.05-1% Thr-MDP, 5% squalane, 2.5% Pluronic
L121 and 0.2% polysorbate 80). It can also be used without the
Thr-MDP, as in the "AF" adjuvant [39] (5% squalane, 1.25% Pluronic
L121 and 0.2% polysorbate 80). Microfluidisation is preferred.
[0151] An emulsion comprising squalene, an aqueous solvent, a
polyoxyethylene alkyl ether hydrophilic nonionic surfactant (e.g.
polyoxyethylene (12) cetostearyl ether) and a hydrophobic nonionic
surfactant (e.g. a sorbitan ester or mannide ester, such as
sorbitan monoleate or `Span 80`). The emulsion is preferably
thermoreversible and/or has at least 90% of the oil droplets (by
volume) with a size less than 200 nm [40]. The emulsion may also
include one or more of: alditol; a cryoprotective agent (e.g. a
sugar, such as dodecylmaltoside and/or sucrose); and/or an
alkylpolyglycoside. The emulsion may include a TLR4 agonist [41].
Such emulsions may be lyophilized. [0152] An emulsion of squalene,
poloxamer 105 and Abil-Care [42]. The final concentration (weight)
of these components in adjuvanted vaccines are 5% squalene, 4%
poloxamer 105 (pluronic polyol) and 2% Abil-Care 85
(Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone; caprylic/capric
triglyceride). [0153] An emulsion having from 0.5-50% of an oil,
0.1-10% of a phospholipid, and 0.05-5% of a non-ionic surfactant.
As described in reference 43, preferred phospholipid components are
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidylglycerol, phosphatidic acid,
sphingomyelin and cardiolipin. Submicron droplet sizes are
advantageous. [0154] A submicron oil-in-water emulsion of a
non-metabolisable oil (such as light mineral oil) and at least one
surfactant (such as lecithin, Tween 80 or Span 80). Additives may
be included, such as QuilA saponin, cholesterol, a
saponin-lipophile conjugate (such as GPI-0100, described in
reference 44, produced by addition of aliphatic amine to
desacylsaponin via the carboxyl group of glucuronic acid),
dimethyidioctadecylammonium bromide and/or N,N-dioctadecyl-N,N-bis
(2-hydroxyethyl)propanediamine. [0155] An emulsion in which a
saponin (e.g. QuilA or QS21) and a sterol (e.g. a cholesterol) are
associated as helical micelles [45]. [0156] An emulsion comprising
a mineral oil, a non-ionic lipophilic ethoxylated fatty alcohol,
and a non-ionic hydrophilic surfactant (e.g. an ethoxylated fatty
alcohol and/or polyoxyethylene-polyoxypropylene block copolymer)
[46]. [0157] An emulsion comprising a mineral oil, a non-ionic
hydrophilic ethoxylated fatty alcohol, and a non-ionic lipophilic
surfactant (e.g. an ethoxylated fatty alcohol and/or
polyoxyethylene-polyoxypropylene block copolymer) [46].
[0158] In some embodiments an emulsion may be mixed with antigen(s)
extemporaneously, at the time of delivery, and thus the adjuvant
and antigen(s) may be kept separately in a packaged or distributed
vaccine, ready for final formulation at the time of use. In other
embodiments an emulsion is mixed with antigen during manufacture,
and thus the composition is packaged in a liquid adjuvanted form.
The antigen will generally be in an aqueous form, such that the
vaccine is finally prepared by mixing two liquids. The volume ratio
of the two liquids for mixing can vary (e.g. between 5:1 and 1:5)
but is generally about 1:1. Where concentrations of components are
given in the above descriptions of specific emulsions, these
concentrations are typically for an undiluted composition, and the
concentration after mixing with an antigen solution will thus
decrease.
[0159] C. Saponin Formulations [Chapter 22 of Ref 29]
[0160] Saponin formulations may also be used as adjuvants in the
invention. Saponins are a heterogeneous group of sterol glycosides
and triterpenoid glycosides that are found in the bark, leaves,
stems, roots and even flowers of a wide range of plant species.
Saponin from the bark of the Quillaia saponaria Molina tree have
been widely studied as adjuvants. Saponin can also be commercially
obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata
(brides veil), and Saponaria officianalis (soap root). Saponin
adjuvant formulations include purified formulations, such as QS21,
as well as lipid formulations, such as ISCOMs. QS21 is marketed as
Stimulon.TM..
[0161] Saponin compositions have been purified using HPLC and
RP-HPLC. Specific purified fractions using these techniques have
been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and
QH-C. Preferably, the saponin is QS21. A method of production of
QS21 is disclosed in ref. 47. Saponin formulations may also
comprise a sterol, such as cholesterol [48].
[0162] Combinations of saponins and cholesterols can be used to
form particles called ISCOMs (chapter 23 of ref. 29). ISCOMs
typically also include a phospholipid such as
phosphatidylethanolamine or phosphatidylcholine. Any known saponin
can be used in ISCOMs. Preferably, the ISCOM includes one or more
of QuilA, QHA & QHC. ISCOMs are further described in refs.
48-50. Optionally, the ISCOMS may be devoid of additional detergent
[51].
[0163] A review of the development of saponin based adjuvants can
be found in refs. 52 & 53.
[0164] D. Bacterial or Microbial Derivatives
[0165] Adjuvants suitable for use in the invention include
bacterial or microbial derivatives such as non-toxic derivatives of
enterobacterial lipopolysaccharide (LPS), Lipid A derivatives,
immunostimulatory oligonucleotides and ADP-ribosylating toxins and
detoxified derivatives thereof.
[0166] Non-toxic derivatives of LPS include monophosphoryl lipid A
(MPL) and 3-0-deacylated MPL (3 dMPL). 3 dMPL is a mixture of 3
de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated
chains. A preferred "small particle" form of 3 De-O-acylated
monophosphoryl lipid A is disclosed in ref. 54. Such "small
particles" of 3 dMPL are small enough to be sterile filtered
through a 0.22 .mu.m membrane [54]. Other non-toxic LPS derivatives
include monophosphoryl lipid A mimics, such as aminoalkyl
glucosaminide phosphate derivatives e.g. RC-529 (see below).
[0167] Lipid A derivatives include derivatives of lipid A from
Escherichia coli such as OM-174. OM-174 is described for example in
refs. 55 & 56.
[0168] Immunostimulatory oligonucleotides suitable for use as
adjuvants in the invention include nucleotide sequences containing
a CpG motif (a dinucleotide sequence containing an unmethylated
cytosine linked by a phosphate bond to a guanosine).
Double-stranded RNAs and oligonucleotides containing palindromic or
poly(dG) sequences have also been shown to be
immunostimulatory.
[0169] The CpG's can include nucleotide modifications/analogs such
as phosphorothioate modifications and can be double-stranded or
single-stranded. References 57 , 58 and 59 disclose possible analog
substitutions e.g. replacement of guanosine with
2'-deoxy-7-deazaguanosine. The adjuvant effect of CpG
oligonucleotides is further discussed in refs. 60-65.
[0170] The CpG sequence may be directed to TLR9, such as the motif
GTCGTT or TTCGTT [66]. The CpG sequence may be specific for
inducing a Th1 immune response, such as a CpG-A ODN, or it may be
more specific for inducing a B cell response, such a CpG-B ODN.
CpG-A and CpG-B ODNs are discussed in refs. 67-69. Preferably, the
CpG is a CpG-A ODN.
[0171] Preferably, the CpG oligonucleotide is constructed so that
the 5' end is accessible for receptor recognition. Optionally, two
CpG oligonucleotide sequences may be attached at their 3' ends to
form "immunomers". See, for example, refs. 66 & 70-72.
[0172] A useful CpG adjuvant is CpG7909, also known as ProMune.TM.
(Coley Pharmaceutical Group, Inc.). Another is CpG1826. As an
alternative, or in addition, to using CpG sequences, TpG sequences
can be used [ 73 ], and these oligonucleotides may be free from
unmethylated CpG motifs. The immunostimulatory oligonucleotide may
be pyrimidine-rich. For example, it may comprise more than one
consecutive thymidine nucleotide (e.g. TTTT, as disclosed in ref.
73), and/or it may have a nucleotide composition with >25%
thymidine (e.g. >35%, >40%, >50%, >60%, >80%, etc.).
For example, it may comprise more than one consecutive cytosine
nucleotide (e.g. CCCC, as disclosed in ref. 73), and/or it may have
a nucleotide composition with >25% cytosine (e.g. >35%,
>40%, >50%, >60%, >80%, etc.). These oligonucleotides
may be free from unmethylated CpG motifs. Immunostimulatory
oligonucleotides will typically comprise at least 20 nucleotides.
They may comprise fewer than 100 nucleotides.
[0173] A particularly useful adjuvant based around
immunostimulatory oligonucleotides is known as IC-31.TM. [74 ].
Thus an adjuvant used with the invention may comprise a mixture of
(i) an oligonucleotide (e.g. between 15-40 nucleotides) including
at least one (and preferably multiple) CpI motifs (i.e. a cytosine
linked to an inosine to form a dinucleotide), and (ii) a
polycationic polymer, such as an oligopeptide (e.g. between 5-20
amino acids) including at least one (and preferably multiple)
Lys-Arg-Lys tripeptide sequence(s). The oligonucleotide may be a
deoxynucleotide comprising 26-mer sequence 5'-(IC).sub.13-3' (SEQ
ID NO: 41). The polycationic polymer may be a peptide comprising
11-mer amino acid sequence KLKLLLLLKLK (SEQ ID NO: 42). The
oligonucleotide and polymer can form complexes e.g. as disclosed in
references 75 & 76.
[0174] Bacterial ADP-ribosylating toxins and detoxified derivatives
thereof may be used as adjuvants in the invention. Preferably, the
protein is derived from E.coli (E.coli heat labile enterotoxin
"LT"), cholera ("CT"), or pertussis ("PT"). The use of detoxified
ADP-ribosylating toxins as mucosal adjuvants is described in ref.
77 and as parenteral adjuvants in ref. 78. The toxin or toxoid is
preferably in the form of a holotoxin, comprising both A and B
subunits. Preferably, the A subunit contains a detoxifying
mutation; preferably the B subunit is not mutated. Preferably, the
adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and
LT-G192. The use of ADP-ribosylating toxins and detoxified
derivatives thereof, particularly LT-K63 and LT-R72, as adjuvants
can be found in refs. 79-86. A useful CT mutant is or CT-E29H [87].
Numerical reference for amino acid substitutions is preferably
based on the alignments of the A and B subunits of ADP-ribosylating
toxins set forth in ref. 88, specifically incorporated herein by
reference in its entirety.
[0175] E. TLR Agonists
[0176] Compositions can include a TLR agonist i.e. a compound which
can agonise a Toll-like receptor.
[0177] Most preferably, a TLR agonist is an agonist of a human TLR.
The TLR agonist can activate any of TLR1, TLR2, TLR3, TLR4, TLRS,
TLR6, TLR7, TLR8, TLR9 or TLR11; preferably it can activate human
TLR4 or human TLR7.
[0178] Agonist activity of a compound against any particular
Toll-like receptor can be determined by standard assays. Companies
such as Imgenex and Invivogen supply cell lines which are stably
co-transfected with human TLR genes and NFKB, plus suitable
reporter genes, for measuring TLR activation pathways. They are
designed for sensitivity, broad working range dynamics and can be
used for high-throughput screening. Constitutive expression of one
or two specific TLRs is typical in such cell lines. See also
reference 89. Many TLR agonists are known in the art e.g. reference
90 describes certain lipopeptide molecules that are TLR2 agonists,
references 91 to 94 each describe classes of small molecule
agonists of TLR7, and references 95 & 96 describe TLR7 and TLR8
agonists for treatment of diseases.
[0179] A TLR agonist used with the invention ideally includes at
least one adsorptive moiety. The inclusion of such moieties in TLR
agonists allows them to adsorb to insoluble aluminium salts (e.g.
by ligand exchange or any other suitable mechanism) and improves
their immunological behaviour [97]. Phosphorus-containing
adsorptive moieties are particularly useful, and so an adsorptive
moiety may comprise a phosphate, a phosphonate, a phosphinate, a
phosphonite, a phosphinite, etc. Preferably the TLR agonist
includes at least one phosphonate group.
[0180] Thus, in preferred embodiments, a composition includes a TLR
agonist (more preferably a TLR7 agonist) which includes a
phosphonate group. This phosphonate group can allow adsorption of
the agonist to an insoluble aluminium salt [97].
[0181] TLR agonists useful with the invention may include a single
adsorptive moiety, or may include more than one e.g. between 2 and
15 adsorptive moieties. Typically a compound will include 1, 2 or 3
adsorptive moieties.
[0182] Useful phosphorus-containing TLR agonists can be represented
by formula (A1):
##STR00001## [0183] wherein: [0184] R.sup.X and R.sup.Y are
independently selected from H and C.sub.1-C.sub.6 alkyl; [0185] X
is selected from a covalent bond, O and NH; [0186] Y is selected
from a covalent bond, O, C(O), S and NH; [0187] L is a linker e.g.
selected from, C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene,
arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and
--((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally
substituted with 1 to 4 substituents independently selected from
halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and
--P(O)(OH).sub.2; [0188] each p is independently selected from 1,
2, 3, 4, 5 and 6; [0189] q is selected from 1, 2, 3 and 4; [0190] n
is selected from 1, 2 and 3; and [0191] A is a TLR agonist
moiety.
[0192] In one embodiment, the TLR agonist according to formula (Al)
is as follows: R.sup.X and R.sup.Y are H; X is O; L is selected
from C.sub.1-C.sub.6 alkylene and
--((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally
substituted with 1 to 2 halogen atoms; p is selected from 1, 2 and
3; q is selected from 1 and 2; and n is 1. Thus in these
embodiments the adsorptive moiety comprises a phosphate group.
[0193] Other useful TLR agonists of formula (Al) are disclosed on
pages 6-13 of reference 98.
[0194] Compositions can include an imidazoquinolone compound, such
as Imiquimod ("R-837") [99,100], Resiquimod ("R-848") [101], and
their analogs; and salts thereof (e.g. the hydrochloride salts).
Further details about immunostimulatory imidazoquinolines can be
found in references 102 to 106.
[0195] Compositions can include a TLR4 agonist, and most preferably
an agonist of human TLR4. TLR4 is expressed by cells of the innate
immune system, including conventional dendritic cells and
macrophages [107]. Triggering via TLR4 induces a signalling cascade
that utilizes both the MyD88 - and TRIF-dependent pathways, leading
to NF-.kappa.B and IRF3/7 activation, respectively. TLR4 activation
typically induces robust IL-12p70 production and strongly enhances
Th1-type cellular and humoral immune responses.
[0196] Various useful TLR4 agonists are known in the art, many of
which are analogs of endotoxin or lipopolysaccharide (LPS). For
instance, the TLR4 agonist can be: 3d-MPL (i.e. 3-O-deacylated
monophosphoryl lipid A; present in GSK's `AS04` adjuvant, with
further details in references 108 to 111 glucopyranosyl lipid A
(GLA) [112] or its ammonium salt; an aminoalkyl glucosaminide
phosphate, such as RC-529 or CRX-524 [113-115]; E5564 [116,117]; or
a compound of formula I, II or III as defined in reference 118, or
a salt thereof, such as compounds `ER 803058`, `ER 803732`, `ER
804053`, `ER 804058`, `ER 804059`, `ER 804442`, `ER 804680`, `ER
803022`, `ER 804764` or `ER 804057` (also known as E6020).
[0197] The invention is particularly useful when using human TLR7
agonists, such as a compound of formula (K). These agonists are
discussed in detail in reference 119:
##STR00002##
[0198] wherein: [0199] R.sup.1 is H, C.sub.1-C.sub.6alkyl,
--C(R.sup.5).sub.2OH, -L.sup.1R.sup.5, -L.sup.1R.sup.6,
-L.sup.2R.sup.5, -L.sup.2R.sup.6, -0L.sup.2R.sup.5, or
-0L.sup.2R.sup.6; [0200] L.sup.1 is --C(O)-- or --O--; [0201]
L.sup.2 is C.sub.1-C.sub.6alkylene, C.sub.2-C.sub.6alkenylene,
arylene, heteroarylene or
--((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p--, wherein the
C.sub.1-C.sub.6alkylene and C.sub.2-C.sub.6alkenylene of L.sup.2
are optionally substituted with 1 to 4 fluoro groups; [0202] each
L.sup.3 is independently selected from C.sub.1-C.sub.6alkylene and
--((CR.sup.4R.sup.4).sub.pO).sub.q(CH.sub.2).sub.p--, wherein the
C.sub.1-C.sub.6alkylene of L.sup.3 is optionally substituted with 1
to 4 fluoro groups; [0203] L.sup.4 is arylene or heteroarylene;
[0204] R.sup.2 is H or C.sub.1-C.sub.6alkyl; [0205] R.sup.3 is
selected from C.sub.1-C.sub.4alkyl, -L.sup.3R.sup.5,
-L.sup.1R.sup.5, -L.sup.3R.sup.7, -L.sup.3L.sup.4L.sup.3R.sup.7,
-L.sup.3L.sup.4R.sup.5, -L.sup.3L.sup.4L.sup.3R.sup.5, -
OL.sup.3R.sup.5, -OL.sup.3R.sup.7, -OL.sup.3L.sup.4R.sup.7,
-OL.sup.3L.sup.4L.sup.3R.sup.7, --OR.sup.8,
-OL.sup.3L.sup.4R.sup.5, -OL.sup.3L.sup.4L.sup.3R.sup.5 and
--C(R.sup.5).sub.2OH; [0206] each R.sup.4 is independently selected
from H and fluoro; [0207] R.sup.5 is --P(O)(OR.sup.9).sub.2, [0208]
R.sup.6 is --CF.sub.2P(O)(OR.sup.9).sub.2 or --C(O)OR.sup.10;
[0209] R.sup.7 is --CF.sub.2P(O)(O.sup.9).sub.2 or --C(O)OR.sup.10;
[0210] R.sup.8 is H or C.sub.1-C.sub.4alkyl; [0211] each R.sup.9 is
independently selected from H and C.sub.1-C.sub.6alkyl; [0212]
R.sup.10 is H or C.sub.1-C.sub.4alkyl; [0213] each p is
independently selected from 1, 2, 3, 4, 5 and 6, and
[0214] q is 1, 2, 3 or 4.
[0215] The compound of formula (K) is preferably of formula
(K'):
##STR00003##
wherein: [0216] P.sup.1 is selected from H, C.sub.1-C.sub.6alkyl
optionally substituted with COOH and
--Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0217] P.sup.2 is selected from
H, C.sub.1-C.sub.6alkyl, C.sub.1-C.sub.6alkoxy and
--Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0218] with the proviso that at
least one of P.sup.1 and P.sup.2 is
--Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); [0219] R.sup.B is selected from
H and C.sub.1-C.sub.6alkyl; [0220] R.sup.X and R.sup.Y are
independently selected from H and C.sub.1-C.sub.6alkyl; [0221] X is
selected from a covalent bond, O and NH; [0222] Y is selected from
a covalent bond, O, C(O), S and NH; [0223] L is selected from, a
covalent bond C.sub.1-C.sub.6alkylene, C.sub.1-C.sub.6alkenylene,
arylene, heteroarylene, C.sub.1-C.sub.6alkyleneoxy and
--((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally
substituted with 1 to 4 substituents independently selected from
halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and
'P(O)(OH).sub.2; [0224] each p is independently selected from 1, 2,
3, 4, 5 and 6; and [0225] q is selected from 1, 2, 3 and 4.
[0226] In some embodiments of formula (K'): P.sup.1 is selected
from C.sub.1-C.sub.6alkyl optionally substituted with COOH and
--Y-L-X--P(O)(OR.sup.X)(OR.sup.Y); P.sup.2 is selected from
C.sub.1-C.sub.6alkoxy and --Y-L-X--P(O)(OR.sup.X)(OR.sup.Y);
R.sup.B is C.sub.1-C.sub.6alkyl; X is a covalent bond; L is
selected from C.sub.1-C.sub.6alkylene and
--((CH.sub.2).sub.pO).sub.q(CH.sub.2).sub.p-- each optionally
substituted with 1 to 4 substituents independently selected from
halo, OH, C.sub.1-C.sub.4alkyl, --OP(O)(OH).sub.2 and
--P(0)(OH).sub.2; each p is independently selected from 1, 2 and 3;
q is selected from 1 and 2.
[0227] A preferred compound of formula (K) for use with the
invention is 3-(5-amino-2-(2-methyl-4-(2-(2-(2-phosphonoethoxy)
ethoxy)ethoxy)phenethyl)benzo[f][1,7]naphthyridin-8-yl)propanoic
acid, or compound `K1`:
##STR00004##
This compound can be used as free base or in the form of a
pharmaceutically acceptable salt e.g. an arginine salt [120].
[0228] F. Microparticles
[0229] Microparticles may also be used as adjuvants in the
invention. Microparticles (i.e. a particle of .about.100 nm to
.about.150 .mu.m in diameter, more preferably .about.200 nm to
.about.30 .mu.m in diameter, and most preferably .about.500 nm to
.about.10 .mu.m in diameter) formed from materials that are
biodegradable and non-toxic (e.g. a poly(.alpha.-hydroxy acid), a
polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a
polycaprolactone, etc.), with poly(lactide-co-glycolide) are
preferred, optionally treated to have a negatively-charged surface
(e.g. with SDS) or a positively-charged surface (e.g. with a
cationic detergent, such as CTAB).
Adjuvant Combinations
[0230] The individual adjuvants listed above may also be included
in combinations. For instance, a combination of an aluminium
hydroxide and an aluminium phosphate adjuvant can be used.
Similarly, a combination of aluminium phosphate and 3 dMPL may be
used.
[0231] A particularly preferred adjuvant combination is an
insoluble metal salt (e.g. an aluminium salt, such as an aluminium
hydroxide) and a TLR agonist (e.g. a human TLR7 agonist, such as
compound `K1` identified above), as disclosed in references 5 and
97. So, in particular, said adjuvant is selected from the group
consisting of: [0232] aluminium salts, particularly aluminium
hydroxides and aluminium phosphates; [0233] human TLR agonists,
particularly TLR7 agonists; and [0234] a mixture thereof.
[0235] The TLR agonist is preferably adsorbed to the metal salt,
and the S.aureus antigen(s) can also be adsorbed to the metal
salt.
[0236] A composition including a TLR agonist of the invention
adsorbed to a metal salt can also include a buffer (e.g. a
phosphate or a histidine or a Tris buffer). When such a composition
includes a phosphate buffer, however, it is preferred that the
concentration of phosphate ions in the buffer should be less than
50 mM e.g. <40 mM, <30 mM, <20 mM, <10 mM, or <5 mM,
or between 1-15 mM. A histidine buffer is preferred e.g. between
1-50 mM, between 5-25 mM, or about 10 mM.
[0237] A composition can include a mixture of both an aluminium
oxyhydroxide and an aluminium hydroxyphosphate, and a TLR agonist
may be adsorbed to one or both of these salts.
[0238] As mentioned above, a maximum of 0.85 mg/dose Al.sup.+++ is
preferred. Because the inclusion of a TLR agonist can improve the
adjuvant effect of aluminium salts then the invention
advantageously permits lower amounts of Al.sup.+++ per dose, and so
a composition can usefully include between 10 and 250.mu.g of
Al.sup.+++ per unit dose. Current pediatric vaccines typically
include at least 300 .mu.g Al.sup.+++. In concentration terms, a
composition may have an Al.sup.+++ concentration between 10 and 500
.mu.g/ml e.g. between 10-300 .mu.g/ml, between 10-200 .mu.g/ml, or
between 10-100 .mu.g/ml.
[0239] In general, when a composition includes both a TLR agonist
and an aluminium salt, the weight ratio of agonist to Al.sup.+++
will be less than 5:1 e.g. less than 4:1, less than 3:1, less than
2:1, or less than 1:1. Thus, for example, with an Al.sup.+++
concentration of 0.5 mg/ml the maximum concentration of TLR agonist
would be 1.5 mg/ml. But higher or lower levels can be used.
[0240] Where a composition includes a TLR agonist and an insoluble
metal salt, it is preferred that at least 50% (by mass) of the
agonist in the composition is adsorbed to the metal salt e.g.
.gtoreq.60%, .gtoreq.70%, .gtoreq.80%, .gtoreq.85%, .gtoreq.90%,
.gtoreq.92%, .gtoreq.94%, .gtoreq.95%, .gtoreq.96%, .gtoreq.97%,
.gtoreq.98%, .gtoreq.99%, or even 100%.
[0241] Thus, in one embodiment, the invention uses an immunogenic
composition comprising: [0242] an aluminium hydroxide adjuvant;
[0243] a TLR7 agonist of formula (K), such as compound K1; [0244] a
first polypeptide comprising SEQ ID NO: 6, or a modified amino acid
sequence which differs from SEQ ID NO: 6 by up to 5 single amino
changes provided that the modified sequence can elicit antibodies
which bind to a polypeptide consisting of SEQ ID NO: 6; [0245] a
second polypeptide comprising SEQ ID NO: 13, or a modified amino
acid sequence which differs from SEQ ID NO: 13 by up to 5 single
amino changes provided that the modified sequence can elicit
antibodies which bind to a polypeptide consisting of SEQ ID NO: 13;
[0246] a third polypeptide comprising SEQ ID NO: 31, or a modified
amino acid sequence which differs from SEQ ID NO: 31 by up to 5
single amino changes provided that the modified sequence can elicit
antibodies which bind to a polypeptide consisting of SEQ ID NO: 31;
[0247] a fourth polypeptide comprising SEQ ID NO: 33, or a modified
amino acid sequence which differs from SEQ ID NO: 33 by up to 5
single amino changes provided that the modified sequence can elicit
antibodies which bind to a polypeptide consisting of SEQ ID NO: 33;
and [0248] a fifth polypeptide comprising SEQ ID NO: 45, or a
modified amino acid sequence which differs from SEQ ID NO: 45 by up
to 5 single amino changes provided that the modified sequence can
elicit antibodies which bind to a polypeptide consisting of SEQ ID
NO: 43; and, [0249] in which the TLR7 agonist and/or at least one
of the polypeptides is/are adsorbed to the aluminium hydroxide
adjuvant.
[0250] For example, as explained in more detail elsewhere herein:
the first polypeptide can comprise SEQ ID NO: 34; the second
polypeptide can comprise SEQ ID NO: 13; the third polypeptide can
comprise SEQ ID NO: 40; the fourth polypeptide can comprise SEQ ID
NO: 36; and the fifth polypeptide can comprise SEQ ID NO: 45,
optionally modified by up to 3 amino acid substitutions (other than
at positions which are X in SEQ ID NO: 44). Thus the composition
can use a mixture of five polypeptides having SEQ ID NOs: 37, 27,
38, 39, and 45 (except that SEQ ID NO: 45 can be modified by up to
3 amino acid substitutions as discussed above).
Chemical Groups
[0251] Unless specifically defined elsewhere, the chemical groups
discussed herein have the following meaning when used in present
specification:
[0252] The term "alkyl" includes saturated hydrocarbon residues
including: [0253] linear groups up to 10 atoms (C.sub.1-C.sub.10),
or of up to 6 atoms (C.sub.1-C.sub.6), or of up to 4 atoms
(C.sub.1-C.sub.4). Examples of such alkyl groups include, but are
not limited, to C.sub.1- methyl, C.sub.2- ethyl, C.sub.3- propyl
and C.sub.4- n-butyl. [0254] branched groups of between 3 and 10
atoms (C.sub.3-C.sub.10), or of up to 7 atoms (C.sub.3-C.sub.7), or
of up to 4 atoms (C.sub.3-C.sub.4). Examples of such alkyl groups
include, but are not limited to, C.sub.3- iso-propyl, C.sub.4
-sec-butyl, C.sub.4 - iso-butyl, C.sub.4 - tert-butyl and C.sub.5 -
neo-pentyl.
[0255] The term "alkylene" refers to the divalent hydrocarbon
radical derived from an alkyl group, and shall be construed in
accordance with the definition above.
[0256] The term "alkenyl" includes monounsaturated hydrocarbon
residues including: [0257] linear groups of between 2 and 6 atoms
(C.sub.2-C.sub.6). Examples of such alkenyl groups include, but are
not limited to, C.sub.2-vinyl, C.sub.3-1-propenyl, C.sub.3-allyl,
C.sub.4-2-butenyl [0258] branched groups of between 3 and 8 atoms
(C.sub.3-C.sub.8). Examples of such alkenyl groups include, but are
not limited to, C.sub.4- 2-methyl-2-propenyl and C.sub.6-
2,3-dimethyl-2-butenyl.
[0259] The term alkenylene refers to the divalent hydrocarbon
radical derived from an alkenyl group, and shall be construed in
accordance with the definition above.
[0260] The term "alkoxy" includes O-linked hydrocarbon residues
including: [0261] linear groups of between 1 and 6 atoms
(C.sub.1-C.sub.6), or of between 1 and 4 atoms (C.sub.1-C.sub.4).
Examples of such alkoxy groups include, but are not limited to,
C.sub.1- methoxy, C.sub.2- ethoxy, C.sub.3- n-propoxy and C.sub.4-
n-butoxy. [0262] branched groups of between 3 and 6 atoms
(C.sub.3-C.sub.6) or of between 3 and 4 atoms
(C.sub.3-C.sub.4).
[0263] Examples of such alkoxy groups include, but are not limited
to, C.sub.3- iso-propoxy, and C.sub.4- sec-butoxy and
tert-butoxy.
[0264] Halo is selected from Cl, F, Br and I. Halo is preferably
F.
[0265] The term "aryl" includes a single or fused aromatic ring
system containing 6 or 10 carbon atoms; wherein, unless otherwise
stated, each occurrence of aryl may be optionally substituted with
up to 5 substituents independently selected from
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, OH, halo, CN,
COOR.sup.14, CF.sub.3 and NR.sup.14R.sup.15; as defined above.
Typically, aryl will be optionally substituted with 1, 2 or 3
substituents. Optional substituents are selected from those stated
above. Examples of suitable aryl groups include phenyl and naphthyl
(each optionally substituted as stated above). Arylene refers the
divalent radical derived from an aryl group, and shall be construed
in accordance with the definition above.
[0266] The term "heteroaryl" includes a 5, 6, 9 or 10 membered
mono- or bi-cyclic aromatic ring, containing 1 or 2 N atoms and,
optionally, an NR.sup.14 atom, or one NR.sup.14 atom and an S or an
O atom, or one S atom, or one O atom; wherein, unless otherwise
stated, said heteroaryl may be optionally substituted with 1, 2 or
3 substituents independently selected from (C.sub.1-C.sub.6)alkyl,
(C.sub.1-C.sub.6)alkoxy, OH, halo, CN, COOR.sup.14, CF.sub.3 and
NR.sup.14R.sup.15; as defined below. Examples of suitable
heteroaryl groups include thienyl, furanyl, pyrrolyl, pyrazolyl,
imidazoyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl,
triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl,
pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, benzimidazolyl,
benzotriazolyl, quinolinyl and isoquinolinyl (optionally
substituted as stated above). Heteroarylene refers the divalent
radical derived from heteroaryl, and shall be construed in
accordance with the definition above.
[0267] The term "heterocyclyl" is a C-linked or N-linked 3 to 10
membered non-aromatic, mono- or bi-cyclic ring, wherein said
heterocycloalkyl ring contains, where possible, 1, 2 or 3
heteroatoms independently selected from N, NR.sup.14, S(O).sub.q
and O; and said heterocycloalkyl ring optionally contains, where
possible, 1 or 2 double bonds, and is optionally substituted on
carbon with 1 or 2 substituents independently selected from
(C.sub.1-C.sub.6)alkyl, (C.sub.1-C.sub.6)alkoxy, OH, CN, CF.sub.3,
halo, COOR.sup.14, NR.sup.14R.sup.15 and aryl.
[0268] In the above definitions R.sup.14 and R.sup.15 are
independently selected from H and (C.sub.1-C.sub.6)alkyl.
[0269] When a structural formula is defined with a substituent
attached to the core of the molecule by an unspecified, or
"floating" bond, for example, as for the group P.sup.3 in the case
of formula (C), this definition encompasses the cases where the
unspecified substituent is attached to any of the atoms on the ring
in which the floating bond is located, whilst complying with the
allowable valence for that atom.
[0270] In the case of compounds of the invention which may exist in
tautomeric forms (i.e. in keto or enol forms), for example the
compounds of formula (C) or (H), reference to a particular compound
optionally includes all such tautomeric forms.
General
[0271] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., references 121-128, etc. "GI" numbering
is used above. A GI number, or "GenInfo Identifier", is a series of
digits assigned consecutively to each sequence record processed by
NCBI when sequences are added to its databases. The GI number bears
no resemblance to the accession number of the sequence record. When
a sequence is updated (e.g. for correction, or to add more
annotation or information) then it receives a new GI number. Thus
the sequence associated with a given GI number is never
changed.
[0272] Where the invention concerns an "epitope", this epitope may
be a B-cell epitope and/or a T-cell epitope. Such epitopes can be
identified empirically (e.g. using PEPSCAN [129,130] or similar
methods), or they can be predicted (e.g. using the Jameson-Wolf
antigenic index [131], matrix-based approaches [132], MAPITOPE
[133], TEPITOPE [134,135], neural networks [136], OptiMer &
EpiMer [137, 138], ADEPT [139], Tsites [140], hydrophilicity [141],
antigenic index [142] or the methods disclosed in references 143
-147, etc.). Epitopes are the parts of an antigen that are
recognised by and bind to the antigen binding sites of antibodies
or T-cell receptors, and they may also be referred to as "antigenic
determinants".
[0273] Where an antigen "domain" is omitted, this may involve
omission of a signal peptide, of a cytoplasmic domain, of a
transmembrane domain, of an extracellular domain, etc.
[0274] The term "comprising" encompasses "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional e.g. X+Y.
[0275] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0276] References to a percentage sequence identity between two
amino acid sequences means that, when aligned, that percentage of
amino acids are the same in comparing the two sequences. This
alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example
those described in section 7.7.18 of ref. 148. A preferred
alignment is determined by the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is disclosed in ref. 149.
Percentage identity to any particular sequence (e.g. to a
particular SEQ ID) is ideally calculated over the entire length of
that sequence.
[0277] Binding affinity may be determined by any method known in
the art, including surface plasmon resistance, isothermal titration
calorimetry, competitive binding assays, thermal shift assay, etc.
Although the absolute figures obtained using different methods may
vary, it is envisaged that the determination relative binding
affinity of one protein compared to another should not depend on
the method used.
[0278] Phosphorous-containing adjuvants used with the invention may
exist in a number of protonated and deprotonated forms depending on
the pH of the surrounding environment, for example the pH of the
solvent in which they are dissolved. Therefore, although a
particular form may be illustrated, it is intended that these
illustrations are merely representative and not limiting to a
specific protonated or deprotonated form. For example, in the case
of a phosphate group, this has been illustrated as
--OP(O)(OH).sub.2 but the definition includes the protonated forms
[OP(O)(OH.sub.2)(OH)].sup.+ and --[OP(O)(OH).sub.2].sup.2+ that may
exist in acidic conditions and the deprotonated forms
--[OP(O)(OH)(O)].sup.- and [OP(O)(O).sub.2].sup.2- that may exist
in basic conditions.
[0279] Compounds can exist as pharmaceutically acceptable salts.
Thus, compounds (e.g. adjuvants) may be used in the form of their
pharmaceutically acceptable salts i.e. physiologically or
toxicologically tolerable salt (which includes, when appropriate,
pharmaceutically acceptable base addition salts and
pharmaceutically acceptable acid addition salts).
[0280] The word "substantially" does not exclude "completely" e.g.
a composition which is "substantially free" from Y may be
completely free from Y. Where necessary, the word "substantially"
may be omitted from the definition of the invention.
MODES FOR CARRYING OUT THE INVENTION
SpAkR mutant
[0281] SpA is a crucial virulence factor in S. aureus and it acts
by interfering with opsonophagocytic clearance of the bacterium and
by ablating adaptive immune responses. The wild-type sequence SEQ
ID NO: 43 includes five Ig-binding domains (IgBD), as shown above,
and it is known to mutate amino acid residues within these domains
to abolish Fc/Fab binding activity while retaining immunogenicity
e.g. see reference Error! Bookmark not defined., which replaces
Gln-Gln dipeptides with Lys-Lys and/or replaces Asp-Asp dipeptides
with Ala-Ala.
[0282] A new mutant SpA has been produced in which a further
Gln-Gln dipeptide is mutated to Lys-Arg. This dipeptide was
identified based on bioinformatics analysis and predictions of an
additional site involved in Ig binding. In particular, residues 96
& 97 of SEQ ID NO: 43 were hypothesised to be involved in Ig
binding. This site was overlooked in earlier work because it is
outside the conserved IgBD and is not part of the solved
structures. The new mutant is referred to as `SpAkR` and it has the
following amino acid sequence, where the extra QQ/KR substitution
relative to the previous `SpAkkAA` mutant is boxed:
TABLE-US-00003 (SEQ ID NO: 48) ##STR00005##
[0283] The SpAkR mutant has been confirmed to have reduced affinity
for immunoglobulins relative to known mutants, while retaining
immunogenicity.
[0284] CD and DSC analysis showed that the introduction of the KR
mutant did not materially affect the structure of SpA. The CD
spectra of SpAkkAA and SpAKR were identical. T.sub.m1 and T.sub.m2
from DSC analysis were as follows. T.sub.m1: SpAkkAA 48.9.degree.
C.; SpAKR 51.1.degree. C. T.sub.m2: SpAkkAA 68.1.degree. C.; SpAKR
68.0.degree. C.
[0285] Affinity of wild-type SpA, and of the SpAkkAA and SpAkR
mutants, for human IgG, IgA and IgM was tested using surface
plasmon resonance. The proteins were immobilized on a sensor chip
and human IgG, IgA and IgM were used as analytes. Both mutants
displayed a greatly reduced binding capacity compared to wild-type,
but while SpAkkAA showed residual binding to IgG and IgM, no
detectable interactions with any immunoglobulins were seen with
SpAkR. Residual binding activities of SpAkkAA towards IgG and IgM
were very low (11-12-fold lower than wild-type), but reproducible
and concentration-dependent.
[0286] S. aureus survival in the presence of SpA (WT, SpAkkAA and
SpAkR) was assessed through whole blood survival assay (WBA). 1 ml
whole blood from healthy donors, supplemented with 50 mg/l
lepirudin anticoagulant (10 .mu.l/ml blood), was incubated with
0.15 .mu.M SpA or with PBS, for 15 min at 37.degree. C., before
adding approximately 2.5.times.10.sup.5 CFU of S. aureus USA300 LAC
(OD.sub.600, 0.4) diluted in BHI. Aliquots of this culture were
plated on BHI-agar to determine the input CFU. After incubation at
37.degree. C. for 2 h with shaking (180 RPM), neutrophils were
lysed at 0.5% saponin-PBS for 3 min on ice. The number of viable
bacteria was determined by serial tenfold dilutions in BHI and
plating on nutrient agar plates. Colonies were counted after
incubation of the plates at 37.degree. C. for 18 h.
[0287] Control was the blood sample preincubated with PBS. Relative
survival was calculated based on the input CFU compared to CFU
post-incubation. Experiments were performed in triplicate; there
was little variation between the experiments.
[0288] In the WBA, relative survival of S aureus in human whole
blood was considerably lower when incubated with SpAKR than with
wild type SpA (p<0.001, Mann-Whitney) or with SpAkkAA
(p<0.05). Incubation with SpAwt resulted in increased S aureus
survival compared to control (p<0.001), whereas survival in WBA
incubated with SpAkkAA was comparable to control. Incubation with
SpAkR reduced survival to about half of that of the control.
[0289] SpAkkAA or SpAkR formulated with aluminium hydroxide
adjuvant were found to be weakly immunogenic in mice on their own,
but inclusion of adsorbed TLR7 agonist `K1` significantly increased
antibody titres. Because the mechanism of action associated with
SpA immunisation appears to be mainly driven by antibodies, this is
an important improvement.
S.aureus vaccines
[0290] SpAkR, SpAkkAA, SpAkR E domain alone and SpAkR E domain
fused to HlaH35L were tested in the renal abscess model, adjuvanted
with aluminium hydroxide (Al-H) at 2 mg/ml (total salt). Antigens
were each present at 0 .mu.g in a 100 .mu.L, dose for intramuscular
injection.
[0291] Four or five-week old mice (CD1) were immunized
intramuscularly (IM) with prime-booster injections with a 14-day
interval. Control mice received equal amounts of adjuvants alone.
Serum was collected from mice both pre- and post-vaccination to
document serum antibody titers to each protein component in the
combination vaccine. These titers were measured by Luminex
technology using the recombinant vaccine antigens conjugated to
microspheres.
[0292] Renal abscess model: Immunized animals were challenged on
day 24 by intravenous injection of a sublethal dose of S.aureus
Newman strain (.about.2-6.times.10.sup.7 CFU). On day 28, mice were
euthanized and kidneys were removed and homogenized in 2 mL of PBS
and plated on agar media in duplicate for determination of colony
forming units (CFU).
[0293] A comparable reduction in log.sub.10 CFU/ml (around 1 log
reduction) was obtained on vaccination with SpAKR, SpAkkAA, SpAkR E
domain alone and SpAkR E domain fused to HlaH35L, compared to
adjuvant alone.
Combination S.aureus Vaccines
[0294] A 5-valent and a 6-valent vaccine were prepared. The
5-valent vaccine included antigens consisting of SEQ ID NOs: 7, 8,
27 and 32 (FhuD2, Sta011, Hla-H35L, and EsxAB); the 6-valent
vaccine also included the SpAkR mutant. The vaccines were
adjuvanted with: (i) aluminium hydroxide, Al-H; (ii) Al-H +adsorbed
TLR7 agonist K1; or (iii) the oil-in-water emulsion MF59. Al-H was
used at 2 mg/ml (total salt), K1 was present at 50 .mu.g per dose,
and MF59 was mixed with the antigens at a 1:1 volume ratio.
Antigens were each present at 10 .mu.g in a 100 .mu.L dose for
intramuscular injection.
[0295] Four or five-week old mice (CD1) were immunized with
prime-booster injections with a 14-day interval. Control mice
received equal amounts of adjuvants alone. Serum was collected from
mice both pre- and post-vaccination to document serum antibody
titers to each protein component in the combination vaccine. These
titers were measured by Luminex technology using the recombinant
vaccine antigens conjugated to microspheres.
[0296] Renal abscess model: Immunized animals were challenged on
day 24 by intravenous injection of a sublethal dose of S.aureus
(.about.2-6.times.10.sup.7 CFU, where the specific inoculum varied
depending on the challenge strain). On day 28, mice were euthanized
and kidneys were removed and homogenized in 2 mL of PBS and plated
on agar media in duplicate for determination of colony forming
units (CFU). Kidneys were also processed for histopathology.
[0297] Peritonitis model: Separately, immunized animals were
challenged on day 24 by intraperitoneal injection of a lethal dose
of S.aureus . (.about.2-5.times.10.sup.8 CFU) and then monitored
daily for 14 days.
[0298] Skin infection model: Immunized mice were inoculated by
subcutaneous injection in the shaved right flank with
2.times.10.sup.7 CFU S.aureus LAC strain (USA300 clone, which is
one of the most important clones worldwide and highly associated to
community-acquired cutaneous infections). Mass and abscess
formation (size and dermonecrosis) were monitored at 24-hour
intervals over a course of 7 days. The size of an abscess and
associated overlying dermonecrotic lesion was determined using
image analysis software. Mouse skin and abscesses were harvested on
day 7 post inoculation for CFU enumeration. This model was used
only with the Al-H/K1 adjuvant.
[0299] Results were as follows:
TABLE-US-00004 Adjuvant alone 5-valent 6-valent Al--H Al--H/K1 MF59
Al--H Al--H/K1 MF59 Al--H Al--H/K1 MF59 Renal abscess - log
reduction in median cfu/ml A -- -- -- 1.5** 1.3** 0.93** 1.57**
2.31** 1.07** Peritonitis - % survival B 29 42 33 67* 88** 67* 83**
92** 71** Skin infection - log reduction in median cfu/ml C N/A --
-- -- 3.35 -- -- 4.39 -- *p .ltoreq. 0.05 compared to control; **p
< 0.01 compared to control
[0300] These results demonstrate that the mutant SpAkR improves the
5-valent Combo-1 product in both models. Furthermore, the best
results were seen using the Al-H/K1 adjuvant. Strikingly, results
in the renal abscess model the 6-valent vaccine with Al-H/K1
approximated to sterility.
[0301] In the peritonitis model, the 6-valent vaccine with Al-H/K1
was statistically superior when compared to the negative control
(see table above), and also when compared to the 5-valent Al-H
vaccine. In the abscess model, the 6-valent vaccine with Al-H/K1
was statistically superior when compared to the negative control
(see table above), to the 5-valent Al-H vaccine, and to the
5-valent Al-H/K1 vaccine, thus showing that the contribution of the
mutant SpA goes beyond the enhancement which was due solely to the
K1 agonist.
[0302] In the skin infection model, the 5-valent and 6-valent
vaccines both significantly reduced abscess formation and CFU
counts (see table above). Dermonecrosis was absent in the
vaccinated mice while it was observed in all mice that received
adjuvant alone (`N/A` in table). Furthermore, CFU reduction was
significantly improved by the inclusion of SpAkR in the vaccine,
and fewer mice were observed with distinguishable abscesses at a
macroscopic level (71% vs. 88%).
[0303] Anti-SpA titers were also compared for the three adjuvants.
Median titers using Al-H or MF59 were not significantly different,
but the titer using Al-H/K1 was significantly higher than with Al-H
alone (p=0.0047, Mann-Whitney test) and than with MF59 (p=0.01).
Thus the formulation which performed best in the functional assays
was the one which elicited the highest anti-SpA antibody titres, in
line with the hypothesis that SpA's activity as a vaccine antigen
depends on antibodies, which can bind it and inhibit its
immune-evasion activity.
[0304] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
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TABLE-US-00005 SEQUENCE LISTING SEQ ID NO: 1 (EsxA)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQ SEQ ID NO: 2 (EsxB)
MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELV
QPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 3 (FhuD2)
MKKLLLPLIIMLLVLAACGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVA
VNQQVDQSKVLKDKFKGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVVVDYNKHKYLEQQE
MLGKIVGKEDKVKAWKKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGL
KMQPEQQKLTAKAGWAEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWY
NDPYTLDFMRKDLKEKLIKAAK SEQ ID NO: 4 (Sta011)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKIKKEIENFKFFVQYGDFKNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 5 (Hla)
MKTRIVSSVTTTLLLGSILMNPVANAADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFID
DKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMS
TLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDR
DSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRD
DYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 6 (N-terminally
truncated FhuD2 of SEQ ID NO: 3)
CGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKG
VTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKK
DWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAE
VKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKL
IKAAK SEQ ID NO: 7 (Example FhuD2 sequence)
MASCGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDK
FKGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKA
WKKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAG
WAEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLK
EKLIKAAK SEQ ID NO: 8 (Example Sta011 sequence)
MGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMV
LYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDF
KNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEF
TFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 9 (Example Sta011 sequence)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKLKKEIENFKFFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 10
(Example Sta011 sequence)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKVKKEIENFKFFVQYGDFKNIKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 11
(Example Sta011 sequence)
MMKRLNKLVLGIIFLFLVISITAGCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGT
WIINSEMVIQPNNEDMVAKGMVLYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKE
IKDEKLKKEIENFKFFVQYGDFKNVKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAP
KLLLKGSGNLKGSSVGYKDIEFTFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 12
(N-terminally truncated Hla SEQ ID NO: 5)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 13 (Mature Hla-H35L)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 14 (tetramer) PSGS SEQ ID NO: 15 (Hla-H35L
with PSGS substitution)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMV
NQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNI
DVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 16 (Hla
with PSGS substitution)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTPSGSVQPDFKTILESPTDKKVGWKVIFNNMV
NQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNI
DVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSERYKIDWEKEEMTN SEQ ID NO: 17 (Hla
with Y101 mutation)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 18 (Hla with D152 mutation)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPLFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 19 (Hla with H35 and Y101 mutations)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDLYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 20 (Hla with H53 and D152 mutations)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSE
EGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANV
SIGHTLKYVQPLFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNF
LDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDRSSE
RYKIDWEKEEMTN SEQ ID NO: 21 (Hla fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNK SEQ ID NO: 22
(Hla fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAG SEQ
ID NO: 23 (Hla fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLL SEQ ID NO: 24
(Hla-H35L fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNK SEQ ID NO: 25
(Hla-H35L fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAG SEQ
ID NO: 26 (Hla-H35L fragment)
ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLL SEQ ID NO: 27
(Useful HLA sequence with H35L mutation)
MASADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMLKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRV
YSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIG
ANVSIGHTLKYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAA
DNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWIDR
SSERYKIDWEKEEMTN SEQ ID NO: 28 (EsxAB example)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSMGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQ
LAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP
SEQ ID NO: 29 (EsxBA example)
MGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELV
QPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNPASGGGSMAMIKMSPEEIRAKSQSYGQGSDQIRQILS
DLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEEIKQQLNSTADAVQEQDQQLSNNFGLQ
SEQ ID NO: 30 (linker) ASGGGS SEQ ID NO: 31 (EsxAB example)
AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEE
IKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQLA
EYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP
SEQ ID NO: 32 (EsxAB example)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEACQKQTQQL
AEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP
SEQ ID NO: 33 (N-terminally truncated SEQ ID NO: 4)
GCGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMVL
YMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDFK
NLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFT
FVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 34 (FhuD2)
GNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKFKGV
TKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAWKKD
WEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGWAEV
KQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKEKLI
KAAK SEQ ID NO: 35 (Cys-free EsxB sequence)
GGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLAEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQ
PMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP SEQ ID NO: 36 (Cys-free version
of Sta011 SEQ ID NO: 4)
GIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMVLYM
NRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDFKNL
KNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEFTFV
EKKEENIYFSDSLDYKKSGDV SEQ ID NO: 37 (Cys-free version of FhuD2 SEQ
ID NO: 7)
MASGNQGEKNNKAETKSYKMDDGKTVDIPKDPKRIAVVAPTYAGGLKKLGANIVAVNQQVDQSKVLKDKF
KGVTKIGDGDVEKVAKEKPDLIIVYSTDKDIKKYQKVAPTVVVDYNKHKYLEQQEMLGKIVGKEDKVKAW
KKDWEETTAKDGKEIKKAIGQDATVSLFDEFDKKLYTYGDNWGRGGEVLYQAFGLKMQPEQQKLTAKAGW
AEVKQEEIEKYAGDYIVSTSEGKPTPGYESTNMWKNLKATKEGHIVKVDAGTYWYNDPYTLDFMRKDLKE
KLIKAAK SEQ ID NO: 38 (EsxAB Cys-Ala mutant)
MAMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLE
EIKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAAQKQTQQL
AEYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP
SEQ ID NO: 39 (useful Sta011 sequence)
MGSGIGKEAEVKKSFEKTLSMYPIKNLEDLYDKEGYRDDQFDKNDKGTWIINSEMVIQPNNEDMVAKGMV
LYMNRNTKTTNGYYYVDVTKDEDEGKPHDNEKRYPVKMVDNKIIPTKEIKDEKIKKEIENFKFFVQYGDF
KNLKNYKDGDISYNPEVPSYSAKYQLTNDDYNVKQLRKRYDIPTSKAPKLLLKGSGNLKGSSVGYKDIEF
TFVEKKEENIYFSDSLDYKKSGDV SEQ ID NO: 40 (Cys-free EsxAB example)
AMIKMSPEEIRAKSQSYGQGSDQIRQILSDLTRAQGEIAANWEGQAFSRFEEQFQQLSPKVEKFAQLLEE
IKQQLNSTADAVQEQDQQLSNNFGLQASGGGSGGYKGIKADGGKVDQAKQLAAKTAKDIEAXQKQTQQLA
EYIEGSDWEGQFANKVKDVLLIMAKFQEELVQPMADHQKAIDNLSQNLAKYDTLSIKQGLDRVNP
SEQ ID NO: 41 icicicicicicicicicicicicic SEQ ID NO: 42 KLKLLLLLKLK
SEQ ID NO: 43 (SpA)
MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKD
DPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVL
GEAKKLNESQAPKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLSEAKKLNESQAP
KADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSANLLAEAKKLNDAQAPKADNKFNKEQQN
AFYEILHLPNLTEEQRNGFIQSLKDDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDNNKPGKEDNNKP
GKEDNNKPGKEDNNKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDGNKPGKEDGN
GVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKPGQELVVDKKQPANHADANKAQALPETGEENP
FIGTTVFGGLSLALGAALLAGRRREL SEQ ID NO: 44 (SpAkkAA)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDXXS
AFYEILNMPNLNEAQRNGFIQSLKXXPSQSTNVLGEAKKLNESQAPKADNNFNKEXXNAFYEILNMPNLN
EEQRNGFIQSLKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLPNLNEEQRNGFIQSLK
XXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEILHLPNLTEEQRNGFIQSLKXXPSVSKEILAE
AKKLNDAQAPK SEQ ID NO: 45 (SpAkkAA)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAQQNNFNKDKKS
AFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLN
EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK
AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE
AKKLNDAQAPK SEQ ID NO: 46 (SpAkR)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAXXNNFNKDKKS
AFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLN
EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK
AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE
AKKLNDAQAPK SEQ ID NO: 47 (SpAkR)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKDKKS
AFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNLN
EEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSLK
AAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILAE
AKKLNDAQAPK SEQ ID NO: 48 (SpAkR used in example)
MAQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKDKK
SAFYEILNMPNLNEAQRNGFIQSLKAAPSQSTNVLGEAKKLNESQAPKADNNFNKEKKNAFYEILNMPNL
NEEQRNGFIQSLKAAPSQSANLLSEAKKLNESQAPKADNKFNKEKKNAFYEILHLPNLNEEQRNGFIQSL
KAAPSQSANLLAEAKKLNDAQAPKADNKFNKEKKNAFYEILHLPNLTEEQRNGFIQSLKAAPSVSKEILA
EAKKLNDAQAPK SEQ ID NO: 49 (SpAkR)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAXXNNFNKDXXS
AFYEILNMPNLNEAQRNGFIQSLKXXPSQSTNVLGEAKKLNESQAPKADNNFNKEXXNAFYEILNMPNLN
EEQRNGFIQSLKXXPSQSANLLSEAKKLNESQAPKADNKFNKEXXNAFYEILHLPNLNEEQRNGFIQSLK
XXPSQSANLLAEAKKLNDAQAPKADNKFNKEXXNAFYEILHLPNLTEEQRNGFIQSLKXXPSVSKEILAE
AKKLNDAQAPK SEQ ID NO: 50 (SpAkR E domain)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAXXNNFNKD
SEQ ID NO: 51 (SpAkR E domain)
AQHDEAXXNAFYQVLNMPNLNADQRNGFIQSLKXXPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD
SEQ ID NO: 52 (SpAkR E domain)
AQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD
SEQ ID NO: 53 (E domain of SpAkR used in example)
MAQHDEAKKNAFYQVLNMPNLNADQRNGFIQSLKAAPSQSANVLGEAQKLNDSQAPKADAKRNNFNKD
SEQ ID NO: 54 (SpA E domain)
AQHDEAQQNAFYQVLNMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNNFNKD
Sequence CWU 1
1
54197PRTStaphylococcus aureus 1Met Ala Met Ile Lys Met Ser Pro Glu
Glu Ile Arg Ala Lys Ser Gln 1 5 10 15 Ser Tyr Gly Gln Gly Ser Asp
Gln Ile Arg Gln Ile Leu Ser Asp Leu 20 25 30 Thr Arg Ala Gln Gly
Glu Ile Ala Ala Asn Trp Glu Gly Gln Ala Phe 35 40 45 Ser Arg Phe
Glu Glu Gln Phe Gln Gln Leu Ser Pro Lys Val Glu Lys 50 55 60 Phe
Ala Gln Leu Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala 65 70
75 80 Asp Ala Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly
Leu 85 90 95 Gln 2104PRTStaphylococcus aureus 2Met Gly Gly Tyr Lys
Gly Ile Lys Ala Asp Gly Gly Lys Val Asp Gln 1 5 10 15 Ala Lys Gln
Leu Ala Ala Lys Thr Ala Lys Asp Ile Glu Ala Cys Gln 20 25 30 Lys
Gln Thr Gln Gln Leu Ala Glu Tyr Ile Glu Gly Ser Asp Trp Glu 35 40
45 Gly Gln Phe Ala Asn Lys Val Lys Asp Val Leu Leu Ile Met Ala Lys
50 55 60 Phe Gln Glu Glu Leu Val Gln Pro Met Ala Asp His Gln Lys
Ala Ile 65 70 75 80 Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr Asp Thr
Leu Ser Ile Lys 85 90 95 Gln Gly Leu Asp Arg Val Asn Pro 100
3302PRTStaphylococcus aureus 3Met Lys Lys Leu Leu Leu Pro Leu Ile
Ile Met Leu Leu Val Leu Ala 1 5 10 15 Ala Cys Gly Asn Gln Gly Glu
Lys Asn Asn Lys Ala Glu Thr Lys Ser 20 25 30 Tyr Lys Met Asp Asp
Gly Lys Thr Val Asp Ile Pro Lys Asp Pro Lys 35 40 45 Arg Ile Ala
Val Val Ala Pro Thr Tyr Ala Gly Gly Leu Lys Lys Leu 50 55 60 Gly
Ala Asn Ile Val Ala Val Asn Gln Gln Val Asp Gln Ser Lys Val 65 70
75 80 Leu Lys Asp Lys Phe Lys Gly Val Thr Lys Ile Gly Asp Gly Asp
Val 85 90 95 Glu Lys Val Ala Lys Glu Lys Pro Asp Leu Ile Ile Val
Tyr Ser Thr 100 105 110 Asp Lys Asp Ile Lys Lys Tyr Gln Lys Val Ala
Pro Thr Val Val Val 115 120 125 Asp Tyr Asn Lys His Lys Tyr Leu Glu
Gln Gln Glu Met Leu Gly Lys 130 135 140 Ile Val Gly Lys Glu Asp Lys
Val Lys Ala Trp Lys Lys Asp Trp Glu 145 150 155 160 Glu Thr Thr Ala
Lys Asp Gly Lys Glu Ile Lys Lys Ala Ile Gly Gln 165 170 175 Asp Ala
Thr Val Ser Leu Phe Asp Glu Phe Asp Lys Lys Leu Tyr Thr 180 185 190
Tyr Gly Asp Asn Trp Gly Arg Gly Gly Glu Val Leu Tyr Gln Ala Phe 195
200 205 Gly Leu Lys Met Gln Pro Glu Gln Gln Lys Leu Thr Ala Lys Ala
Gly 210 215 220 Trp Ala Glu Val Lys Gln Glu Glu Ile Glu Lys Tyr Ala
Gly Asp Tyr 225 230 235 240 Ile Val Ser Thr Ser Glu Gly Lys Pro Thr
Pro Gly Tyr Glu Ser Thr 245 250 255 Asn Met Trp Lys Asn Leu Lys Ala
Thr Lys Glu Gly His Ile Val Lys 260 265 270 Val Asp Ala Gly Thr Tyr
Trp Tyr Asn Asp Pro Tyr Thr Leu Asp Phe 275 280 285 Met Arg Lys Asp
Leu Lys Glu Lys Leu Ile Lys Ala Ala Lys 290 295 300
4256PRTStaphylococcus aureus 4Met Met Lys Arg Leu Asn Lys Leu Val
Leu Gly Ile Ile Phe Leu Phe 1 5 10 15 Leu Val Ile Ser Ile Thr Ala
Gly Cys Gly Ile Gly Lys Glu Ala Glu 20 25 30 Val Lys Lys Ser Phe
Glu Lys Thr Leu Ser Met Tyr Pro Ile Lys Asn 35 40 45 Leu Glu Asp
Leu Tyr Asp Lys Glu Gly Tyr Arg Asp Asp Gln Phe Asp 50 55 60 Lys
Asn Asp Lys Gly Thr Trp Ile Ile Asn Ser Glu Met Val Ile Gln 65 70
75 80 Pro Asn Asn Glu Asp Met Val Ala Lys Gly Met Val Leu Tyr Met
Asn 85 90 95 Arg Asn Thr Lys Thr Thr Asn Gly Tyr Tyr Tyr Val Asp
Val Thr Lys 100 105 110 Asp Glu Asp Glu Gly Lys Pro His Asp Asn Glu
Lys Arg Tyr Pro Val 115 120 125 Lys Met Val Asp Asn Lys Ile Ile Pro
Thr Lys Glu Ile Lys Asp Glu 130 135 140 Lys Ile Lys Lys Glu Ile Glu
Asn Phe Lys Phe Phe Val Gln Tyr Gly 145 150 155 160 Asp Phe Lys Asn
Leu Lys Asn Tyr Lys Asp Gly Asp Ile Ser Tyr Asn 165 170 175 Pro Glu
Val Pro Ser Tyr Ser Ala Lys Tyr Gln Leu Thr Asn Asp Asp 180 185 190
Tyr Asn Val Lys Gln Leu Arg Lys Arg Tyr Asp Ile Pro Thr Ser Lys 195
200 205 Ala Pro Lys Leu Leu Leu Lys Gly Ser Gly Asn Leu Lys Gly Ser
Ser 210 215 220 Val Gly Tyr Lys Asp Ile Glu Phe Thr Phe Val Glu Lys
Lys Glu Glu 225 230 235 240 Asn Ile Tyr Phe Ser Asp Ser Leu Asp Tyr
Lys Lys Ser Gly Asp Val 245 250 255 5319PRTStaphylococcus aureus
5Met Lys Thr Arg Ile Val Ser Ser Val Thr Thr Thr Leu Leu Leu Gly 1
5 10 15 Ser Ile Leu Met Asn Pro Val Ala Asn Ala Ala Asp Ser Asp Ile
Asn 20 25 30 Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser Asn Thr Thr
Val Lys Thr 35 40 45 Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn Gly
Met His Lys Lys Val 50 55 60 Phe Tyr Ser Phe Ile Asp Asp Lys Asn
His Asn Lys Lys Leu Leu Val 65 70 75 80 Ile Arg Thr Lys Gly Thr Ile
Ala Gly Gln Tyr Arg Val Tyr Ser Glu 85 90 95 Glu Gly Ala Asn Lys
Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val 100 105 110 Gln Leu Gln
Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr 115 120 125 Pro
Arg Asn Ser Ile Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr 130 135
140 Gly Phe Asn Gly Asn Val Thr Gly Asp Asp Thr Gly Lys Ile Gly Gly
145 150 155 160 Leu Ile Gly Ala Asn Val Ser Ile Gly His Thr Leu Lys
Tyr Val Gln 165 170 175 Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro Thr
Asp Lys Lys Val Gly 180 185 190 Trp Lys Val Ile Phe Asn Asn Met Val
Asn Gln Asn Trp Gly Pro Tyr 195 200 205 Asp Arg Asp Ser Trp Asn Pro
Val Tyr Gly Asn Gln Leu Phe Met Lys 210 215 220 Thr Arg Asn Gly Ser
Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn 225 230 235 240 Lys Ala
Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr 245 250 255
Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr Asn Ile Asp 260
265 270 Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln Leu His Trp Thr
Ser 275 280 285 Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys Trp Ile
Asp Arg Ser 290 295 300 Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys Glu
Glu Met Thr Asn 305 310 315 6285PRTStaphylococcus aureus 6Cys Gly
Asn Gln Gly Glu Lys Asn Asn Lys Ala Glu Thr Lys Ser Tyr 1 5 10 15
Lys Met Asp Asp Gly Lys Thr Val Asp Ile Pro Lys Asp Pro Lys Arg 20
25 30 Ile Ala Val Val Ala Pro Thr Tyr Ala Gly Gly Leu Lys Lys Leu
Gly 35 40 45 Ala Asn Ile Val Ala Val Asn Gln Gln Val Asp Gln Ser
Lys Val Leu 50 55 60 Lys Asp Lys Phe Lys Gly Val Thr Lys Ile Gly
Asp Gly Asp Val Glu 65 70 75 80 Lys Val Ala Lys Glu Lys Pro Asp Leu
Ile Ile Val Tyr Ser Thr Asp 85 90 95 Lys Asp Ile Lys Lys Tyr Gln
Lys Val Ala Pro Thr Val Val Val Asp 100 105 110 Tyr Asn Lys His Lys
Tyr Leu Glu Gln Gln Glu Met Leu Gly Lys Ile 115 120 125 Val Gly Lys
Glu Asp Lys Val Lys Ala Trp Lys Lys Asp Trp Glu Glu 130 135 140 Thr
Thr Ala Lys Asp Gly Lys Glu Ile Lys Lys Ala Ile Gly Gln Asp 145 150
155 160 Ala Thr Val Ser Leu Phe Asp Glu Phe Asp Lys Lys Leu Tyr Thr
Tyr 165 170 175 Gly Asp Asn Trp Gly Arg Gly Gly Glu Val Leu Tyr Gln
Ala Phe Gly 180 185 190 Leu Lys Met Gln Pro Glu Gln Gln Lys Leu Thr
Ala Lys Ala Gly Trp 195 200 205 Ala Glu Val Lys Gln Glu Glu Ile Glu
Lys Tyr Ala Gly Asp Tyr Ile 210 215 220 Val Ser Thr Ser Glu Gly Lys
Pro Thr Pro Gly Tyr Glu Ser Thr Asn 225 230 235 240 Met Trp Lys Asn
Leu Lys Ala Thr Lys Glu Gly His Ile Val Lys Val 245 250 255 Asp Ala
Gly Thr Tyr Trp Tyr Asn Asp Pro Tyr Thr Leu Asp Phe Met 260 265 270
Arg Lys Asp Leu Lys Glu Lys Leu Ile Lys Ala Ala Lys 275 280 285
7288PRTStaphylococcus aureus 7Met Ala Ser Cys Gly Asn Gln Gly Glu
Lys Asn Asn Lys Ala Glu Thr 1 5 10 15 Lys Ser Tyr Lys Met Asp Asp
Gly Lys Thr Val Asp Ile Pro Lys Asp 20 25 30 Pro Lys Arg Ile Ala
Val Val Ala Pro Thr Tyr Ala Gly Gly Leu Lys 35 40 45 Lys Leu Gly
Ala Asn Ile Val Ala Val Asn Gln Gln Val Asp Gln Ser 50 55 60 Lys
Val Leu Lys Asp Lys Phe Lys Gly Val Thr Lys Ile Gly Asp Gly 65 70
75 80 Asp Val Glu Lys Val Ala Lys Glu Lys Pro Asp Leu Ile Ile Val
Tyr 85 90 95 Ser Thr Asp Lys Asp Ile Lys Lys Tyr Gln Lys Val Ala
Pro Thr Val 100 105 110 Val Val Asp Tyr Asn Lys His Lys Tyr Leu Glu
Gln Gln Glu Met Leu 115 120 125 Gly Lys Ile Val Gly Lys Glu Asp Lys
Val Lys Ala Trp Lys Lys Asp 130 135 140 Trp Glu Glu Thr Thr Ala Lys
Asp Gly Lys Glu Ile Lys Lys Ala Ile 145 150 155 160 Gly Gln Asp Ala
Thr Val Ser Leu Phe Asp Glu Phe Asp Lys Lys Leu 165 170 175 Tyr Thr
Tyr Gly Asp Asn Trp Gly Arg Gly Gly Glu Val Leu Tyr Gln 180 185 190
Ala Phe Gly Leu Lys Met Gln Pro Glu Gln Gln Lys Leu Thr Ala Lys 195
200 205 Ala Gly Trp Ala Glu Val Lys Gln Glu Glu Ile Glu Lys Tyr Ala
Gly 210 215 220 Asp Tyr Ile Val Ser Thr Ser Glu Gly Lys Pro Thr Pro
Gly Tyr Glu 225 230 235 240 Ser Thr Asn Met Trp Lys Asn Leu Lys Ala
Thr Lys Glu Gly His Ile 245 250 255 Val Lys Val Asp Ala Gly Thr Tyr
Trp Tyr Asn Asp Pro Tyr Thr Leu 260 265 270 Asp Phe Met Arg Lys Asp
Leu Lys Glu Lys Leu Ile Lys Ala Ala Lys 275 280 285
8234PRTStaphylococcus aureus 8Met Gly Cys Gly Ile Gly Lys Glu Ala
Glu Val Lys Lys Ser Phe Glu 1 5 10 15 Lys Thr Leu Ser Met Tyr Pro
Ile Lys Asn Leu Glu Asp Leu Tyr Asp 20 25 30 Lys Glu Gly Tyr Arg
Asp Asp Gln Phe Asp Lys Asn Asp Lys Gly Thr 35 40 45 Trp Ile Ile
Asn Ser Glu Met Val Ile Gln Pro Asn Asn Glu Asp Met 50 55 60 Val
Ala Lys Gly Met Val Leu Tyr Met Asn Arg Asn Thr Lys Thr Thr 65 70
75 80 Asn Gly Tyr Tyr Tyr Val Asp Val Thr Lys Asp Glu Asp Glu Gly
Lys 85 90 95 Pro His Asp Asn Glu Lys Arg Tyr Pro Val Lys Met Val
Asp Asn Lys 100 105 110 Ile Ile Pro Thr Lys Glu Ile Lys Asp Glu Lys
Ile Lys Lys Glu Ile 115 120 125 Glu Asn Phe Lys Phe Phe Val Gln Tyr
Gly Asp Phe Lys Asn Leu Lys 130 135 140 Asn Tyr Lys Asp Gly Asp Ile
Ser Tyr Asn Pro Glu Val Pro Ser Tyr 145 150 155 160 Ser Ala Lys Tyr
Gln Leu Thr Asn Asp Asp Tyr Asn Val Lys Gln Leu 165 170 175 Arg Lys
Arg Tyr Asp Ile Pro Thr Ser Lys Ala Pro Lys Leu Leu Leu 180 185 190
Lys Gly Ser Gly Asn Leu Lys Gly Ser Ser Val Gly Tyr Lys Asp Ile 195
200 205 Glu Phe Thr Phe Val Glu Lys Lys Glu Glu Asn Ile Tyr Phe Ser
Asp 210 215 220 Ser Leu Asp Tyr Lys Lys Ser Gly Asp Val 225 230
9256PRTStaphylococcus aureus 9Met Met Lys Arg Leu Asn Lys Leu Val
Leu Gly Ile Ile Phe Leu Phe 1 5 10 15 Leu Val Ile Ser Ile Thr Ala
Gly Cys Gly Ile Gly Lys Glu Ala Glu 20 25 30 Val Lys Lys Ser Phe
Glu Lys Thr Leu Ser Met Tyr Pro Ile Lys Asn 35 40 45 Leu Glu Asp
Leu Tyr Asp Lys Glu Gly Tyr Arg Asp Asp Gln Phe Asp 50 55 60 Lys
Asn Asp Lys Gly Thr Trp Ile Ile Asn Ser Glu Met Val Ile Gln 65 70
75 80 Pro Asn Asn Glu Asp Met Val Ala Lys Gly Met Val Leu Tyr Met
Asn 85 90 95 Arg Asn Thr Lys Thr Thr Asn Gly Tyr Tyr Tyr Val Asp
Val Thr Lys 100 105 110 Asp Glu Asp Glu Gly Lys Pro His Asp Asn Glu
Lys Arg Tyr Pro Val 115 120 125 Lys Met Val Asp Asn Lys Ile Ile Pro
Thr Lys Glu Ile Lys Asp Glu 130 135 140 Lys Leu Lys Lys Glu Ile Glu
Asn Phe Lys Phe Phe Val Gln Tyr Gly 145 150 155 160 Asp Phe Lys Asn
Ile Lys Asn Tyr Lys Asp Gly Asp Ile Ser Tyr Asn 165 170 175 Pro Glu
Val Pro Ser Tyr Ser Ala Lys Tyr Gln Leu Thr Asn Asp Asp 180 185 190
Tyr Asn Val Lys Gln Leu Arg Lys Arg Tyr Asp Ile Pro Thr Ser Lys 195
200 205 Ala Pro Lys Leu Leu Leu Lys Gly Ser Gly Asn Leu Lys Gly Ser
Ser 210 215 220 Val Gly Tyr Lys Asp Ile Glu Phe Thr Phe Val Glu Lys
Lys Glu Glu 225 230 235 240 Asn Ile Tyr Phe Ser Asp Ser Leu Asp Tyr
Lys Lys Ser Gly Asp Val 245 250 255 10256PRTStaphylococcus aureus
10Met Met Lys Arg Leu Asn Lys Leu Val Leu Gly Ile Ile Phe Leu Phe 1
5 10 15 Leu Val Ile Ser Ile Thr Ala Gly Cys Gly Ile Gly Lys Glu Ala
Glu 20 25 30 Val Lys Lys Ser Phe Glu Lys Thr Leu Ser Met Tyr Pro
Ile Lys Asn 35 40 45 Leu Glu Asp Leu Tyr Asp Lys Glu Gly Tyr Arg
Asp Asp Gln Phe Asp 50 55 60 Lys Asn Asp Lys Gly Thr Trp Ile Ile
Asn Ser Glu Met Val Ile Gln 65 70 75 80 Pro Asn Asn Glu Asp Met Val
Ala Lys Gly Met Val Leu Tyr Met Asn 85 90 95 Arg Asn Thr Lys Thr
Thr Asn Gly Tyr Tyr Tyr Val Asp Val Thr Lys 100 105 110 Asp Glu Asp
Glu Gly Lys Pro His Asp Asn Glu Lys Arg Tyr Pro Val 115 120 125
Lys
Met Val Asp Asn Lys Ile Ile Pro Thr Lys Glu Ile Lys Asp Glu 130 135
140 Lys Val Lys Lys Glu Ile Glu Asn Phe Lys Phe Phe Val Gln Tyr Gly
145 150 155 160 Asp Phe Lys Asn Ile Lys Asn Tyr Lys Asp Gly Asp Ile
Ser Tyr Asn 165 170 175 Pro Glu Val Pro Ser Tyr Ser Ala Lys Tyr Gln
Leu Thr Asn Asp Asp 180 185 190 Tyr Asn Val Lys Gln Leu Arg Lys Arg
Tyr Asp Ile Pro Thr Ser Lys 195 200 205 Ala Pro Lys Leu Leu Leu Lys
Gly Ser Gly Asn Leu Lys Gly Ser Ser 210 215 220 Val Gly Tyr Lys Asp
Ile Glu Phe Thr Phe Val Glu Lys Lys Glu Glu 225 230 235 240 Asn Ile
Tyr Phe Ser Asp Ser Leu Asp Tyr Lys Lys Ser Gly Asp Val 245 250 255
11256PRTStaphylococcus aureus 11Met Met Lys Arg Leu Asn Lys Leu Val
Leu Gly Ile Ile Phe Leu Phe 1 5 10 15 Leu Val Ile Ser Ile Thr Ala
Gly Cys Gly Ile Gly Lys Glu Ala Glu 20 25 30 Val Lys Lys Ser Phe
Glu Lys Thr Leu Ser Met Tyr Pro Ile Lys Asn 35 40 45 Leu Glu Asp
Leu Tyr Asp Lys Glu Gly Tyr Arg Asp Asp Gln Phe Asp 50 55 60 Lys
Asn Asp Lys Gly Thr Trp Ile Ile Asn Ser Glu Met Val Ile Gln 65 70
75 80 Pro Asn Asn Glu Asp Met Val Ala Lys Gly Met Val Leu Tyr Met
Asn 85 90 95 Arg Asn Thr Lys Thr Thr Asn Gly Tyr Tyr Tyr Val Asp
Val Thr Lys 100 105 110 Asp Glu Asp Glu Gly Lys Pro His Asp Asn Glu
Lys Arg Tyr Pro Val 115 120 125 Lys Met Val Asp Asn Lys Ile Ile Pro
Thr Lys Glu Ile Lys Asp Glu 130 135 140 Lys Leu Lys Lys Glu Ile Glu
Asn Phe Lys Phe Phe Val Gln Tyr Gly 145 150 155 160 Asp Phe Lys Asn
Val Lys Asn Tyr Lys Asp Gly Asp Ile Ser Tyr Asn 165 170 175 Pro Glu
Val Pro Ser Tyr Ser Ala Lys Tyr Gln Leu Thr Asn Asp Asp 180 185 190
Tyr Asn Val Lys Gln Leu Arg Lys Arg Tyr Asp Ile Pro Thr Ser Lys 195
200 205 Ala Pro Lys Leu Leu Leu Lys Gly Ser Gly Asn Leu Lys Gly Ser
Ser 210 215 220 Val Gly Tyr Lys Asp Ile Glu Phe Thr Phe Val Glu Lys
Lys Glu Glu 225 230 235 240 Asn Ile Tyr Phe Ser Asp Ser Leu Asp Tyr
Lys Lys Ser Gly Asp Val 245 250 255 12293PRTStaphylococcus aureus
12Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1
5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu
Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp
Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly
Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala
Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln
Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr
Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr
Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr
Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135
140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro
145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn
Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp
Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn
Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys
Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala
Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln
Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255
Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260
265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu
Lys 275 280 285 Glu Glu Met Thr Asn 290 13293PRTStaphylococcus
aureus 13Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile
Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr
Asp Lys Glu Asn 20 25 30 Gly Met Leu Lys Lys Val Phe Tyr Ser Phe
Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg
Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu
Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe
Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile
Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110
Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115
120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly
His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu
Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile
Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg
Asp Ser Trp Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys
Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp
Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro
Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235
240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr
245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr
Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile
Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290
144PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic replacement tetramer peptide" 14Pro Ser Gly Ser
1 15258PRTStaphylococcus aureus 15Ala Asp Ser Asp Ile Asn Ile Lys
Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr
Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met Leu Lys
Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys
Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60
Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65
70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu
Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp
Thr Pro Ser Gly 100 105 110 Ser Val Gln Pro Asp Phe Lys Thr Ile Leu
Glu Ser Pro Thr Asp Lys 115 120 125 Lys Val Gly Trp Lys Val Ile Phe
Asn Asn Met Val Asn Gln Asn Trp 130 135 140 Gly Pro Tyr Asp Arg Asp
Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu 145 150 155 160 Phe Met Lys
Thr Arg Asn Gly Ser Met Lys Ala Ala Asp Asn Phe Leu 165 170 175 Asp
Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp 180 185
190 Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr
195 200 205 Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln
Leu His 210 215 220 Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys
Asp Lys Trp Ile 225 230 235 240 Asp Arg Ser Ser Glu Arg Tyr Lys Ile
Asp Trp Glu Lys Glu Glu Met 245 250 255 Thr Asn
16258PRTStaphylococcus aureus 16Ala Asp Ser Asp Ile Asn Ile Lys Thr
Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly
Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys
Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys
Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr
Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70
75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val
Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr
Pro Ser Gly 100 105 110 Ser Val Gln Pro Asp Phe Lys Thr Ile Leu Glu
Ser Pro Thr Asp Lys 115 120 125 Lys Val Gly Trp Lys Val Ile Phe Asn
Asn Met Val Asn Gln Asn Trp 130 135 140 Gly Pro Tyr Asp Arg Asp Ser
Trp Asn Pro Val Tyr Gly Asn Gln Leu 145 150 155 160 Phe Met Lys Thr
Arg Asn Gly Ser Met Lys Ala Ala Asp Asn Phe Leu 165 170 175 Asp Pro
Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp 180 185 190
Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr 195
200 205 Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln Leu
His 210 215 220 Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp
Lys Trp Ile 225 230 235 240 Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp
Trp Glu Lys Glu Glu Met 245 250 255 Thr Asn 17293PRTStaphylococcus
aureus 17Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile
Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr
Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys Val Phe Tyr Ser Phe
Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys Leu Leu Val Ile Arg
Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr Arg Val Tyr Ser Glu
Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe
Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val Ala 85 90 95 Gln Ile
Ser Asp Leu Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr 100 105 110
Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp Asp 115
120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly
His 130 135 140 Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu
Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys Val Gly Trp Lys Val Ile
Phe Asn Asn Met Val Asn 165 170 175 Gln Asn Trp Gly Pro Tyr Asp Arg
Asp Ser Trp Asn Pro Val Tyr Gly 180 185 190 Asn Gln Leu Phe Met Lys
Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195 200 205 Asn Phe Leu Asp
Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe 210 215 220 Ser Pro
Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys Ala Ser Lys 225 230 235
240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr
245 250 255 Gln Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr
Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile
Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr Asn 290
18293PRTStaphylococcus aureus 18Ala Asp Ser Asp Ile Asn Ile Lys Thr
Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys Thr Gly
Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His Lys Lys
Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn Lys Lys
Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55 60 Tyr
Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp 65 70
75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val
Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr
Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn
Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile Gly
Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln Pro
Leu Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys Lys
Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln Asn
Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly 180 185 190
Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp 195
200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly
Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys
Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu
Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr Ser Thr Asn
Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile Asp Arg Ser
Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu Glu Met Thr
Asn 290 19293PRTStaphylococcus aureus 19Ala Asp Ser Asp Ile Asn Ile
Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys
Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met Leu
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn
Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln 50 55
60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp
65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu
Val Ala 85 90 95 Gln Ile Ser Asp Leu Tyr Pro Arg Asn Ser Ile Asp
Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly
Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly Gly Leu Ile
Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys Tyr Val Gln
Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160 Thr Asp Lys
Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175 Gln
Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly
180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala
Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu
Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr Met
Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp Val
Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp Thr
Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp Ile
Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285 Glu
Glu Met Thr Asn 290 20293PRTStaphylococcus aureus 20Ala Asp Ser Asp
Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30
Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35
40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly
Gln 50 55 60 Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly
Leu Ala Trp 65 70 75 80 Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro
Asp Asn Glu Val Ala 85 90 95 Gln Ile Ser Asp Tyr Tyr Pro Arg Asn
Ser Ile Asp Thr Lys Glu Tyr 100 105 110 Met Ser Thr Leu Thr Tyr Gly
Phe Asn Gly Asn Val Thr Gly Asp Asp 115 120 125 Thr Gly Lys Ile Gly
Gly Leu Ile Gly Ala Asn Val Ser Ile Gly His 130 135 140 Thr Leu Lys
Tyr Val Gln Pro Leu Phe Lys Thr Ile Leu Glu Ser Pro 145 150 155 160
Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165
170 175 Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr
Gly 180 185 190 Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys
Ala Ala Asp 195 200 205 Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu
Leu Ser Ser Gly Phe 210 215 220 Ser Pro Asp Phe Ala Thr Val Ile Thr
Met Asp Arg Lys Ala Ser Lys 225 230 235 240 Gln Gln Thr Asn Ile Asp
Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr 245 250 255 Gln Leu His Trp
Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270 Lys Trp
Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285
Glu Glu Met Thr Asn 290 2150PRTStaphylococcus aureus 21Ala Asp Ser
Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn
Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25
30 Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His
35 40 45 Asn Lys 50 2263PRTStaphylococcus aureus 22Ala Asp Ser Asp
Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30
Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35
40 45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly
50 55 60 2353PRTStaphylococcus aureus 23Ala Asp Ser Asp Ile Asn Ile
Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys
Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met His
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn
Lys Lys Leu Leu 50 2450PRTStaphylococcus aureus 24Ala Asp Ser Asp
Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30
Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35
40 45 Asn Lys 50 2563PRTStaphylococcus aureus 25Ala Asp Ser Asp Ile
Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr
Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly
Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40
45 Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly 50
55 60 2653PRTStaphylococcus aureus 26Ala Asp Ser Asp Ile Asn Ile
Lys Thr Gly Thr Thr Asp Ile Gly Ser 1 5 10 15 Asn Thr Thr Val Lys
Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30 Gly Met Leu
Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40 45 Asn
Lys Lys Leu Leu 50 27296PRTStaphylococcus aureus 27Met Ala Ser Ala
Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp 1 5 10 15 Ile Gly
Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp 20 25 30
Lys Glu Asn Gly Met Leu Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp 35
40 45 Lys Asn His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr
Ile 50 55 60 Ala Gly Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn
Lys Ser Gly 65 70 75 80 Leu Ala Trp Pro Ser Ala Phe Lys Val Gln Leu
Gln Leu Pro Asp Asn 85 90 95 Glu Val Ala Gln Ile Ser Asp Tyr Tyr
Pro Arg Asn Ser Ile Asp Thr 100 105 110 Lys Glu Tyr Met Ser Thr Leu
Thr Tyr Gly Phe Asn Gly Asn Val Thr 115 120 125 Gly Asp Asp Thr Gly
Lys Ile Gly Gly Leu Ile Gly Ala Asn Val Ser 130 135 140 Ile Gly His
Thr Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu 145 150 155 160
Glu Ser Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn 165
170 175 Met Val Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn
Pro 180 185 190 Val Tyr Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly
Ser Met Lys 195 200 205 Ala Ala Asp Asn Phe Leu Asp Pro Asn Lys Ala
Ser Ser Leu Leu Ser 210 215 220 Ser Gly Phe Ser Pro Asp Phe Ala Thr
Val Ile Thr Met Asp Arg Lys 225 230 235 240 Ala Ser Lys Gln Gln Thr
Asn Ile Asp Val Ile Tyr Glu Arg Val Arg 245 250 255 Asp Asp Tyr Gln
Leu His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn 260 265 270 Thr Lys
Asp Lys Trp Ile Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp 275 280 285
Trp Glu Lys Glu Glu Met Thr Asn 290 295 28207PRTStaphylococcus
aureus 28Met Ala Met Ile Lys Met Ser Pro Glu Glu Ile Arg Ala Lys
Ser Gln 1 5 10 15 Ser Tyr Gly Gln Gly Ser Asp Gln Ile Arg Gln Ile
Leu Ser Asp Leu 20 25 30 Thr Arg Ala Gln Gly Glu Ile Ala Ala Asn
Trp Glu Gly Gln Ala Phe 35 40 45 Ser Arg Phe Glu Glu Gln Phe Gln
Gln Leu Ser Pro Lys Val Glu Lys 50 55 60 Phe Ala Gln Leu Leu Glu
Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala 65 70 75 80 Asp Ala Val Gln
Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly Leu 85 90 95 Gln Ala
Ser Gly Gly Gly Ser Met Gly Gly Tyr Lys Gly Ile Lys Ala 100 105 110
Asp Gly Gly Lys Val Asp Gln Ala Lys Gln Leu Ala Ala Lys Thr Ala 115
120 125 Lys Asp Ile Glu Ala Cys Gln Lys Gln Thr Gln Gln Leu Ala Glu
Tyr 130 135 140 Ile Glu Gly Ser Asp Trp Glu Gly Gln Phe Ala Asn Lys
Val Lys Asp 145 150 155 160 Val Leu Leu Ile Met Ala Lys Phe Gln Glu
Glu Leu Val Gln Pro Met 165 170 175 Ala Asp His Gln Lys Ala Ile Asp
Asn Leu Ser Gln Asn Leu Ala Lys 180 185 190 Tyr Asp Thr Leu Ser Ile
Lys Gln Gly Leu Asp Arg Val Asn Pro 195 200 205
29207PRTStaphylococcus aureus 29Met Gly Gly Tyr Lys Gly Ile Lys Ala
Asp Gly Gly Lys Val Asp Gln 1 5 10 15 Ala Lys Gln Leu Ala Ala Lys
Thr Ala Lys Asp Ile Glu Ala Cys Gln 20 25 30 Lys Gln Thr Gln Gln
Leu Ala Glu Tyr Ile Glu Gly Ser Asp Trp Glu 35 40 45 Gly Gln Phe
Ala Asn Lys Val Lys Asp Val Leu Leu Ile Met Ala Lys 50 55 60 Phe
Gln Glu Glu Leu Val Gln Pro Met Ala Asp His Gln Lys Ala Ile 65 70
75 80 Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr Asp Thr Leu Ser Ile
Lys 85 90 95 Gln Gly Leu Asp Arg Val Asn Pro Ala Ser Gly Gly Gly
Ser Met Ala 100 105 110 Met Ile Lys Met Ser Pro Glu Glu Ile Arg Ala
Lys Ser Gln Ser Tyr 115 120 125 Gly Gln Gly Ser Asp Gln Ile Arg Gln
Ile Leu Ser Asp Leu Thr Arg 130 135 140 Ala Gln Gly Glu Ile Ala Ala
Asn Trp Glu Gly Gln Ala Phe Ser Arg 145 150 155 160 Phe Glu Glu Gln
Phe Gln Gln Leu Ser Pro Lys Val Glu Lys Phe Ala 165 170 175 Gln Leu
Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala Asp Ala 180 185 190
Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly Leu Gln 195 200
205 306PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic linker peptide" 30Ala Ser Gly Gly Gly Ser 1 5
31205PRTStaphylococcus aureus 31Ala Met Ile Lys Met Ser Pro Glu Glu
Ile Arg Ala Lys Ser Gln Ser 1 5 10 15 Tyr Gly Gln Gly Ser Asp Gln
Ile Arg Gln Ile Leu Ser Asp Leu Thr 20 25 30 Arg Ala Gln Gly Glu
Ile Ala Ala Asn Trp Glu Gly Gln Ala Phe Ser 35 40 45 Arg Phe Glu
Glu Gln Phe Gln Gln Leu Ser Pro Lys Val Glu Lys Phe 50 55 60 Ala
Gln Leu Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala Asp 65 70
75 80 Ala Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly Leu
Gln 85 90 95 Ala Ser Gly Gly Gly Ser Gly Gly Tyr Lys Gly Ile Lys
Ala Asp Gly 100 105 110 Gly Lys Val Asp Gln Ala Lys Gln Leu Ala Ala
Lys Thr Ala Lys Asp 115 120 125 Ile Glu Ala Cys Gln Lys Gln Thr Gln
Gln Leu Ala Glu Tyr Ile Glu 130 135 140 Gly Ser Asp Trp Glu Gly Gln
Phe Ala Asn Lys Val Lys Asp Val Leu 145 150 155 160 Leu Ile Met Ala
Lys Phe Gln Glu Glu Leu Val Gln Pro Met Ala Asp 165 170 175 His Gln
Lys Ala Ile Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr Asp 180 185 190
Thr Leu Ser Ile Lys Gln Gly Leu Asp Arg Val Asn Pro 195 200 205
32206PRTStaphylococcus aureus 32Met Ala Met Ile Lys Met Ser Pro Glu
Glu Ile Arg Ala Lys Ser Gln 1 5 10 15 Ser Tyr Gly Gln Gly Ser Asp
Gln Ile Arg Gln Ile Leu Ser Asp Leu 20 25 30 Thr Arg Ala Gln Gly
Glu Ile Ala Ala Asn Trp Glu Gly Gln Ala Phe 35 40 45 Ser Arg Phe
Glu Glu Gln Phe Gln Gln Leu Ser Pro Lys Val Glu Lys 50 55 60 Phe
Ala Gln Leu Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala 65 70
75 80 Asp Ala Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly
Leu 85 90 95 Gln Ala Ser Gly Gly Gly Ser Gly Gly Tyr Lys Gly Ile
Lys Ala Asp 100 105 110 Gly Gly Lys Val Asp Gln Ala Lys Gln Leu Ala
Ala Lys Thr Ala Lys 115 120 125 Asp Ile Glu Ala Cys Gln Lys Gln Thr
Gln Gln Leu Ala Glu Tyr Ile 130 135 140 Glu Gly Ser Asp Trp Glu Gly
Gln Phe Ala Asn Lys Val Lys Asp Val 145 150 155 160 Leu Leu Ile Met
Ala Lys Phe Gln Glu Glu Leu Val Gln Pro Met Ala 165 170 175 Asp His
Gln Lys Ala Ile Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr 180 185 190
Asp Thr Leu Ser Ile Lys Gln Gly Leu Asp Arg Val Asn Pro 195 200 205
33233PRTStaphylococcus aureus 33Gly Cys Gly Ile Gly Lys Glu Ala Glu
Val Lys Lys Ser Phe Glu Lys 1 5 10 15 Thr Leu Ser Met Tyr Pro Ile
Lys Asn Leu Glu Asp Leu Tyr Asp Lys 20 25 30 Glu Gly Tyr Arg Asp
Asp Gln Phe Asp Lys Asn Asp Lys Gly Thr Trp 35 40 45 Ile Ile Asn
Ser Glu Met Val Ile Gln Pro Asn Asn Glu Asp Met Val 50 55 60 Ala
Lys Gly Met Val Leu Tyr Met Asn Arg Asn Thr Lys Thr Thr Asn 65 70
75 80 Gly Tyr Tyr Tyr Val Asp Val Thr Lys Asp Glu Asp Glu Gly Lys
Pro 85 90 95 His Asp Asn Glu Lys Arg Tyr Pro Val Lys Met Val Asp
Asn Lys Ile 100 105 110 Ile Pro Thr Lys Glu Ile Lys Asp Glu Lys Ile
Lys Lys Glu Ile Glu 115 120 125 Asn Phe Lys Phe Phe Val Gln Tyr Gly
Asp Phe Lys Asn Leu Lys Asn 130 135 140 Tyr Lys Asp Gly Asp Ile Ser
Tyr Asn Pro Glu Val Pro Ser Tyr Ser 145 150 155 160 Ala Lys Tyr Gln
Leu Thr Asn Asp Asp Tyr Asn Val Lys Gln Leu Arg 165 170 175 Lys Arg
Tyr Asp Ile Pro Thr Ser Lys Ala Pro Lys Leu Leu Leu Lys 180 185 190
Gly Ser Gly Asn Leu Lys Gly Ser Ser Val Gly Tyr Lys Asp Ile Glu 195
200 205 Phe Thr Phe Val Glu Lys Lys Glu Glu Asn Ile Tyr Phe Ser Asp
Ser 210 215 220 Leu Asp Tyr Lys Lys Ser Gly Asp Val 225 230
34284PRTStaphylococcus aureus 34Gly Asn Gln Gly Glu Lys Asn Asn Lys
Ala Glu Thr Lys Ser Tyr Lys 1 5 10 15 Met Asp Asp Gly Lys Thr Val
Asp Ile Pro Lys Asp Pro Lys Arg Ile 20 25 30 Ala Val Val Ala Pro
Thr Tyr Ala Gly Gly Leu Lys Lys Leu Gly Ala 35 40 45 Asn Ile Val
Ala Val Asn Gln Gln Val Asp Gln Ser Lys Val Leu Lys 50 55 60 Asp
Lys Phe Lys Gly Val Thr Lys Ile Gly Asp Gly Asp Val Glu Lys 65 70
75 80 Val Ala Lys Glu Lys Pro Asp Leu Ile Ile Val Tyr Ser Thr Asp
Lys 85 90 95 Asp Ile Lys Lys Tyr Gln Lys Val Ala Pro Thr Val Val
Val Asp Tyr 100 105 110 Asn Lys His Lys Tyr Leu Glu Gln Gln Glu Met
Leu Gly Lys Ile Val 115 120 125 Gly Lys Glu Asp Lys Val Lys Ala Trp
Lys Lys Asp
Trp Glu Glu Thr 130 135 140 Thr Ala Lys Asp Gly Lys Glu Ile Lys Lys
Ala Ile Gly Gln Asp Ala 145 150 155 160 Thr Val Ser Leu Phe Asp Glu
Phe Asp Lys Lys Leu Tyr Thr Tyr Gly 165 170 175 Asp Asn Trp Gly Arg
Gly Gly Glu Val Leu Tyr Gln Ala Phe Gly Leu 180 185 190 Lys Met Gln
Pro Glu Gln Gln Lys Leu Thr Ala Lys Ala Gly Trp Ala 195 200 205 Glu
Val Lys Gln Glu Glu Ile Glu Lys Tyr Ala Gly Asp Tyr Ile Val 210 215
220 Ser Thr Ser Glu Gly Lys Pro Thr Pro Gly Tyr Glu Ser Thr Asn Met
225 230 235 240 Trp Lys Asn Leu Lys Ala Thr Lys Glu Gly His Ile Val
Lys Val Asp 245 250 255 Ala Gly Thr Tyr Trp Tyr Asn Asp Pro Tyr Thr
Leu Asp Phe Met Arg 260 265 270 Lys Asp Leu Lys Glu Lys Leu Ile Lys
Ala Ala Lys 275 280 35103PRTStaphylococcus
aureusMOD_RES(30)..(30)Any amino acid which lacks a free thiol or
absent 35Gly Gly Tyr Lys Gly Ile Lys Ala Asp Gly Gly Lys Val Asp
Gln Ala 1 5 10 15 Lys Gln Leu Ala Ala Lys Thr Ala Lys Asp Ile Glu
Ala Xaa Gln Lys 20 25 30 Gln Thr Gln Gln Leu Ala Glu Tyr Ile Glu
Gly Ser Asp Trp Glu Gly 35 40 45 Gln Phe Ala Asn Lys Val Lys Asp
Val Leu Leu Ile Met Ala Lys Phe 50 55 60 Gln Glu Glu Leu Val Gln
Pro Met Ala Asp His Gln Lys Ala Ile Asp 65 70 75 80 Asn Leu Ser Gln
Asn Leu Ala Lys Tyr Asp Thr Leu Ser Ile Lys Gln 85 90 95 Gly Leu
Asp Arg Val Asn Pro 100 36231PRTStaphylococcus aureus 36Gly Ile Gly
Lys Glu Ala Glu Val Lys Lys Ser Phe Glu Lys Thr Leu 1 5 10 15 Ser
Met Tyr Pro Ile Lys Asn Leu Glu Asp Leu Tyr Asp Lys Glu Gly 20 25
30 Tyr Arg Asp Asp Gln Phe Asp Lys Asn Asp Lys Gly Thr Trp Ile Ile
35 40 45 Asn Ser Glu Met Val Ile Gln Pro Asn Asn Glu Asp Met Val
Ala Lys 50 55 60 Gly Met Val Leu Tyr Met Asn Arg Asn Thr Lys Thr
Thr Asn Gly Tyr 65 70 75 80 Tyr Tyr Val Asp Val Thr Lys Asp Glu Asp
Glu Gly Lys Pro His Asp 85 90 95 Asn Glu Lys Arg Tyr Pro Val Lys
Met Val Asp Asn Lys Ile Ile Pro 100 105 110 Thr Lys Glu Ile Lys Asp
Glu Lys Ile Lys Lys Glu Ile Glu Asn Phe 115 120 125 Lys Phe Phe Val
Gln Tyr Gly Asp Phe Lys Asn Leu Lys Asn Tyr Lys 130 135 140 Asp Gly
Asp Ile Ser Tyr Asn Pro Glu Val Pro Ser Tyr Ser Ala Lys 145 150 155
160 Tyr Gln Leu Thr Asn Asp Asp Tyr Asn Val Lys Gln Leu Arg Lys Arg
165 170 175 Tyr Asp Ile Pro Thr Ser Lys Ala Pro Lys Leu Leu Leu Lys
Gly Ser 180 185 190 Gly Asn Leu Lys Gly Ser Ser Val Gly Tyr Lys Asp
Ile Glu Phe Thr 195 200 205 Phe Val Glu Lys Lys Glu Glu Asn Ile Tyr
Phe Ser Asp Ser Leu Asp 210 215 220 Tyr Lys Lys Ser Gly Asp Val 225
230 37287PRTStaphylococcus aureus 37Met Ala Ser Gly Asn Gln Gly Glu
Lys Asn Asn Lys Ala Glu Thr Lys 1 5 10 15 Ser Tyr Lys Met Asp Asp
Gly Lys Thr Val Asp Ile Pro Lys Asp Pro 20 25 30 Lys Arg Ile Ala
Val Val Ala Pro Thr Tyr Ala Gly Gly Leu Lys Lys 35 40 45 Leu Gly
Ala Asn Ile Val Ala Val Asn Gln Gln Val Asp Gln Ser Lys 50 55 60
Val Leu Lys Asp Lys Phe Lys Gly Val Thr Lys Ile Gly Asp Gly Asp 65
70 75 80 Val Glu Lys Val Ala Lys Glu Lys Pro Asp Leu Ile Ile Val
Tyr Ser 85 90 95 Thr Asp Lys Asp Ile Lys Lys Tyr Gln Lys Val Ala
Pro Thr Val Val 100 105 110 Val Asp Tyr Asn Lys His Lys Tyr Leu Glu
Gln Gln Glu Met Leu Gly 115 120 125 Lys Ile Val Gly Lys Glu Asp Lys
Val Lys Ala Trp Lys Lys Asp Trp 130 135 140 Glu Glu Thr Thr Ala Lys
Asp Gly Lys Glu Ile Lys Lys Ala Ile Gly 145 150 155 160 Gln Asp Ala
Thr Val Ser Leu Phe Asp Glu Phe Asp Lys Lys Leu Tyr 165 170 175 Thr
Tyr Gly Asp Asn Trp Gly Arg Gly Gly Glu Val Leu Tyr Gln Ala 180 185
190 Phe Gly Leu Lys Met Gln Pro Glu Gln Gln Lys Leu Thr Ala Lys Ala
195 200 205 Gly Trp Ala Glu Val Lys Gln Glu Glu Ile Glu Lys Tyr Ala
Gly Asp 210 215 220 Tyr Ile Val Ser Thr Ser Glu Gly Lys Pro Thr Pro
Gly Tyr Glu Ser 225 230 235 240 Thr Asn Met Trp Lys Asn Leu Lys Ala
Thr Lys Glu Gly His Ile Val 245 250 255 Lys Val Asp Ala Gly Thr Tyr
Trp Tyr Asn Asp Pro Tyr Thr Leu Asp 260 265 270 Phe Met Arg Lys Asp
Leu Lys Glu Lys Leu Ile Lys Ala Ala Lys 275 280 285
38206PRTStaphylococcus aureus 38Met Ala Met Ile Lys Met Ser Pro Glu
Glu Ile Arg Ala Lys Ser Gln 1 5 10 15 Ser Tyr Gly Gln Gly Ser Asp
Gln Ile Arg Gln Ile Leu Ser Asp Leu 20 25 30 Thr Arg Ala Gln Gly
Glu Ile Ala Ala Asn Trp Glu Gly Gln Ala Phe 35 40 45 Ser Arg Phe
Glu Glu Gln Phe Gln Gln Leu Ser Pro Lys Val Glu Lys 50 55 60 Phe
Ala Gln Leu Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala 65 70
75 80 Asp Ala Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly
Leu 85 90 95 Gln Ala Ser Gly Gly Gly Ser Gly Gly Tyr Lys Gly Ile
Lys Ala Asp 100 105 110 Gly Gly Lys Val Asp Gln Ala Lys Gln Leu Ala
Ala Lys Thr Ala Lys 115 120 125 Asp Ile Glu Ala Ala Gln Lys Gln Thr
Gln Gln Leu Ala Glu Tyr Ile 130 135 140 Glu Gly Ser Asp Trp Glu Gly
Gln Phe Ala Asn Lys Val Lys Asp Val 145 150 155 160 Leu Leu Ile Met
Ala Lys Phe Gln Glu Glu Leu Val Gln Pro Met Ala 165 170 175 Asp His
Gln Lys Ala Ile Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr 180 185 190
Asp Thr Leu Ser Ile Lys Gln Gly Leu Asp Arg Val Asn Pro 195 200 205
39234PRTStaphylococcus aureus 39Met Gly Ser Gly Ile Gly Lys Glu Ala
Glu Val Lys Lys Ser Phe Glu 1 5 10 15 Lys Thr Leu Ser Met Tyr Pro
Ile Lys Asn Leu Glu Asp Leu Tyr Asp 20 25 30 Lys Glu Gly Tyr Arg
Asp Asp Gln Phe Asp Lys Asn Asp Lys Gly Thr 35 40 45 Trp Ile Ile
Asn Ser Glu Met Val Ile Gln Pro Asn Asn Glu Asp Met 50 55 60 Val
Ala Lys Gly Met Val Leu Tyr Met Asn Arg Asn Thr Lys Thr Thr 65 70
75 80 Asn Gly Tyr Tyr Tyr Val Asp Val Thr Lys Asp Glu Asp Glu Gly
Lys 85 90 95 Pro His Asp Asn Glu Lys Arg Tyr Pro Val Lys Met Val
Asp Asn Lys 100 105 110 Ile Ile Pro Thr Lys Glu Ile Lys Asp Glu Lys
Ile Lys Lys Glu Ile 115 120 125 Glu Asn Phe Lys Phe Phe Val Gln Tyr
Gly Asp Phe Lys Asn Leu Lys 130 135 140 Asn Tyr Lys Asp Gly Asp Ile
Ser Tyr Asn Pro Glu Val Pro Ser Tyr 145 150 155 160 Ser Ala Lys Tyr
Gln Leu Thr Asn Asp Asp Tyr Asn Val Lys Gln Leu 165 170 175 Arg Lys
Arg Tyr Asp Ile Pro Thr Ser Lys Ala Pro Lys Leu Leu Leu 180 185 190
Lys Gly Ser Gly Asn Leu Lys Gly Ser Ser Val Gly Tyr Lys Asp Ile 195
200 205 Glu Phe Thr Phe Val Glu Lys Lys Glu Glu Asn Ile Tyr Phe Ser
Asp 210 215 220 Ser Leu Asp Tyr Lys Lys Ser Gly Asp Val 225 230
40205PRTStaphylococcus aureusMOD_RES(132)..(132)Any amino acid
which lacks a free thiol or absent 40Ala Met Ile Lys Met Ser Pro
Glu Glu Ile Arg Ala Lys Ser Gln Ser 1 5 10 15 Tyr Gly Gln Gly Ser
Asp Gln Ile Arg Gln Ile Leu Ser Asp Leu Thr 20 25 30 Arg Ala Gln
Gly Glu Ile Ala Ala Asn Trp Glu Gly Gln Ala Phe Ser 35 40 45 Arg
Phe Glu Glu Gln Phe Gln Gln Leu Ser Pro Lys Val Glu Lys Phe 50 55
60 Ala Gln Leu Leu Glu Glu Ile Lys Gln Gln Leu Asn Ser Thr Ala Asp
65 70 75 80 Ala Val Gln Glu Gln Asp Gln Gln Leu Ser Asn Asn Phe Gly
Leu Gln 85 90 95 Ala Ser Gly Gly Gly Ser Gly Gly Tyr Lys Gly Ile
Lys Ala Asp Gly 100 105 110 Gly Lys Val Asp Gln Ala Lys Gln Leu Ala
Ala Lys Thr Ala Lys Asp 115 120 125 Ile Glu Ala Xaa Gln Lys Gln Thr
Gln Gln Leu Ala Glu Tyr Ile Glu 130 135 140 Gly Ser Asp Trp Glu Gly
Gln Phe Ala Asn Lys Val Lys Asp Val Leu 145 150 155 160 Leu Ile Met
Ala Lys Phe Gln Glu Glu Leu Val Gln Pro Met Ala Asp 165 170 175 His
Gln Lys Ala Ile Asp Asn Leu Ser Gln Asn Leu Ala Lys Tyr Asp 180 185
190 Thr Leu Ser Ile Lys Gln Gly Leu Asp Arg Val Asn Pro 195 200 205
4126DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic immunostimulatory
oligonucleotide"modified_base(1)..(1)Inosinemodified_base(3)..(3)Inosinem-
odified_base(5)..(5)Inosinemodified_base(7)..(7)Inosinemodified_base(9)..(-
9)Inosinemodified_base(11)..(11)Inosinemodified_base(13)..(13)Inosinemodif-
ied_base(15)..(15)Inosinemodified_base(17)..(17)Inosinemodified_base(19)..-
(19)Inosinemodified_base(21)..(21)Inosinemodified_base(23)..(23)Inosinemod-
ified_base(25)..(25)Inosine 41ncncncncnc ncncncncnc ncncnc
264211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic cationic oligopeptide" 42Lys Leu Lys Leu Leu Leu
Leu Leu Lys Leu Lys 1 5 10 43516PRTStaphylococcus aureus 43Met Lys
Lys Lys Asn Ile Tyr Ser Ile Arg Lys Leu Gly Val Gly Ile 1 5 10 15
Ala Ser Val Thr Leu Gly Thr Leu Leu Ile Ser Gly Gly Val Thr Pro 20
25 30 Ala Ala Asn Ala Ala Gln His Asp Glu Ala Gln Gln Asn Ala Phe
Tyr 35 40 45 Gln Val Leu Asn Met Pro Asn Leu Asn Ala Asp Gln Arg
Asn Gly Phe 50 55 60 Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser
Ala Asn Val Leu Gly 65 70 75 80 Glu Ala Gln Lys Leu Asn Asp Ser Gln
Ala Pro Lys Ala Asp Ala Gln 85 90 95 Gln Asn Asn Phe Asn Lys Asp
Gln Gln Ser Ala Phe Tyr Glu Ile Leu 100 105 110 Asn Met Pro Asn Leu
Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser 115 120 125 Leu Lys Asp
Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys 130 135 140 Lys
Leu Asn Glu Ser Gln Ala Pro Lys Ala Asp Asn Asn Phe Asn Lys 145 150
155 160 Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu Asn Met Pro Asn Leu
Asn 165 170 175 Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp
Asp Pro Ser 180 185 190 Gln Ser Ala Asn Leu Leu Ser Glu Ala Lys Lys
Leu Asn Glu Ser Gln 195 200 205 Ala Pro Lys Ala Asp Asn Lys Phe Asn
Lys Glu Gln Gln Asn Ala Phe 210 215 220 Tyr Glu Ile Leu His Leu Pro
Asn Leu Asn Glu Glu Gln Arg Asn Gly 225 230 235 240 Phe Ile Gln Ser
Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu 245 250 255 Ala Glu
Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn 260 265 270
Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu 275
280 285 Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu
Lys 290 295 300 Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala
Lys Lys Leu 305 310 315 320 Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp
Asn Asn Lys Pro Gly Lys 325 330 335 Glu Asp Asn Asn Lys Pro Gly Lys
Glu Asp Asn Asn Lys Pro Gly Lys 340 345 350 Glu Asp Asn Asn Lys Pro
Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys 355 360 365 Glu Asp Gly Asn
Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly Lys 370 375 380 Glu Asp
Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly Lys 385 390 395
400 Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Lys Pro Gly Lys
405 410 415 Glu Asp Gly Asn Gly Val His Val Val Lys Pro Gly Asp Thr
Val Asn 420 425 430 Asp Ile Ala Lys Ala Asn Gly Thr Thr Ala Asp Lys
Ile Ala Ala Asp 435 440 445 Asn Lys Leu Ala Asp Lys Asn Met Ile Lys
Pro Gly Gln Glu Leu Val 450 455 460 Val Asp Lys Lys Gln Pro Ala Asn
His Ala Asp Ala Asn Lys Ala Gln 465 470 475 480 Ala Leu Pro Glu Thr
Gly Glu Glu Asn Pro Phe Ile Gly Thr Thr Val 485 490 495 Phe Gly Gly
Leu Ser Leu Ala Leu Gly Ala Ala Leu Leu Ala Gly Arg 500 505 510 Arg
Arg Glu Leu 515 44291PRTStaphylococcus aureusMOD_RES(7)..(8)Any
amino acid except GlnMOD_RES(34)..(35)Any amino acid except
AspMOD_RES(68)..(69)Any amino acid except GlnMOD_RES(95)..(96)Any
amino acid except AspMOD_RES(126)..(127)Any amino acid except
GlnMOD_RES(153)..(154)Any amino acid except
AspMOD_RES(184)..(185)Any amino acid except
GlnMOD_RES(211)..(212)Any amino acid except
AspMOD_RES(242)..(243)Any amino acid except
GlnMOD_RES(269)..(270)Any amino acid except Asp 44Ala Gln His Asp
Glu Ala Xaa Xaa Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro
Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30
Lys Xaa Xaa Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35
40 45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Asn Asn
Phe 50 55 60 Asn Lys Asp Xaa Xaa Ser Ala Phe Tyr Glu Ile Leu Asn
Met Pro Asn 65 70 75 80 Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln
Ser Leu Lys Xaa Xaa 85 90 95 Pro Ser Gln Ser Thr Asn Val Leu Gly
Glu Ala Lys Lys Leu Asn Glu 100 105 110 Ser Gln Ala Pro Lys Ala Asp
Asn Asn Phe Asn Lys Glu Xaa Xaa Asn 115 120 125 Ala Phe Tyr Glu Ile
Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Gly Phe
Ile Gln Ser Leu Lys Xaa Xaa Pro Ser Gln Ser Ala Asn 145 150
155 160 Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys
Ala 165 170 175 Asp Asn Lys Phe Asn Lys Glu Xaa Xaa Asn Ala Phe Tyr
Glu Ile Leu 180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser 195 200 205 Leu Lys Xaa Xaa Pro Ser Gln Ser Ala
Asn Leu Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala
Pro Lys Ala Asp Asn Lys Phe Asn Lys 225 230 235 240 Glu Xaa Xaa Asn
Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr 245 250 255 Glu Glu
Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Xaa Xaa Pro Ser 260 265 270
Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275
280 285 Ala Pro Lys 290 45291PRTStaphylococcus aureus 45Ala Gln His
Asp Glu Ala Lys Lys Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met
Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25
30 Lys Ala Ala Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys
35 40 45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Asn
Asn Phe 50 55 60 Asn Lys Asp Lys Lys Ser Ala Phe Tyr Glu Ile Leu
Asn Met Pro Asn 65 70 75 80 Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile
Gln Ser Leu Lys Ala Ala 85 90 95 Pro Ser Gln Ser Thr Asn Val Leu
Gly Glu Ala Lys Lys Leu Asn Glu 100 105 110 Ser Gln Ala Pro Lys Ala
Asp Asn Asn Phe Asn Lys Glu Lys Lys Asn 115 120 125 Ala Phe Tyr Glu
Ile Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Gly
Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn 145 150 155
160 Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala
165 170 175 Asp Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu
Ile Leu 180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly
Phe Ile Gln Ser 195 200 205 Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn
Leu Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro
Lys Ala Asp Asn Lys Phe Asn Lys 225 230 235 240 Glu Lys Lys Asn Ala
Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Thr 245 250 255 Glu Glu Gln
Arg Asn Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser 260 265 270 Val
Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275 280
285 Ala Pro Lys 290 46291PRTStaphylococcus
aureusMOD_RES(60)..(61)Any amino acid except Gln 46Ala Gln His Asp
Glu Ala Lys Lys Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro
Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30
Lys Ala Ala Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35
40 45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Xaa Xaa Asn Asn
Phe 50 55 60 Asn Lys Asp Lys Lys Ser Ala Phe Tyr Glu Ile Leu Asn
Met Pro Asn 65 70 75 80 Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln
Ser Leu Lys Ala Ala 85 90 95 Pro Ser Gln Ser Thr Asn Val Leu Gly
Glu Ala Lys Lys Leu Asn Glu 100 105 110 Ser Gln Ala Pro Lys Ala Asp
Asn Asn Phe Asn Lys Glu Lys Lys Asn 115 120 125 Ala Phe Tyr Glu Ile
Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Gly Phe
Ile Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn 145 150 155 160
Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 165
170 175 Asp Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile
Leu 180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser 195 200 205 Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn Leu
Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro Lys
Ala Asp Asn Lys Phe Asn Lys 225 230 235 240 Glu Lys Lys Asn Ala Phe
Tyr Glu Ile Leu His Leu Pro Asn Leu Thr 245 250 255 Glu Glu Gln Arg
Asn Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser 260 265 270 Val Ser
Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275 280 285
Ala Pro Lys 290 47291PRTStaphylococcus aureus 47Ala Gln His Asp Glu
Ala Lys Lys Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30 Lys
Ala Ala Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35 40
45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Lys Arg Asn Asn Phe
50 55 60 Asn Lys Asp Lys Lys Ser Ala Phe Tyr Glu Ile Leu Asn Met
Pro Asn 65 70 75 80 Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser
Leu Lys Ala Ala 85 90 95 Pro Ser Gln Ser Thr Asn Val Leu Gly Glu
Ala Lys Lys Leu Asn Glu 100 105 110 Ser Gln Ala Pro Lys Ala Asp Asn
Asn Phe Asn Lys Glu Lys Lys Asn 115 120 125 Ala Phe Tyr Glu Ile Leu
Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Gly Phe Ile
Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn 145 150 155 160 Leu
Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 165 170
175 Asp Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile Leu
180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile
Gln Ser 195 200 205 Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn Leu Leu
Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala
Asp Asn Lys Phe Asn Lys 225 230 235 240 Glu Lys Lys Asn Ala Phe Tyr
Glu Ile Leu His Leu Pro Asn Leu Thr 245 250 255 Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro Ser 260 265 270 Val Ser Lys
Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275 280 285 Ala
Pro Lys 290 48292PRTStaphylococcus aureus 48Met Ala Gln His Asp Glu
Ala Lys Lys Asn Ala Phe Tyr Gln Val Leu 1 5 10 15 Asn Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser 20 25 30 Leu Lys
Ala Ala Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln 35 40 45
Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Lys Arg Asn Asn 50
55 60 Phe Asn Lys Asp Lys Lys Ser Ala Phe Tyr Glu Ile Leu Asn Met
Pro 65 70 75 80 Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser
Leu Lys Ala 85 90 95 Ala Pro Ser Gln Ser Thr Asn Val Leu Gly Glu
Ala Lys Lys Leu Asn 100 105 110 Glu Ser Gln Ala Pro Lys Ala Asp Asn
Asn Phe Asn Lys Glu Lys Lys 115 120 125 Asn Ala Phe Tyr Glu Ile Leu
Asn Met Pro Asn Leu Asn Glu Glu Gln 130 135 140 Arg Asn Gly Phe Ile
Gln Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala 145 150 155 160 Asn Leu
Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys 165 170 175
Ala Asp Asn Lys Phe Asn Lys Glu Lys Lys Asn Ala Phe Tyr Glu Ile 180
185 190 Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe Ile
Gln 195 200 205 Ser Leu Lys Ala Ala Pro Ser Gln Ser Ala Asn Leu Leu
Ala Glu Ala 210 215 220 Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala
Asp Asn Lys Phe Asn 225 230 235 240 Lys Glu Lys Lys Asn Ala Phe Tyr
Glu Ile Leu His Leu Pro Asn Leu 245 250 255 Thr Glu Glu Gln Arg Asn
Gly Phe Ile Gln Ser Leu Lys Ala Ala Pro 260 265 270 Ser Val Ser Lys
Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala 275 280 285 Gln Ala
Pro Lys 290 49291PRTStaphylococcus aureusMOD_RES(7)..(8)Any amino
acid except GlnMOD_RES(34)..(35)Any amino acid except
AspMOD_RES(60)..(61)Any amino acid except GlnMOD_RES(68)..(69)Any
amino acid except GlnMOD_RES(95)..(96)Any amino acid except
AspMOD_RES(126)..(127)Any amino acid except
GlnMOD_RES(153)..(154)Any amino acid except
AspMOD_RES(184)..(185)Any amino acid except
GlnMOD_RES(211)..(212)Any amino acid except
AspMOD_RES(242)..(243)Any amino acid except
GlnMOD_RES(269)..(270)Any amino acid except Asp 49Ala Gln His Asp
Glu Ala Xaa Xaa Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro
Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30
Lys Xaa Xaa Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35
40 45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Xaa Xaa Asn Asn
Phe 50 55 60 Asn Lys Asp Xaa Xaa Ser Ala Phe Tyr Glu Ile Leu Asn
Met Pro Asn 65 70 75 80 Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln
Ser Leu Lys Xaa Xaa 85 90 95 Pro Ser Gln Ser Thr Asn Val Leu Gly
Glu Ala Lys Lys Leu Asn Glu 100 105 110 Ser Gln Ala Pro Lys Ala Asp
Asn Asn Phe Asn Lys Glu Xaa Xaa Asn 115 120 125 Ala Phe Tyr Glu Ile
Leu Asn Met Pro Asn Leu Asn Glu Glu Gln Arg 130 135 140 Asn Gly Phe
Ile Gln Ser Leu Lys Xaa Xaa Pro Ser Gln Ser Ala Asn 145 150 155 160
Leu Leu Ser Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Ala 165
170 175 Asp Asn Lys Phe Asn Lys Glu Xaa Xaa Asn Ala Phe Tyr Glu Ile
Leu 180 185 190 His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly Phe
Ile Gln Ser 195 200 205 Leu Lys Xaa Xaa Pro Ser Gln Ser Ala Asn Leu
Leu Ala Glu Ala Lys 210 215 220 Lys Leu Asn Asp Ala Gln Ala Pro Lys
Ala Asp Asn Lys Phe Asn Lys 225 230 235 240 Glu Xaa Xaa Asn Ala Phe
Tyr Glu Ile Leu His Leu Pro Asn Leu Thr 245 250 255 Glu Glu Gln Arg
Asn Gly Phe Ile Gln Ser Leu Lys Xaa Xaa Pro Ser 260 265 270 Val Ser
Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln 275 280 285
Ala Pro Lys 290 5067PRTStaphylococcus aureusMOD_RES(7)..(8)Any
amino acid except GlnMOD_RES(34)..(35)Any amino acid except
AspMOD_RES(60)..(61)Any amino acid except Gln 50Ala Gln His Asp Glu
Ala Xaa Xaa Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30 Lys
Xaa Xaa Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35 40
45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Xaa Xaa Asn Asn Phe
50 55 60 Asn Lys Asp 65 5167PRTStaphylococcus
aureusMOD_RES(7)..(8)Any amino acid except GlnMOD_RES(34)..(35)Any
amino acid except Asp 51Ala Gln His Asp Glu Ala Xaa Xaa Asn Ala Phe
Tyr Gln Val Leu Asn 1 5 10 15 Met Pro Asn Leu Asn Ala Asp Gln Arg
Asn Gly Phe Ile Gln Ser Leu 20 25 30 Lys Xaa Xaa Pro Ser Gln Ser
Ala Asn Val Leu Gly Glu Ala Gln Lys 35 40 45 Leu Asn Asp Ser Gln
Ala Pro Lys Ala Asp Ala Lys Arg Asn Asn Phe 50 55 60 Asn Lys Asp 65
5267PRTStaphylococcus aureus 52Ala Gln His Asp Glu Ala Lys Lys Asn
Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met Pro Asn Leu Asn Ala Asp
Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25 30 Lys Ala Ala Pro Ser
Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys 35 40 45 Leu Asn Asp
Ser Gln Ala Pro Lys Ala Asp Ala Lys Arg Asn Asn Phe 50 55 60 Asn
Lys Asp 65 5368PRTStaphylococcus aureus 53Met Ala Gln His Asp Glu
Ala Lys Lys Asn Ala Phe Tyr Gln Val Leu 1 5 10 15 Asn Met Pro Asn
Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser 20 25 30 Leu Lys
Ala Ala Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln 35 40 45
Lys Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Lys Arg Asn Asn 50
55 60 Phe Asn Lys Asp 65 5467PRTStaphylococcus aureus 54Ala Gln His
Asp Glu Ala Gln Gln Asn Ala Phe Tyr Gln Val Leu Asn 1 5 10 15 Met
Pro Asn Leu Asn Ala Asp Gln Arg Asn Gly Phe Ile Gln Ser Leu 20 25
30 Lys Asp Asp Pro Ser Gln Ser Ala Asn Val Leu Gly Glu Ala Gln Lys
35 40 45 Leu Asn Asp Ser Gln Ala Pro Lys Ala Asp Ala Gln Gln Asn
Asn Phe 50 55 60 Asn Lys Asp 65
* * * * *