U.S. patent application number 13/846166 was filed with the patent office on 2013-08-08 for peptide vaccines against group a streptococci.
This patent application is currently assigned to Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Edwin W. Ades, Bernard W. Beall, George M. Carlone, Jacquelyn S. Sampson.
Application Number | 20130202635 13/846166 |
Document ID | / |
Family ID | 23122046 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130202635 |
Kind Code |
A1 |
Beall; Bernard W. ; et
al. |
August 8, 2013 |
PEPTIDE VACCINES AGAINST GROUP A STREPTOCOCCI
Abstract
This invention, in one aspect, relates to synthetic
immunoreactive peptides. These peptides are approximately 20-25
amino acids in length which are portions of the N termini of the M
proteins of the most prevalent United States (U.S.) Group A
Streptococcus (GAS) serotypes. At least some of the synthetic
peptides can be recognized by M type-specific antibodies and are
capable of eliciting functional opsonic antibodies and/or
anti-attachment antibodies without eliciting tissue cross-reactive
antibodies. In another aspect, it relates to compositions or
vaccines comprising these synthetic serotype-specific peptides,
including polypeptides and proteins. The invention may also be
isolated antibodies which are raised in response to the peptides,
compositions or vaccines. The invention further relates to kits for
using the peptides, compositions, or antibodies. In still further
aspects, the invention also relates to methods for using the
peptides, compositions, vaccines, or antibodies and methods for
tailoring vaccines.
Inventors: |
Beall; Bernard W.;
(Doraville, GA) ; Carlone; George M.; (Stone
Mountain, GA) ; Sampson; Jacquelyn S.; (College Park,
GA) ; Ades; Edwin W.; (Atlanta, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Department of Health and Human Services; The United States of
America, as represented by the Secretary, |
Atlanta |
GA |
US |
|
|
Assignee: |
Services
Atlanta
GA
The United States of America, as represented by the Secretary,
Department of Health and Human
|
Family ID: |
23122046 |
Appl. No.: |
13/846166 |
Filed: |
March 18, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13427477 |
Mar 22, 2012 |
8420107 |
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13846166 |
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12973247 |
Dec 20, 2010 |
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13427477 |
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12144461 |
Jun 23, 2008 |
7883710 |
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12973247 |
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10477955 |
Mar 15, 2004 |
7407664 |
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PCT/US2002/015909 |
May 20, 2002 |
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12144461 |
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60291835 |
May 18, 2001 |
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Current U.S.
Class: |
424/192.1 ;
530/326 |
Current CPC
Class: |
A61P 31/04 20180101;
C07K 14/315 20130101; A61K 39/00 20130101; A61K 39/092 20130101;
A61P 37/04 20180101; C07K 2319/00 20130101; C07K 14/001
20130101 |
Class at
Publication: |
424/192.1 ;
530/326 |
International
Class: |
A61K 39/09 20060101
A61K039/09; C07K 14/00 20060101 C07K014/00 |
Claims
1. A composition comprising a polypeptide comprising the amino acid
sequence of SEQ ID NO: 32.
2. The composition of claim 1, wherein the polypeptide is a
chimeric fusion protein.
3. The composition of claim 2, wherein the chimeric fusion protein
comprises an amino acid sequence of one or more heterologous Group
A Streptococcus (GAS) M-type peptides.
4. The composition of claim 1, wherein the polypeptide is linked to
a peptide backbone.
5. The composition of claim 1, further comprising a
pharmaceutically acceptable vehicle or carrier.
6. The composition of claim 1, further comprising additional
immune-stimulatory molecules.
7. The composition of claim 6, wherein the additional
immune-stimulatory molecules comprise GAS-based peptides, non-GAS
vaccines/immunogens selected from the group consisting of
Hemophilus influenza, pertussis, N. meningitidis, pneumococcus, and
Influenzae, or adjuvants.
8. The composition of claim 1, wherein the polypeptide consists
essentially of the amino acid sequence of SEQ ID NO: 32.
9. The composition of claim 1, wherein the polypeptide consists of
the amino acid sequence of SEQ ID NO: 32.
10. An immunogenic composition for eliciting an immune response to
Group A Streptococcus comprising an immunogenic amount of the
composition of claim 1.
11. The immunogenic composition of claim 10, wherein the
immunogenic composition is capable of eliciting functional opsonic
antibodies and does not contain epitopes that cross-react with
tissues.
12. The immunogenic composition of claim 10, wherein the
immunogenic composition is effective in decreasing the
nasopharyngeal reservoir of GAS when administered.
13. A method for inducing an immune response against the emm12
serotype of Group A Streptococcus, comprising administering an
immunogenic amount of the immunogenic composition of claim 1 to a
subject.
14. The method of claim 13, wherein the administration is via
injection or a mucosal delivery method.
15. The method of claim 13, wherein the immune response comprises
induction of opsonic antibodies.
16. An isolated polypeptide comprising the amino acid sequence
according to SEQ ID NO: 32.
17. The polypeptide of claim 16, wherein the polypeptide is a
chimeric fusion protein.
18. The polypeptide of claim 17, wherein the chimeric fusion
protein comprises an amino acid sequence of one or more
heterologous Group A Streptococcus (GAS) M-type peptides.
19. The isolated polypeptide of claim 16, wherein the polypeptide
consists essentially of the amino acid sequence of SEQ ID NO:
32.
20. The isolated polypeptide of claim 16, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO: 32.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. patent application Ser. No.
13/427,477, filed Mar. 22, 2012, which is a divisional of U.S.
patent application Ser. No. 12/973,247, filed Dec. 20, 2010, now
abandoned, which is a divisional of U.S. patent application Ser.
No. 12/144,461, filed Jun. 23, 2008, now U.S. Pat. No. 7,883,710,
issued Feb. 8, 2011, which is a continuation of U.S. patent
application Ser. No. 10/477,955, filed Mar. 15, 2004, now U.S. Pat.
No. 7,407,664, issued Aug. 5, 2008, which is the .sctn.371 U.S.
National Stage of International Application PCT/US2002/015909,
filed May 20, 2002, which claims the benefit of U.S. Provisional
Application 60/291,835, filed May 18, 2001, all of which are
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] This invention relates generally to immunoreactive
molecules, compositions, and methods related thereto. Specifically,
those related to Group A Streptococci. More specifically, it
relates to synthetic Group A strep immunoreactive peptides,
compositions comprising the peptide sequences, vaccines, isolated
antibodies elicited by the peptides, kits comprising the peptides
or antibodies, and methods of using the peptides, compositions,
vaccines and antibodies.
BACKGROUND OF THE INVENTION
[0003] Group A streptococci (GAS) are responsible for a wide
variety of diseases. These range from umcomplicated pharyngitis to
more serious invasive diseases such as necrotizing fasciitis
("flesh eating syndrome") and streptococcal toxic shock syndrome.
Additionally, approximately 3% of GAS infections that go untreated
will result in acute rheumatic fever. (Brandt, E. R., Good, M. F.
1999. Vaccine strategies to prevent rheumatic fever. Immunol. Res.
19:89-103) All ages are susceptible to GAS attack, but those
particularly vulnerable are the elderly, children under 2 years,
and African Americans. (Emerging Infections Programs (EIP),
supported by the National Center for Infectious Diseases for
isolates resulting from active surveillance 1995-1997. California
EIP: Arthur Reingold; Connecticut EIP: Matt Carter; Georgia EIP,
Monica Farley; Minnesota EIP, Kristine MacDonald; Oregon EIP, Paul
Cieslak; Centers for Disease Control and Prevention (CDC), K.
O'Brien, B. Beall, K. Deaver-Robinson, R. Facklam, A. Kraus, A.
Schuchat, B. Schwartz) Recently, there has been a significant
increase in the number of streptococcal infections (Davies, H. D.,
McGeer, A., Schwartz, B., et al. 1996. Invasive group A
streptococcal infections in Ontario, Canada. Ontario group A
streptococcal study group. N. Engl. J. Med. 335:547-54) as well as
rheumatic fever (Veasey, L. G., Wiedneier, S. W., Osmond, G. S., et
al. Resurgence of acute rheumatic fever in the intermountain region
of the United States. N. Engl. J. Med. 316:42-7). Based on recent
active surveillance, it is estimated that there are approximately
8,500 cases and 1,300 deaths annually in the United States from
invasive GAS disease, (EIP supported by the National Center for
Infectious Diseases for isolates resulting from active surveillance
1995-1997. California EIP: Arthur Reingold; Connecticut EIP: Matt
Carter; Georgia EIP, Monica Farley; Minnesota EIP, Kristine
MacDonald; Oregon EIP, Paul Cieslak; CDC, K. O'Brien, B. Beall, K.
Deaver-Robinson, R. Facklam, A. Kraus, A. Schuchat, B.
Schwartz).
[0004] A vaccine against GAS could eliminate millions of dollars in
health care costs and numerous physician visits.
[0005] There are a number of strategies that have been used towards
designing an effective streptococcal vaccine (Salvadori, L. G.,
Blake, M. S., McCarty, M., Tai, J. Y., Zabriskie, J. B. 1995. Group
A streptococcus-liposome ELISA antibody titers to group A
polysaccharide and opsonophagocytic capabilities of the antibodies.
J. Infect. Dis. 171:593-600; Ji, Y. Carlson, B., Kondagunta, A.,
Cleary, P. P. 1997. Intranasal immunization with C5a peptidase
prevents nasopharyngeal colonization of mice by group A
streptococcus. Infect. Immun. 65:2080-2087; Kapur, V. Maffei, J.
T., Greer R. S., Li, L. L., Adams, G. J., Musser, J. M. 1994.
Vaccination with streptococcal cysteine protease protects mice
against challenge with heterologous group A streptococci. Microb.
Pathogenesis. 16:443-450; Dale, J. B., Baird, R. W., Courtney, H.
S., Hasty, D. L., Bronze, M. S. 1994. Passive protection of mice
against group A streptococcal pharyngeal infection by lipoteichoic
acid. J. Infect. Dis. 169:319-323; Dale, J. B., Washburn, R. G.,
Marques, M. B., Wessels, M. R. 1996. Hyuaronated capsule and
surface M protein in resistance to opsonization of group A
streptococci. Infect. Immun. 64:1495-1501; Fischetti, V. A. 1989.
Streptococcal M protein: molecular design and biological behavior.
Clin. Microbiol. 2:285-314; Lancefield, R. C. 1962. Current
knowledge of the type-specific M antigens of group A streptococci.
J. Immun. 89:307-313; Lancefield, R. C. 1959. Persistence of
type-specific antibodies in man following infection with group A
streptococci. J. Exp. Med. 110:271-283).
[0006] There are difficulties associated with a vaccine strategy
involving the M protein, such as the large number of serologic M
(emm) types (over 100 serotypes) and the observation that some M
proteins contain epitopes that cross-react with human tissues. In
addition to the large number of serotypes, every population has a
different subset of GAS serotypes which are the most prevalent. In
order to deal with these difficulties, different approaches have
been tried. For example, observation that the M protein's
C-terminus is conserved while the N-terminus is variable has led
some workers to try to focus on the C-terminus for broader
protection and others to focus on the N-terminus where the most
variability is.
[0007] Even though some M protein-based vaccines have been
designed, for the above reasons, a need still exists for a
flexible, effective, multivalent GAS vaccine.
SUMMARY OF THE INVENTION
[0008] In accordance with the purpose(s) of this invention, as
embodied and broadly described herein, this invention, in one
aspect, relates to immunoreactive peptides. In another aspect, it
relates to compositions or vaccines comprising the peptides,
including polypeptides and proteins.
[0009] The synthetic peptides of the invention are approximately
20-25 amino acids in length which are portions of the N termini of
the M proteins of the most prevalent United States (U.S.) GAS
serotypes and which are immunoreactive. At least some of the
synthetic peptides can be recognized by M type-specific antibodies
and are capable of eliciting functional opsonic antibodies and/or
anti-attachment antibodies without eliciting tissue cross-reactive
antibodies.
[0010] The invention is also a composition or a vaccine comprised
of these synthetic serotype-specific peptides of 20-25 amino acids
in length from GAS M proteins. The peptides can be used, for
example, individually, in a mixture, or in a polypeptide or
protein. Examples of ways the polypeptide or protein can be created
include fusing or linking the peptides to each other, synthesizing
the polypeptide or protein based on the peptide sequences, and
linking or fusing the peptides to a backbone. Also, a liposome may
be prepared with the peptides conjugated to it or integrated within
it. The compositions or vaccines may further comprise additional
components, including but not limited to, carriers, vehicles (e.g.,
encapsulated, liposomes), and other immune-stimulatory molecules
(e.g., adjuvants, other vaccines). Additionally, a DNA vaccine
comprising DNA encoding the peptides or compositions of the present
invention is disclosed.
[0011] The invention may also be isolated antibodies which are
elicited in response to the peptides, compositions or vaccines.
[0012] In further aspects, the invention also relates to methods
for using the peptides, compositions, vaccines, or antibodies and
methods for tailoring vaccines. The invention still further relates
to kits for using the peptides or antibodies, which can, for
example, be used for diagnostic purposes.
[0013] Additional aspects of the invention will be set forth in
part in the description which follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The aspects of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The accompanying drawing, which is incorporated in and
constitute a part of this specification, illustrates several
aspects of the invention and together with the description, serves
to explain the principles of the invention.
[0015] FIG. 1 shows a bar graph of a population-based U.S. sterile
site invasive isolate distribution among the 24 most prevalent emm
types (2321 (88.9%) of the entire 2612 isolate sample) of Group A
strep. The 10 most prevalent isolates (emm1, emm3, emm28, emm12,
emm4, emm11, emm89, st2967, emm77/27L, emm6) account for 65% of the
disease.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0016] Before the present compounds, compositions, articles,
devices, and/or methods are disclosed and described, it is to be
understood that this invention is not limited to specific peptides,
specific synthetic methods, specific compositions, specific
vaccines, specific antibodies, specific kits, specific methods of
use, as such may, of course, vary. It is also to be understood that
the terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting.
[0017] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a peptide" includes mixtures of
peptides, reference to "a carrier" includes mixtures of two or more
carriers, and the like.
[0018] Ranges may be expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. It will be further understood that the
endpoints of each of the ranges are significant both in relation to
the other endpoint, and independently of the other endpoint.
[0019] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0020] The terms "peptide", "polypeptide" and "protein" are used
interchangeably and as used herein refer to more than one amino
acid joined by a peptide bond.
[0021] "Optional" or "optionally" means that the subsequently
described event or circumstance may or may not occur, and that the
description includes instances where said event or circumstance
occurs and instances where it does not.
[0022] By the term "effective amount" of a compound as provided
herein is meant a nontoxic but sufficient amount of the compound to
provide the desired biological effect. As will be pointed out
below, the exact amount required will vary from subject to subject,
depending on the species, age, and general condition of the
subject, the particular compound used, its mode of administration,
and the like. Thus, it is not possible to specify an exact
"effective amount." However, an appropriate effective amount may be
determined by one of ordinary skill in the art using only routine
experimentation.
[0023] By "pharmaceutically acceptable" is meant a material that is
not biologically or otherwise undesirable, i.e., the material may
be administered to an individual without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the composition in which it is
contained.
[0024] "Synthetic" is meant to encompass items, e.g., peptides,
which are not naturally occurring, in that they are isolated,
synthesized, or otherwise manipulated by man.
[0025] "Immunoreactive" as used herein is meant to encompass
materials which are capable of reacting with a specific antigen.
"Antigenic" and "immunogenic" are terms which fall within the scope
of the term "immunoreactive".
[0026] "Composition" as used throughout the specification and
claims is meant to include any composition of matter, including the
peptides, polypeptides, proteins, mixtures, vaccines, antibodies or
other forms of matter of the present invention. It is meant to be
used generically and interchangeably with the other composition of
matter terms, and if used in addition to the other terms, it is
used for sake of completeness. "Composition" is a broad term
overlapping the coverage of these more specific terms and when used
in addition to these more specific terms it is not meant as an
indication that it is necessarily different from these more
specific terms.
[0027] In one aspect, the present invention provides synthetic
peptides, compositions, and a vaccine made therefrom and isolated
antibodies elicited by administration thereof. The invention also
provides methods for using the peptides, compositions, vaccines, or
antibodies such as, vaccination of recipients. The invention
further provides a method for tailoring vaccines. The invention
additionally provides kits for using the peptides or
antibodies.
Peptides
[0028] The invention is synthetic peptides of approximately 20-25
amino acids in length selected from a section of approximately 45
amino acids from the most N terminal region of the M proteins of
the most prevalent U.S. Group A Streptococcus (GAS) serotypes which
are immunoreactive. At least some of the peptides are capable of
eliciting opsonic antibodies and/or anti-attachment antibodies to
the GAS serotypes without eliciting tissue cross-reactive
antibodies. In one aspect of the invention, the synthetic peptides
are from the most prevalent invasive U.S. GAS serotypes which are
immunoreactive. The prevalence data in FIG. 1 includes data from
invasive isolates. The most frequently occurring invasive types
reflect the incidence rate of the same types found in non-invasive
isolates. Specific peptides of the present invention are shown
below in Table 1. One aspect of the invention is a peptide
consisting essentially of the amino acid sequence of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID
NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ
ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39,
SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ
ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID
NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ
ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67,
SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID
NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ
ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81,
SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ
ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95,
SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104,
SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113,
SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122,
SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131,
SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID
NO:136, SEQ ID NO:137, or SEQ ID NO:138.
[0029] Examples of the peptides of the invention, several for each
of the 25 most common serotypes (138 peptides), are as follows:
TABLE-US-00001 TABLE 1 Synthesized Type-Specific Peptides. Seq
Peptide ID design- No: Serotype ation Peptide 1 M1 M1-1
CNGDGNPREVIEDLAANNPAIQ 2 M1-2 CIQNIRLRHENKDLKARLENA 3 M1-3
CIRLRHENKDLKARLENAMEV 4 M1-4 CNGDGNPREVIEDLAANNPAME 5 M1-5
CIRNIRLRHENKDLKARLENA 6 M1-6 CNGDGNPREVIEDLAANNPVIQ 7 M1-7
CNGDGNPRVVIEDLAANNPAIQ 8 M1-8 CIRLRHHENKDLKARLENAMEV 9 M2 M2-1
CNSKNPVPVKKEAKLSEAELHDK 10 M2-2 CKKEAKLSEAELHDKIKNLEEEK 11 M2-3
CELHDKIKNLEEEKAELFEKLD 12 M2-4 CELFEKLDKVEEEHKKVEEEHKK 13 M3 M3-1
CDARSVNGEFPRHVKLKNEIE 14 M3-2 CGEFPRHVKLKNEIENLLDQV 15 M3-3
CLDQVTQLYTKHNSNYQQYNA 16 M3-4 CLDQVTQLYNKHNSNYQQYSA 17 M3-5
CLDQVTQLYTKHNSNYQQYSA 18 M3-6 CLNQVTQLYTKHNSNYQQYNA 19 M3-7
CLAQVTQLYTKHNSNYQQYNA 20 M3-8 CLNQVTQLHTKHNSNYQQYNA 21 M3-9
CRSDARSVNGEFPRHVKLKNE 22 M3-10 CQLYTKHIYTKHNSNYQQYNAQ 23 M3-11
CTQLYTKHNSNYQQYNAQAGR 24 M4 M4-1 CAEIKKPQADSAWNWPKEYNA 25 M4-2
CDSAWNWPKEYNALLKENEEL 26 M4-3 CKENEELKVEREKYLSYADDK 27 M4-4
CEELKVEREKYLSYADDKEKDPQ 28 M11 M11-1 CAGQSAPKGTNVSADLYNSLWDE 29
M11-2 CKGTNVSADLYNSLWDENKT 30 M11-3 CDENKTLREKQEEYITKIQNE 31 M11-4
CTEVKAAGQSAPKGTNVSADL 32 M12 M12-1 CDHSDLVAEKQRLEDLGQKFE 33 M12-2
CAEKQRLEDLGQKFERLKQRS 34 M12-3 CLEDLGQKFERLKQRSELYLQ 35 M12-4
CKFERLKQRSELYLHQYYDNK 36 M12-5 CKFERLKRRSELYLQQYYDNK 37 M12-6
CKQRSELYLQQYYDNKSNRYK 38 M12-7 CSELYLQQYYDNKSNGYKGDW 39 M22 M22-1
CESSNNAESSNISQESKLINT 40 M22-2 CESSNISQESKLINTLTDENEK 41 M22-3
CESKLINTLTDENEKLREELQQ 42 M22-4 CNTLNTLTDENEKLREELQQ 43 M22-5
CESSNISQESKLINTLTDENEK 44 M22-6 CEKLREELQQYYALSDAKEEE 45 M28 M28-1
CAESPKSTETSANGADKLAD 46 M28-2 CKSTETSANGADKLADAYNTL 47 M28-3
CDKLADAYNTLLTEHEKLRDE 48 M28-4 CTEHEKLRDEYYTLIDAKLEEK 49 M28-5
CTEHEKLRDEYYTLIDAKEEE 50 M77 M77-1 CEGVSVGSDASLHNRITDLEEEREK 51
M77-2 CSDASLHNRITDLEEEREKLLNK 52 M77-3 CDLEEEREKLLNKLDKVEEEHKKD 53
M77-4 CDLEEERGKLLNKLDKVEEEHK 54 M77-5 CLNKLDKVEEEHKKDHEQLEK 55 M89
M89-1 CDSDNINRSVSVKDNEKELHNK 56 M89-2 CDNINRSVSVKDNEKELHNKIAD 57
M89-3 CSVKDNEKELHNKIADLEEER 58 M89-4 CELHNEIADLEEERGEHLDKID 59
M89-5 CELHNKIADLEEERGAHLDKID 60 M89-6 CDSDNINRFVSVKDNEKELHN 61
M89-7 CDSDNSDNINRSVSVKDNEKE 62 M89-8 CLEEERGEHLDKIDELKEELK 63
st2967 st2967-1 CNSKNPAPAPASAVPVKKEATK 64 st2967-2
CVPVKKEATKLSEAELYNKIQ 65 st2967-3 CKKEATKLSEAELYNKIQELEE 66
st2967-4 CNSKNPAPAPAVPVKKEATKL 67 st2967-5 CNSKNPAPAVPVKKEATKLSE 68
st2967-6 CAELYNKIQELEEGKAELFDK 69 M6 M6-1 CRVFPRGTVENPDKARELLNK 70
M6-2 CRGTVENPDKARELLNKYDVEN 71 M6-3 CENPDKARELLNKYDVENSMLQ 72 M6-4
CENSMLQANNDNLTDQNKNLTD 73 M6-5 CNSMLQANNDKLTTENKNLTD 74 M82 M82-1
CDSSSRDITEAGVSKFWKSKFD 75 M82-2 CRDITEAGVSKFWKSKFDAEQN 76 M82-3
CEAGVSKFWKSKFDAEQNRANE 77 M82-4 CDAEQNRANELEKKLSGYEKD 78 M43 M43-1
CEEHPDVVAARESVLNNVR 79 M43-2 CHPDVVAARESVLNNVRVPGT 80 M43-3
CRVPGTLWLRQKEENDKLKLEK 81 M43-4 CLRQKEENDKLKLEKKGLETE 82 M75 M75-1
CEEERTFTELPYEARYKAWKSE 83 M75-2 CELPYEARYKAWKSENDELREN 84 M75-3
CNDELRENYRRTLDKFNTEQ 85 M75-4 CKAWKSENDELRENYRKTLDK 86 M75-5
CRENYRRTLDKFNTEQGKTTR 87 M33 M33-1 CEEHEKVTQAREAVIREMQQR 88 M33-2
CHEKVTQAREAVIREMQQRGT 89 M33-3 CEMQQRGTNFGPLLASTMRDNH 90 M92 M92-1
CDDRSVSTNSGSVSTPYNNLLNE 91 M92-2 CRSVSTNSGSVSTPYNNLLNE 92 M92-3
CEYDDLLAKHGELLSEYDALK 93 M92-4 CDLLAKHGELLSEYDALKEKQDK 94 M5 M5-1
CTVTRGTINDPQRAKEALDKYE 95 M5-2 CDPQRAKEALDKYELENHDLK 96 M5-3
CENHDLKTKNEGLKTENEGLK 97 M5-4 CQRAKAALDKYELENHDLKTKN 98 M5-5
CTVTRGTVNDPQRAKEALDKYE 99 M5-6 CTVTRGTVNDPQRAKETLDKYE 100 M5-7
CTVTRGTINDPQRAKEVIDKYE 101 M5-8 CTVTRSTINDPQRAKEALDKYE 102 M5-9
CHDLKTKNEGLKTENEGLKTEN 103 M94 M94-1 CEEASNNGQLTLQHKNNALTSE 104
(formerly M94-2 CQHKNNALTSENESLRREKDR 105 emm13W) M94-3
CESLRREKDRYLYEKEELEKK 106 M94-4 CRREEKDRYLYEKEELEKKNK 107 M73 M73-1
CDNQSPAPVKKEAKKLNEAE 108 M73-2 CKKEAKKLNEAELYNKIQELE 109 M73-3
CELYNKIQELEEGKAELFDKLEK 110 M73-4 CDNQSPALVKKEAKKLNEAEL 111 M73-5
CDNQSPAPAPVKKEAKKLNEAE 112 M73-6 CQELEEGKAELFDKLEKVEEE 113 M18
M18-1 CAAPLTRATADNKDELIKRAND 114 M18-2 CRATADNKDELIKRANDYEIQ 115
M18-3 CEIQNHQLTVENKKLKTDKEQ 116 M18-4 CRATADNKDELIKRANGYEIQ 117
M18-5 CKDELIKRKELTIIEIQNHQL 118 M18-6 CNHQLTVENKKLKTDKEQLTKE 119
M58 M58-1 CDSSREVTNELTASMWKAQAD 120 M58-2 CREVTNELTASMWKAQADSAK 121
M58-3 CKAKELEKQVEEYKKNYETLEK 122 M58-4 CDSSREVTNELAASMWKAQAD 123
M58-5 CDSSRDSSREVTNELTASMWK 124 M58-6 CKAKELEKQVEEYKKNYETLEK 125
M59 M59-1 CEQAKNNNGELTLQQKYDALT 126 M59-2 CELTLQQKYDALTNENKSLRRE
127 M59-3 CNENKSLRRERDNYLNYLYEK 128 M59-4 CRRERDNYLNYLYEKEELEKK 129
M101 M101-1 CADHPSYTAAKDEVLSKFSVPGH 130 (formerly M101-2
CKDEVLSKFSVPGHVWAHERE 131 stNS5) M101-3 CHEREKNDKLSSENEGLK 132
M101-4 CDKLRLEKEELKTDLQKKERE 133 M101-5 CKNDKLSSENEGLKAGLQEKE 134
M41 M41-1 CEGNARLAQAQEEALRDVLNN 135 M41-2 CRLAQAQEEALRDVLNNTPHN 136
M41-3 CQAQEEALRDVLNNTPHNQLRD 137 M41-4 CDVLNNTPHNQLRDAYAGAFRR 138
M41-5 CQLRDPYAGAFRRNNELEKIIQE
[0030] It is important to note that a single peptide representing
each of the 25 M serotypes represented is predicted to protect
against the majority of invasive GAS within each of these serotypes
in the U.S. For the majority of these types, the invention provides
a peptide that actually matches the sequences of all GAS of these
types that we have encountered. We include additional peptides that
encompass the extent of M protein gene allelic variation that we
have encountered to date within each type from various geographic
locations. It is important to note that for the majority of these
types, at least one peptide is conserved among all allelic variants
that we have encountered.
[0031] The small size of the peptides used in the current invention
allows a flexible approach for formulating compositions or
vaccines. The formulations can be readily and inexpensively changed
to account for changes in GAS serotype frequencies in the target
population. The adaptability includes frequency changes between
populations, years, and the like. Any population for which there is
frequency data can have a vaccine formulation customized for it by
the methods of the present invention, which are discussed
below.
[0032] Recently a rapid M protein gene-based subtyping system has
been initiated which predicts the type-specific portion of the M
protein with very high efficiency. (Beall, B., Facklam, R., Hoenes,
T., Schwartz, B. 1997. A survey of emm gene sequences from systemic
Streptococcus pyogenes infection isolates collected in San
Francisco, Calif.; Atlanta, Ga.; and Connecticut state in 1994 and
1995. J. Clin. Microbiol. 35:1231-1235; Beall, B., Facklam, R.,
Elliot, J., Franklin, A., Hoenes, T., Jackson, D., Laclaire, L.,
Thompson, T., Viswanathan, R. 1998. Streptococcal emm types
associated with T-agglutination types and the use of conserved emm
restriction fragment patterns for subtyping group A Streptococci.
J. Med. Micro. 47:1-6). A Centers for Disease Control and
Prevention (CDC) surveillance system used this rapid gene based M
subtyping system to gather epidemiological data which showed that
the 30 most prevalent invasive serotypes account for approximately
95% of the total invasive isolates in the U.S.
[0033] In addition, these peptides should have direct use in
formulating vaccines for countries other than the U.S. For example,
the 25 serotypes represented in Table 1 also appear to encompass
the majority of GAS pediatric pharyngitis isolates in Rome, Italy
(91/114=80%), 85% (367/430) of a mixture of sterile and non-sterile
GAS isolates recovered in Mexican patients, and 80% (110/137) of
primarily invasive isolates recently recovered from patients in
Argentina.
[0034] It is also important to note that data indicates that these
25 serotypes would have less coverage in other geographic areas
such as Malaysia, India, New Guinea, Nepal, and Egypt. For example,
out of 136 pharyngitis and impetigo isolates recently recovered in
Egypt, only 62 (46%) were of one of these 25 types. While type emm1
is by far the most prevalent type recovered from invasive and
noninvasive U.S. isolates (about 20%), only 5/136 (4%) Egypt
isolates were type emm1. Thus, the methods of the present invention
could be used to tailor vaccines or compositions with the serotypes
most prevalent in these areas.
[0035] The peptides are synthesized by any of the techniques known
in the art, as the method of making them is not critical. One
technique is through recombinant methods. Another is manual or
automated chemical synthesis using individual amino acids, such as
solid phase peptide synthesis. Other methods for synthesizing
peptides may be readily apparent to one of ordinary skill in the
art.
[0036] One of ordinary skill in the art would be able to determine
through routine experimentation which of the immunoreactive
peptides are capable of eliciting opsonic and/or anti-attachment
antibodies.
[0037] Though it is known generally in the art that even single
substitutions may have a great impact on immunogenicity of a
molecule, due to allelic variants which exist for any particular
GAS serotype, there are expected to be allowable substitutions
within the peptides corresponding with each serotype which maintain
immunogenicity. As discussed above, the example peptides in Table 1
include allelic variants of the peptides for a given serotype. For
example, up to approximately 3 substitutions within each peptide
which correspond with variants of a given serotype may create
peptides which also are immunoreactive. That a given substitution
results in an immunoreactive peptide can be determined by routine
experimentation by making a proposed substitution then testing the
immunoreactivity by one of many known assays including those
described herein. A variant within a serotype can be identified on
the basis of sequence. Any variation within 50 N-terminal residues
of mature protein of M protein gene type strain is considered a
variant. Isolates within an emm type share about .gtoreq.84%
deduced amino acid sequence identity [as determined by the
Wisconsin Package Version 10.1, Genetics Computer Group (GCG),
Madison Wisc. FASTA program] within the mature amino terminal 45
amino acids compared to the reference type strain sequence. The
first three peptides indicated for each serotype in Table 1 are
considered to be the peptides from the majority of isolates of the
serotype. The additional peptides given in Table 1 for each
serotype in some instances represent the majority of isolates in
the type, and in other instances represent known variants of these
types.
[0038] At least some of the individual peptides are capable of
protecting a recipient against its corresponding serotype. A
composition comprising a mixture of peptides from more than one
serotype is able to protect against those corresponding serotypes.
A mixture can be tailored such that it contains the most prevalent
serotypes in an area (population), thus making the mixture able to
protect against the most important serotypes. The tailoring is
accomplished by matching the serotype-specific peptides to
epidemiological data regarding the prevalence of the serotypes for
the population of recipients desired to be protected.
[0039] Though each peptide will be immunoreactive for the serotype
upon which it is based, the peptides of the present invention may
even provide non-serotype-specific effects. It is believed that it
is possible that certain prevalent N-terminal fragments may evoke
cross-protective opsonic antibodies. This is demonstrated in
Example 5 below. It is expected that the present peptides,
compositions or vaccines will evoke cross-type opsonization.
Compositions, Vaccines, and Kits
[0040] The invention is also polypeptides, proteins, compositions,
or vaccines comprising the peptides or sequences of the peptides.
The peptides, in addition to being used individually, can be used
as a mixture of peptides. One aspect of the invention is a
composition comprising the peptides of the present invention as
described above. A composition comprising a mixture of peptides is
readily prepared by methods well known in the art. Alternatively,
to using the peptides individually or in a mixture, the peptides
may be joined together into a polypeptide or protein. One aspect of
the invention is a polypeptide comprising the sequences of peptides
of the present invention. Another aspect of the invention is a
protein comprising the sequences of the peptides of the present
invention. Standard techniques known in the art may be used to, for
example, link the synthesized peptides, synthesize a polypeptide or
protein which contains segments corresponding to the desired
synthetic peptides, or link the synthetic peptides to a backbone or
a liposome. Examples of backbones include, for example, keyhole
limpet hemocyanin, bovine serum albumin, tetanus toxoid, diphtheria
toxoid, bacterial outer membrane proteins, and artificial amino
acid backbones. It is well known to one of ordinary skill in the
art how to covalently bond peptides to a backbone or liposome or
how to create polypeptides or proteins using recombinant
techniques.
[0041] As noted above, a vaccine comprising these synthetic
peptides is within the scope of the invention. In one aspect, the
vaccine comprises an immunogenic amount of the peptide immunogens
of the present invention. The data from a CDC surveillance system
showing the epidemiological data, as noted above, showed that the
30 most prevalent invasive M types account for approximately 95% of
the total invasive isolates in the U.S. An aspect of the present
invention is the development of a multi-antigenic peptide (MAP)
vaccine representing these most prevalent serotypes. The peptides
of the invention may be conveniently formulated into vaccine
compositions comprising one or more of the peptides alone or in
association with a pharmaceutically acceptable carrier. See, e.g.,
Remington's Pharmaceutical Sciences, latest edition, by E. W.
Martin Mack Pub. Co., Easton, Pa., which discloses typical carriers
and conventional methods of preparing pharmaceutical compositions
that may be used in conjunction with the preparation of
formulations of the inventive peptides and which is incorporated by
reference herein. A benefit of the vaccine is it can eliminate over
85% of Group A Streptococci infections and reduce by 85% the
nasopharyngeal reservoir of Group A Streptococci in the United
States with the correct tailoring. The reservoir of GAS is expected
to be reduced for the population, not just an individual. Reduction
in GAS would have an effect on carriage of the organism, thereby
affecting the reservoir in the population. Reduction in carriage of
the organism subsequently reduces the exposure rate, thereby
increasing herd immunity.
[0042] The vaccine comprises and can be made by providing
immunogenic amounts of the peptides alone or in a pharmaceutically
acceptable vehicle or carrier. Carriers include water, saline,
dextrose, and glycerol, for example. The vaccine can further
comprise additional immune-stimulatory molecules, including other
GAS immunogens, vaccines of other species (such as H. influenza,
pertussis, N. meningitidis, pneumococcus, or Influenzae), and
adjuvants or mixture of adjuvants. One of ordinary skill in the art
would be able to identify vehicles, carriers, other antigens or
immunogens, and immunomodulators, such as adjuvants or cytokines,
appropriate for the present invention. Additional additives would
also be readily apparent to one of skill in the art, such as
wetting agents or preservatives.
[0043] A DNA vaccine is also within the scope of the present
invention. One aspect of the invention is a DNA vaccine comprising
DNA encoding immunoreactive peptides or compositions of the present
invention. Methods for making DNA sequences suitable for DNA
vaccines are known in the art. One of ordinary skill would be able
to determine appropriate promoters or other regulatory sequences
which may be used in the DNA construct encoding the immunoreactive
compositions. DNA vaccines may further comprise other components as
in the vaccines and compositions described above and below, such as
carriers and agents which increase levels of immunity, such as
liposomes. DNA vaccines may be administered by routes similar to
other vaccines. Administration of a DNA vaccine results in
expression of antigens which produce a protective immune
response.
[0044] Though the vaccine of the present invention is expected be
most effective with multiple serotype-specific peptides, it could
contain from one serotype-specific peptide to multiple
serotype-specific peptides for every identified serotype of GAS.
One of skill in the art would be able to determine the most
cost-effective and clinically therapeutic combination based on
epidemiological data, using the tailoring method provided herein.
In one aspect of the invention, the vaccine contains at least 3
serotype-specific peptides from 3 different serotypes. For example,
a vaccine comprising serotype-specific peptides for emm1, emm3, and
emm12 is expected to protect against approximately 38% of invasive
GAS disease in the U.S. More specifically, this vaccine can
comprise the following peptide combinations from Table 1:
TABLE-US-00002 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1
M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1
M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-2 M3-2 M3-2
M3-2 M3-2 M3-2 M3-2 M3-2 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1
M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3
M3-3 M3-3 M3-3 M3-3 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1
M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-4 M3-4 M3-4 M3-4 M3-4
M3-4 M3-4 M3-4 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1
M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5
M3-5 M3-5 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2
M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6
M3-6 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3
M1-4 M1-5 M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7
M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4
M1-5 M1-6 M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-1
M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-1 M12-1
M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-1
M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11
M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M12-1 M1-1 M1-2 M1-3 M1-4
M1-5 M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-2 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-2 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-2
M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M12-2 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-3 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-3 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-3
M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M12-3 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-4 M12-4 M12-4
M12-4 M12-1 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-4 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-4 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-4
M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M12-4 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-5 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-5 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-5
M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M12-5 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-6 M12-6 M12-6
M12-6 M12-1 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-6 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-6 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-6
M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M12-6 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M3-5 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M3-9 M12-7 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M12-7 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6
M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M12-7
M12-7 M12-7 M12-7 M12-7 M12-7 M12-7 M12-7
In another aspect of the invention, the vaccine comprises about 10
serotype-specific peptides, each peptide corresponding to one of
the 10 most prevalent serotypes in the U.S., thus making it
expected to immunize against approximately 65% of GAS disease in
the U.S. More specifically, this vaccine can comprise combinations
of 10 peptides wherein one peptide comes from each of the M1, M3,
M28, M12, M4, M11, M89, st2967, M77/27L, M6 peptides from Table 1.
As demonstrated above, the combinations can be generated and tested
according the procedures described in this application to determine
those which are effective. In a further aspect of the invention,
the vaccine comprises about 30 serotype-specific peptides of the 30
most prevalent serotypes, thus making it expected to immunize
against approximately 95% of GAS disease in the U.S. More
specifically, this vaccine can comprise combinations of 30 peptides
wherein one peptide comes from each of the 30 most prevalent
serotypes. As demonstrated above, the combinations can be generated
and tested according the procedures described in this application
to determine those which are effective. In a still further aspect
of the invention, the vaccine can comprise at least one
serotype-specific peptide from any identified serotype of GAS. A
vaccine covering approximately 60% of GAS disease would be expected
to be commercially viable. FIG. 1 shows the most prevalent
serotypes in the U.S. currently from which the serotype-specific
peptides could be chosen to target. Similar data from any targeted
population could be used to tailor the vaccine for the prevalent
serotypes and a given percentage of disease. This strategy towards
a safe and effective vaccine against GAS offers the advantage of
being easily modified to fit the needs of a particular region
according to the predominant M types located there.
[0045] As indicated above, based on the current epidemiological
data, similar serotype-specific peptides would be expected to be
effective in vaccines or compositions in the U.S., Italy, Mexico
and Argentina, for example. The epidemiological data of Malaysia,
India, New Guinea, Nepal and Egypt indicate that vaccines or
compositions tailored to these areas may require a different subset
of GAS serotype-specific peptides. Based on the teaching herein,
such a vaccine is easily within the grasp of the skilled
person.
[0046] Another strategy for designing a vaccine would be to make it
selective for specific GAS illnesses, as all GAS do not cause the
same illnesses. For example, the most severe GAS diseases are often
considered to be necrotizing fasciitis and toxic shock syndrome
which are most frequently caused by M1 and M3. Thus, selecting
immunogenic molecules specific to these serotypes would tailor the
vaccine to this strategy. More specifically, the combinations could
be, for example,
TABLE-US-00003 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-1 M3-1
M3-1 M3-1 M3-1 M3-1 M3-1 M3-1 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7
M1-8 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M3-2 M1-1 M1-2 M1-3 M1-4
M1-5 M1-6 M1-7 M1-8 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M3-3 M1-1
M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-4 M3-4 M3-4 M3-4 M3-4 M3-4
M3-4 M3-4 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-5 M3-5 M3-5
M3-5 M3-5 M3-5 M3-5 M3-5 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8
M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M3-6 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M3-7 M1-1 M1-2
M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8 M3-8
M3-8 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-9 M3-9 M3-9 M3-9
M3-9 M3-9 M3-9 M3-9 M1-1 M1-2 M1-3 M1-4 M1-5 M1-6 M1-7 M1-8 M3-10
M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M3-10 M1-1 M1-2 M1-3 M1-4 M1-5
M1-6 M1-7 M1-8 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11 M3-11
[0047] The peptides, compositions, vaccines or antibodies
(discussed below) of the present invention may be administered by
any mode of administration capable of delivering a desired dosage
to a desired location for a desired biological effect which are
known to those of ordinary skill in the art. One of ordinary skill
would be able to determine these dosages and routes by routine
experimentation. Routes or modes include, for example, orally,
parenterally (e.g., intravenously, by intramuscular injection, by
intraperitoneal injection), or the like, although subcutaneous
administration is preferred. Though the vaccine is envisioned as an
injectable, such as subcutaneous or intramuscularly, the vaccine
may be formulated in such a way as to render it mucosally
deliverable without the peptides being broken down before providing
systemic or mucosal immunity, such as, orally, inhalationally,
intranasally, or rectally. The amount of active compound
administered will, of course, be dependent, for example, on the
subject being treated, the subject's weight, the manner of
administration and the judgment of the prescribing physician.
Immunogenic amounts can be determined by standard procedures.
Examples of other peptide vaccines are known in the art. Dosages of
the present invention are expected to be in similar ranges.
[0048] Depending on the intended mode of administration, the
compositions or vaccines may be in the form of solid, semi-solid or
liquid dosage forms, such as, for example, tablets, suppositories,
pills, capsules, powders, liquids, suspensions, or the like,
preferably in unit dosage form suitable for single administration
of a precise dosage. The compositions or vaccines may include, as
noted above, an effective amount of the selected immunogens in
combination with a pharmaceutically acceptable carrier and, in
addition, may include other medicinal agents, pharmaceutical
agents, carriers, adjuvants, diluents, etc.
[0049] A more recently revised approach for parental administration
involves use of a slow release or sustained release system, such
that a constant level of dosage is maintained. See, e.g., U.S. Pat.
No. 3,710,795, which is incorporated by reference herein. A system
using slow release or sustained release may be used with oral
administration as well. The vaccine or composition may be
administered in liposomes, encapsulated, or otherwise protected or
formulated for slower or sustained release.
[0050] A subject can be inoculated to generate an active immune
response to the presence of the immunogenic composition which can
later protect the subject from the organism. A passive immune
response may be accomplished by any method known in the art.
[0051] Kits using peptides or antibodies produced by the present
invention may be made. A kit comprises packaging and the antibodies
or peptides. A kit may further comprise a solid phase or substrate
to which the antibodies or peptides may be attached.
Antibodies
[0052] Antibodies are also within the scope of the invention. For
example, isolated antibodies which selectively bind with the
peptides of the present invention are an aspect of the present
invention. These antibodies can be used, for example, in diagnosis,
treatment, or vaccination techniques. The antibodies can be
monoclonal or specific antibodies. The antibodies can be opsonic
antibodies or anti-attachment antibodies. The antibodies are made
and isolated by methods well known in the art. Modified antibodies,
fragments and humanized antibodies are also within the scope of
this invention. It is well known in the art how to make and use
modified antibodies, fragments or humanized antibodies.
Methods and Uses
[0053] The peptides, compositions, vaccines, and antibodies of the
present invention may be used in a variety of applications. For
example, preventative/prophylactic, therapeutic, or diagnostic
methods; affinity chromatography for separating/purifying
antibodies or antigens; active/passive immunotherapy; and use of
antibodies generated in passive immunotherapy.
[0054] An example of a method of preventing GAS infection comprises
administering a prophylactically effective amount of vaccine, or of
an anti-idotype antibody to the peptides of the present invention,
to a subject. Also, the antibodies against the peptides of the
present invention may be administered in a prophylactically
effective amount.
[0055] An example of a method of treating a GAS infection comprises
administering a therapeutically effective amount of antibodies of
the present invention to a subject.
[0056] An example of a diagnostic method is determining the
serotype of GAS organism responsible for an infection by contacting
a sample with multiple serotype-specific antibodies of the present
invention and determining which of these serotype-specific
antibodies are actually bound with the infecting organism. An
example of another diagnostic method is contacting a sample with
multiple serotype-specific peptides of the present invention and
determining which serotype-specific peptides are actually bound
with antibodies in the sample.
[0057] A method of measuring the amount of GAS organism in a sample
comprising contacting a sample with antibodies of the present
invention and measuring the amount of immunocomplexes formed.
[0058] Affinity chromatography is frequently used for separating
and/or purifying antibodies or antigens. By binding the
corresponding antibody or antigen to a substrate, a sample can be
passed through a column containing the immunoadsorbent and then the
column eluted to collect the isolated corresponding antigen or
antibody. More specifically, the peptides of the invention can be
bound on a column to purify anti-GAS antibodies. Likewise, anti-GAS
antibodies generated in accordance with the invention can be bound
to a column and used to purify GAS from a sample.
[0059] Immunotherapy is another use for the peptides, compositions,
vaccines or antibodies of the present invention. As known in the
art, active immunotherapy is achieved by activating a subject's own
immune system. By administering the peptides, compositions or
vaccines of the present invention, an active immune response may be
elicited.
[0060] As known in the art, passive immunotherapy is achieved by
supplementing a subject's immune system with agents such as
antibodies. By administering the antibodies of the present
invention, a passive immune response may be elicited.
[0061] The method for tailoring vaccines comprises a) identifying a
population of recipients for the vaccine; b) gathering prevalence
data on serotypes of the targeted organism from a sample within
that population of recipients; c) choosing a set of the most
prevalent serotypes from the gathered data; d) identifying proteins
from the chosen serotypes responsible for evading
opsonophagocytosis; e) identifying small peptides within the
identified proteins which protect for the chosen serotypes; f)
synthesizing the identified peptides; g) formulating a vaccine
comprising the peptides identified in step e). Specifically, the
small peptides may be those of about 20-25 amino acids and
protection may be by elicitation of opsonic or anti-attachment
antibodies.
[0062] Other uses for or variations of the above methods using the
above peptides, compositions, vaccines or antibodies may be readily
apparent to one of ordinary skill in the art.
[0063] The approach of employing a mixture of defined synthetic N
terminal M protein segments protecting against prevalent U.S. Group
A streptococcal (GAS) strains will favorably compare against any of
the prior art approaches. The present approach has found excellent
immunogenicity and type-specific opsonic antibody titers with the
peptides assessed. Animal studies have indicated that individual
peptides protect in a type-specific manner not only against
systemic infection, but against nasopharyngeal carriage of GAS.
Many N terminal M protein segments have already been demonstrated
to not evoke antibodies cross-reactive with human tissues. There is
no evidence that chemically linking the current peptides to
carriers or backbones will increase the risk of undesirable cross
reactions. The methodology can be proven for each of the most
common M types found in U.S. invasive disease isolates. The
strategy can be expanded to less frequently occurring GAS types.
This allows the vaccine to be quickly and precisely adapted to
changes in individual strain frequencies in a given geographic area
or demographic population by addition or deletion of individual
peptide components.
Examples
Experimental
[0064] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, articles, devices,
and/or methods claimed herein are made and evaluated, and are
intended to be purely exemplary of the invention and are not
intended to limit the scope of what the inventors regard as their
invention. Efforts have been made to ensure accuracy with respect
to numbers (e.g., amounts, temperature, etc.) but some errors and
deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, temperature is in .degree. C. or is at
ambient temperature, and pressure is at or near atmospheric.
Materials and Methods
[0065] A CDC surveillance system used a rapid gene-based M
subtyping system to gather epidemiological data and showed that the
30 most prevalent invasive M types account for approximately 95% of
the total invasive isolates in the U.S.
Peptide Synthesis and Purification
[0066] Synthetic peptides (approximately 20-25-mers), representing
epitopes of the most prevalent GAS M types were synthesized. Their
sequences were derived from the M protein N-terminal region that
confers type-specific immunity.
[0067] Peptides were synthesized on a Model ACT396 Omega multiple
peptide synthesizer (Advanced Chemtech, Louisville, Ky.) by
Fmoc-chemistry using a HOBT/DIC strategy with double couplings.
Matrix-assisted laser desorption/ionization, time-of-flight mass
spectroscopy (MALDI-TOF MS) (Bruker REFLEX, Billerica, Mass.) was
utilized to determine the mass to charge (m/z) of the crude
peptides.
Abbreviations:
[0068] Fmoc (9-fluorenylmethyloxycarbonyl)
[0069] HOBT (1-hydroxybenzotriazole)
[0070] DIC (Diisopropylcarbodiimide)
Enzyme-Linked Immunosorbent Assay (ELISA)
[0071] Overlapping peptides representing each M type were assessed
for their immunogenicity using ELISA and dot-immunoblot.
[0072] Levels of anti-M protein antibodies in mouse sera were
determined by an enzyme-linked immunosorbent assay (ELISA) using
either synthetic peptides or whole M protein as antigen. Microtiter
plates (DYNEX Immulon 2 HB) were coated with 10 .mu.g/ml antigen in
10 mM phosphate-buffered saline (PBS) at 4.degree. C. overnight.
Plates were rinsed three times with wash buffer (0.05% Tween 20 in
10 mM PBS, pH 7.2) and blocked with 1% BSA in PBS for one hour at
37.degree. C. The appropriate test sera were applied at a starting
dilution of 1:1000 and serially diluted two fold down the plate in
PBS. Plates were incubated at 37.degree. C. for 30 minutes and
subsequently rinsed three times. Goat anti-rabbit or goat
anti-mouse Ig-peroxidase conjugate diluted 1:10,000 in PBS was
added to all wells and incubated for two hours at 37.degree. C.
Plates were rinsed three times with wash buffer and freshly
prepared TMB peroxidase (Kirkegaard and Perry Laboratories) was
added. Reactions were quenched with 0.18 M H.sub.2SO.sub.4 after 30
minutes at room temperature and OD.sub.450 was measured with a
Labsystems Multiskan plate reader using Ascent software. ELISA
titers are expressed as the reciprocal of the last dilution which
gave a reading OD.sub.450>0.10.
Immunization
[0073] Mice (6 week old female Swiss Webster, Harley-Sprague) were
immunized subcutaneously (s.c.) with either the peptide in alum or
alum alone for control groups. Peptides were rotated in the alum
mixture for 2 hours at 4.degree. C. and stored at 4.degree. C.
overnight prior to use. Initial injections (100 .mu.l) consisted of
50 .mu.g peptide in alum (25 .mu.g for M3-2 peptide) with identical
booster injections given at 2 and 4 weeks (except for the M3-2
peptide which was only given as an initial dose and single booster
at 2 weeks).
Opsonophagocytosis
[0074] Opsonophagocytosis assays were performed as previously
described in Lancefield, R.C., Persistence of type-specific
antibodies in man following infection with group A streptococci,
1959, J. Immunol., 89:307. Briefly, diluted serum (50 pd) was added
to mid-log phase GAS (10.sup.3 CFU) in Todd-Hewitt broth (50 .mu.l)
and whole, heparinized blood (500 .mu.l) from a nonopsonic human
donor. Mixtures were briefly vortexed and placed at 37.degree. C.
in a shaker for 3 hours. Dilutions were then plated on trypticase
soy agar plates (5% sheep blood) to quantitate viable organisms.
The % killing is expressed as [(CFU control-CFU test)/CFU
control].times.100.
NP Challenge
[0075] Mice were challenged intranasally one week after the final
booster injection was administered. Prior to being challenged, mice
were anesthetized with a ketamine/xylazine mixture. Mice were then
given 10.sup.4-10.sup.5 CFU (10 .mu.l) of streptococci intranasally
via a microliter pipette. They were sacrificed 24 hours after the
challenge and the nasopharyngeal passages were washed with
approximately 100 .mu.l physiological saline which was collected
and immediately placed on ice. Dilutions of the wash were then
plated on trypticase soy agar plates (5% sheep blood) and incubated
at 37.degree. C. for 18 hours to quantitate viable organisms.
Nasopharyngeal colonization in immunized mice was compared to
non-immunized controls. Statistical analysis of the NP data was
accomplished using the t-test and rank sum test.
Detection of Heart Cross-Reactive Antibodies
[0076] Mouse sera were screened for heart cross-reactive antibodies
with an indirect immunofluorescence assay (IFA). Glass slides
containing formaline-fixed human heart tissue were deparaffinized
before use and stored in dH.sub.2O. Slides were air-dried 10
minutes and incubated in a moist chamber with 1:500 mouse test sera
at room temperature for 30 minutes. Slides were rinsed and soaked
in PBS for 5 minutes. Slides were then incubated in a moist chamber
with 1:500 goat anti-mouse FITC-labeled globulin at room
temperature for 30 minutes. Slides were washed as described
previously and allowed to air-dry. One drop of mounting fluid
(DAKO) was applied to each slide followed by a glass cover slip
(Corning 24.times.40 mm). Slides were immediately examined under a
fluorescent microscope. The positive control was a 1:500 mouse
anticlonal antibody to human HLA (Caltag).
Example 1
Peptide Recognition by Anti M-Protein Rabbit Sera
[0077] The immunoreactivity of several synthetic peptides was
determined by ELISA using rabbit sera prepared against whole M
protein. The synthetic peptides are representative of relatively
small portions of the M protein N-terminus; therefore, it was
advantageous to determine if the epitopes contained within
particular peptides were immunoreactive with sera prepared against
whole M protein. It was shown that the whole anti-M protein rabbit
sera of the respective serotypes could bind to each of the
synthetic peptides within that serotype. However, some peptides
were more highly immunoreactive than others, thus being better
suited for use in the animal studies. The peptides showing the
highest reactivity in each serotype group tested were M1-4, M3-2
and M12-1. This indicated that immunoreactive epitopes were
contained within the amino acid sequences of the synthetic
peptides.
[0078] The immunoreactivity of peptides with SEQ ID NO: 1, SEQ ID
NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38,
SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID
NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ
ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID
NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ
ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66,
SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ
ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80,
SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID
NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ
ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94,
SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103,
SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112,
SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID
NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121,
SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID
NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130,
SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID
NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 are confirmed
by ELISA using rabbit sera prepared against whole M protein.
Example 2
Immunogenicity of Synthetic Peptides in Swiss Webster Mice
[0079] Studies were then carried out in vivo in a mouse model to
determine the ability of the tested synthetic peptides to protect
against GAS challenge and to evaluate their immunogenicity as a
single MAP formulation.
[0080] Mice were immunized s.c. with selected peptides using
aluminum hydroxide or aluminum phosphate as an adjuvant. Serology
results indicated that aluminum hydroxide elicited higher antibody
levels and, therefore, it was used for all subsequent
immunizations. ELISA indicated that antibodies were elicited to the
M type peptides.
TABLE-US-00004 TABLE 2 ELISA results determining elicitation of
antibodies. Peptide ELISA M1-4 no M1-2 no M1-3 yes M3-1 yes M3-2
yes M3-4 no M12-1 yes M12-2 yes (weak) M12-3 no
[0081] Five of the nine synthetic peptides shown in Table 2 were
able to elicit an antibody response, with each of the three
serotypes under investigation being represented by at least one
immunogenic peptide. The titers of the immunogenic peptides were as
follows:
TABLE-US-00005 TABLE 3 Titers of the immunogenic peptides in
immunized mice. Peptide Titer M1-3 1:16,000 M3-1 1:8,000 M3-2
1:160,000 M12-1 1:160,000 M12-2 1:1000
[0082] Mice are immunized s.c. with peptides and various
combinations of peptides with SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,
SEQ ID NO:138.
ELISA is used to confirm antibody elicitation to at least some of
the peptides and peptide combinations. Titers are determined for
the peptides and combinations of peptides.
Example 3
Opsonophagocytosis
[0083] The mouse sera that were shown to contain anti-M antibodies
by the ELISA assay were then tested for functional activity with
the in vitro opsonophagocytosis assay (Lancefield 1959), Table 4.
Each of the sera tested positive in the assay and was able to
reduce the amount of viable bacteria relative to controls. The
values for the reduction of bacteria in the opsonophagocytosis
assays were as shown in Table 5.
TABLE-US-00006 TABLE 4 Sera results for opsonophagocytosis assay.
Peptide Opsono (in vitro) M1-4 nd M1-2 nd M1-3 yes M3-1 yes M3-2
yes M3-4 nd M12-1 yes M12-2 nd M12-3 nd
TABLE-US-00007 TABLE 5 Percentage reduction of viable GAS bacteria.
Peptide Reduction M1-3 50% M3-1 80-90% M3-2 60-80% M12-1 70-80%
It is interesting to note that within the M3 serotype, while the
M3-1 peptide induced an antibody response that was more than a
magnitude lower than that of the M3-2 peptide, it was able to
opsonophagocytize bacteria more effectively.
[0084] Mouse sera is tested that is shown to contain anti-M
antibodies from the peptides and combinations of peptides selected
from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ
ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,
SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ
ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33,
SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID
NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ
ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47,
SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID
NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ
ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61,
SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID
NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ
ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75,
SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID
NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ
ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89,
SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID
NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ
ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107,
SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116,
SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID
NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125,
SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID
NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134,
SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138 for
functional activity with an in vitro opsonophagocytosis assay. The
sera test positive in the assay and are able to reduce the amount
of viable bacteria relative to controls.
Example 4
Nasopharyngeal Colonization Challenge
[0085] Nasopharyngeal (NP) colonization challenge experiments were
performed on mice vaccinated (s.c.) with either M3-1 or M3-2
peptide in alum in comparison to control animals which received
alum only. The nasopharyngeal colonization challenge was performed
by doing a nasal wash 24 hours after challenge. The subjects were
administered 10.sup.4 CFUs 1 week after final boost. Dilutions of
the wash were plated. Both peptides were able to induce an
effective in vivo immune response that reduced colonization in the
vaccinated group relative to the unvaccinated group.
TABLE-US-00008 TABLE 6 Reduction of nasopharyngeal colonization in
vaccinated mice relative to control mice. Reduction of Peptide
colonization P M3-1 87% P = 0.010 M3-2 67% P = 0.029
The M3-1 peptide reduced NP colonization by 87% (P<0.010), while
the M3-2 peptide was able to reduce NP colonization by 67%
(P<0.029) relative to the control group. No deaths were recorded
in any group. To our knowledge this is the first example of in vivo
reduction of nasopharyngeal colonization of GAS bacteria by
immunization with a type-specific synthetic peptide.
[0086] NP colonization challenges are performed on mice vaccinated
s.c. with the peptides and peptide combinations selected from SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ
ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34,
SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID
NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ
ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48,
SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID
NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ
ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62,
SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID
NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ
ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76,
SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID
NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ
ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90,
SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID
NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ
ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108,
SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117,
SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID
NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126,
SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID
NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135,
SEQ ID NO:136, SEQ ID NO:137, and SEQ ID NO:138. Peptides and
peptide combinations are confirmed to induce an effective in vivo
immune response that reduced colonization in the vaccinated group
relative to the unvaccinated group. No deaths are reported in any
group.
Example 5
Cross-Type Protection by Peptides
[0087] Some of the peptides tested in the previous examples were
tested for cross-type protection using the methods described above.
Table 7 shows in vitro data on reduction in bacteria versus a
control. The peptide tested and serotype against which it was
tested is indicated. This example clearly shows that peptides M3-1
and M3-2 were able to significantly reduce M43 type bacteria in
addition to M3 type bacteria.
TABLE-US-00009 TABLE 7 Functional antibody bactericidal activity in
vitro showing cross-protection against heterologous M type. GAS M
type used % CFU Peptide in assay reduction M1-3 M1 80-95% M3-1 M3
70-90% M3-2 M3 90-95% M12-1 M12 70-80% M3-1 M43 80-90% M3-2 M43
60-80%
[0088] Some of the peptides were tested for reduction of
colonization of GAS in mice. The second line of data shows mice
immunized with peptides M1-3, M3-2 and M12-1. The peptides were
administered together but were not chemically combined in any way.
The third line of data shows mice that were inoculated with M3-2.
Some of the mice were colonized by M3, and some of the mice were
colonized by M43. The % CFU reduction is the reduction overall for
the mice which received M3-2. This again demonstrates that M3-2 was
capable in vivo of cross-type protection.
TABLE-US-00010 TABLE 8 Reduction of colonization in mice.
Colonizing GAS % CFU reduction Peptides M type (P value) M1-3 M1
90% (P < 0.10) M1-3, M1 74% (P < 0.25) M3-2, M12-1 M3-2 M3 or
M43 67% (P < 0.30)
[0089] The cross-type protection is confirmed for peptides and
peptide combinations selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ
ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ
ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36,
SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ
ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ
ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64,
SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78,
SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ
ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92,
SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101,
SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID
NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110,
SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119,
SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128,
SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137,
and SEQ ID NO:138. Reductions in bacteria are shown in vitro and in
vivo.
Example 6
Detection of Heart Cross-Reactive Antibodies
[0090] Mouse immune sera that contained functional antibody were
examined for the presence of heart cross-reactive antibodies with
an IFA. The mouse sera were tested in parallel with a positive
control, mouse monoclonal antibody to human leukocyte antigen
(HLA). None of the anti-M peptide sera from immunized mice reacted
with the heart tissue.
[0091] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0092] It will be apparent to those skilled in the art that various
modifications and variations can be made in the present invention
without departing from the scope or spirit of the invention. Other
embodiments of the invention will be apparent to those skilled in
the art from consideration of the specification and practice of the
invention disclosed herein. It is intended that the specification
and examples be considered as exemplary only, with a true scope and
spirit of the invention being indicated by the following claims.
Sequence CWU 1
1
138122PRTArtificial SequenceDescription of Artificial Sequence;
NOTE = Synthetic Construct 1Cys Asn Gly Asp Gly Asn Pro Arg Glu Val
Ile Glu Asp Leu Ala Ala 1 5 10 15 Asn Asn Pro Ala Ile Gln 20
221PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 2Cys Ile Gln Asn Ile Arg Leu Arg His Glu Asn
Lys Asp Leu Lys Ala 1 5 10 15 Arg Leu Glu Asn Ala 20
321PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 3Cys Ile Arg Leu Arg His Glu Asn Lys Asp Leu
Lys Ala Arg Leu Glu 1 5 10 15 Asn Ala Met Glu Val 20
422PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 4Cys Asn Gly Asp Gly Asn Pro Arg Glu Val Ile
Glu Asp Leu Ala Ala 1 5 10 15 Asn Asn Pro Ala Met Glu 20
521PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 5Cys Ile Arg Asn Ile Arg Leu Arg His Glu Asn
Lys Asp Leu Lys Ala 1 5 10 15 Arg Leu Glu Asn Ala 20
622PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 6Cys Asn Gly Asp Gly Asn Pro Arg Glu Val Ile
Glu Asp Leu Ala Ala 1 5 10 15 Asn Asn Pro Val Ile Gln 20
722PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 7Cys Asn Gly Asp Gly Asn Pro Arg Val Val Ile
Glu Asp Leu Ala Ala 1 5 10 15 Asn Asn Pro Ala Ile Gln 20
822PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 8Cys Ile Arg Leu Arg His His Glu Asn Lys Asp
Leu Lys Ala Arg Leu 1 5 10 15 Glu Asn Ala Met Glu Val 20
923PRTArtificial SequenceDescription of Artificial Sequence; NOTE =
Synthetic Construct 9Cys Asn Ser Lys Asn Pro Val Pro Val Lys Lys
Glu Ala Lys Leu Ser 1 5 10 15 Glu Ala Glu Leu His Asp Lys 20
1023PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 10Cys Lys Lys Glu Ala Lys Leu Ser Glu Ala Glu
Leu His Asp Lys Ile 1 5 10 15 Lys Asn Leu Glu Glu Glu Lys 20
1122PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 11Cys Glu Leu His Asp Lys Ile Lys Asn Leu Glu
Glu Glu Lys Ala Glu 1 5 10 15 Leu Phe Glu Lys Leu Asp 20
1223PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 12Cys Glu Leu Phe Glu Lys Leu Asp Lys Val Glu
Glu Glu His Lys Lys 1 5 10 15 Val Glu Glu Glu His Lys Lys 20
1321PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 13Cys Asp Ala Arg Ser Val Asn Gly Glu Phe Pro
Arg His Val Lys Leu 1 5 10 15 Lys Asn Glu Ile Glu 20
1421PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 14Cys Gly Glu Phe Pro Arg His Val Lys Leu Lys
Asn Glu Ile Glu Asn 1 5 10 15 Leu Leu Asp Gln Val 20
1521PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 15Cys Leu Asp Gln Val Thr Gln Leu Tyr Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Asn Ala 20
1621PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 16Cys Leu Asp Gln Val Thr Gln Leu Tyr Asn Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Ser Ala 20
1721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 17Cys Leu Asp Gln Val Thr Gln Leu Tyr Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Ser Ala 20
1821PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 18Cys Leu Asn Gln Val Thr Gln Leu Tyr Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Asn Ala 20
1921PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 19Cys Leu Ala Gln Val Thr Gln Leu Tyr Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Asn Ala 20
2021PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 20Cys Leu Asn Gln Val Thr Gln Leu His Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Asn Ala 20
2121PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 21Cys Arg Ser Asp Ala Arg Ser Val Asn Gly Glu
Phe Pro Arg His Val 1 5 10 15 Lys Leu Lys Asn Glu 20
2222PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 22Cys Gln Leu Tyr Thr Lys His Ile Tyr Thr Lys
His Asn Ser Asn Tyr 1 5 10 15 Gln Gln Tyr Asn Ala Gln 20
2321PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 23Cys Thr Gln Leu Tyr Thr Lys His Asn Ser Asn
Tyr Gln Gln Tyr Asn 1 5 10 15 Ala Gln Ala Gly Arg 20
2421PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 24Cys Ala Glu Ile Lys Lys Pro Gln Ala Asp Ser
Ala Trp Asn Trp Pro 1 5 10 15 Lys Glu Tyr Asn Ala 20
2521PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 25Cys Asp Ser Ala Trp Asn Trp Pro Lys Glu Tyr
Asn Ala Leu Leu Lys 1 5 10 15 Glu Asn Glu Glu Leu 20
2621PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 26Cys Lys Glu Asn Glu Glu Leu Lys Val Glu Arg
Glu Lys Tyr Leu Ser 1 5 10 15 Tyr Ala Asp Asp Lys 20
2723PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 27Cys Glu Glu Leu Lys Val Glu Arg Glu Lys Tyr
Leu Ser Tyr Ala Asp 1 5 10 15 Asp Lys Glu Lys Asp Pro Gln 20
2823PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 28Cys Ala Gly Gln Ser Ala Pro Lys Gly Thr Asn
Val Ser Ala Asp Leu 1 5 10 15 Tyr Asn Ser Leu Trp Asp Glu 20
2920PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 29Cys Lys Gly Thr Asn Val Ser Ala Asp Leu Tyr
Asn Ser Leu Trp Asp 1 5 10 15 Glu Asn Lys Thr 20 3021PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 30Cys Asp Glu Asn Lys Thr Leu Arg Glu Lys Gln Glu Glu Tyr
Ile Thr 1 5 10 15 Lys Ile Gln Asn Glu 20 3121PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 31Cys Thr Glu Val Lys Ala Ala Gly Gln Ser Ala Pro Lys Gly
Thr Asn 1 5 10 15 Val Ser Ala Asp Leu 20 3221PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 32Cys Asp His Ser Asp Leu Val Ala Glu Lys Gln Arg Leu Glu
Asp Leu 1 5 10 15 Gly Gln Lys Phe Glu 20 3321PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 33Cys Ala Glu Lys Gln Arg Leu Glu Asp Leu Gly Gln Lys Phe
Glu Arg 1 5 10 15 Leu Lys Gln Arg Ser 20 3421PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 34Cys Leu Glu Asp Leu Gly Gln Lys Phe Glu Arg Leu Lys Gln
Arg Ser 1 5 10 15 Glu Leu Tyr Leu Gln 20 3521PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 35Cys Lys Phe Glu Arg Leu Lys Gln Arg Ser Glu Leu Tyr Leu
His Gln 1 5 10 15 Tyr Tyr Asp Asn Lys 20 3621PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 36Cys Lys Phe Glu Arg Leu Lys Arg Arg Ser Glu Leu Tyr Leu
Gln Gln 1 5 10 15 Tyr Tyr Asp Asn Lys 20 3721PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 37Cys Lys Gln Arg Ser Glu Leu Tyr Leu Gln Gln Tyr Tyr Asp
Asn Lys 1 5 10 15 Ser Asn Arg Tyr Lys 20 3821PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 38Cys Ser Glu Leu Tyr Leu Gln Gln Tyr Tyr Asp Asn Lys Ser
Asn Gly 1 5 10 15 Tyr Lys Gly Asp Trp 20 3921PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 39Cys Glu Ser Ser Asn Asn Ala Glu Ser Ser Asn Ile Ser Gln
Glu Ser 1 5 10 15 Lys Leu Ile Asn Thr 20 4022PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 40Cys Glu Ser Ser Asn Ile Ser Gln Glu Ser Lys Leu Ile Asn
Thr Leu 1 5 10 15 Thr Asp Glu Asn Glu Lys 20 4122PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 41Cys Glu Ser Lys Leu Ile Asn Thr Leu Thr Asp Glu Asn Glu
Lys Leu 1 5 10 15 Arg Glu Glu Leu Gln Gln 20 4220PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 42Cys Asn Thr Leu Asn Thr Leu Thr Asp Glu Asn Glu Lys Leu
Arg Glu 1 5 10 15 Glu Leu Gln Gln 20 4322PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 43Cys Glu Ser Ser Asn Ile Ser Gln Glu Ser Lys Leu Ile Asn
Thr Leu 1 5 10 15 Thr Asp Glu Asn Glu Lys 20 4421PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 44Cys Glu Lys Leu Arg Glu Glu Leu Gln Gln Tyr Tyr Ala Leu
Ser Asp 1 5 10 15 Ala Lys Glu Glu Glu 20 4520PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 45Cys Ala Glu Ser Pro Lys Ser Thr Glu Thr Ser Ala Asn Gly
Ala Asp 1 5 10 15 Lys Leu Ala Asp 20 4621PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 46Cys Lys Ser Thr Glu Thr Ser Ala Asn Gly Ala Asp Lys Leu
Ala Asp 1 5 10 15 Ala Tyr Asn Thr Leu 20 4721PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 47Cys Asp Lys Leu Ala Asp Ala Tyr Asn Thr Leu Leu Thr Glu
His Glu 1 5 10 15 Lys Leu Arg Asp Glu 20 4822PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 48Cys Thr Glu His Glu Lys Leu Arg Asp Glu Tyr Tyr Thr Leu
Ile Asp 1 5 10 15 Ala Lys Leu Glu Glu Lys 20 4921PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 49Cys Thr Glu His Glu Lys Leu Arg Asp Glu Tyr Tyr Thr Leu
Ile Asp 1 5 10 15 Ala Lys Glu Glu Glu 20 5025PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 50Cys Glu Gly Val Ser Val Gly Ser Asp Ala Ser Leu His Asn
Arg Ile 1 5 10 15 Thr Asp Leu Glu Glu Glu Arg Glu Lys 20 25
5123PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 51Cys Ser Asp Ala Ser Leu His Asn Arg Ile Thr
Asp Leu Glu Glu Glu 1 5 10 15 Arg Glu Lys Leu Leu Asn Lys 20
5224PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 52Cys Asp Leu Glu Glu Glu Arg Glu Lys Leu Leu
Asn Lys Leu Asp Lys 1 5 10 15 Val Glu Glu Glu His Lys Lys Asp 20
5322PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 53Cys Asp Leu Glu Glu Glu Arg Gly Lys Leu Leu
Asn Lys Leu Asp Lys 1 5 10 15 Val Glu Glu Glu His Lys 20
5421PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 54Cys Leu Asn Lys Leu Asp Lys Val Glu Glu Glu
His Lys Lys Asp His 1 5 10 15 Glu Gln Leu Glu Lys 20
5522PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 55Cys Asp Ser Asp Asn Ile Asn Arg Ser Val Ser
Val Lys Asp Asn Glu 1 5 10 15 Lys Glu Leu His Asn Lys 20
5623PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 56Cys Asp Asn Ile Asn Arg Ser Val Ser Val Lys
Asp Asn Glu Lys Glu 1 5 10 15 Leu His Asn Lys Ile Ala Asp 20
5721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 57Cys Ser Val Lys Asp Asn Glu Lys Glu Leu His
Asn Lys Ile Ala Asp 1 5 10 15 Leu Glu Glu Glu Arg 20
5822PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 58Cys Glu Leu His Asn Glu Ile Ala Asp Leu Glu
Glu Glu Arg Gly Glu 1 5 10 15 His Leu Asp Lys Ile Asp 20
5922PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 59Cys Glu Leu His Asn Lys Ile Ala Asp Leu Glu
Glu Glu Arg Gly Ala 1 5 10 15 His Leu Asp Lys Ile Asp 20
6021PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 60Cys Asp Ser Asp Asn Ile Asn Arg Phe Val Ser
Val Lys Asp Asn Glu 1 5 10 15 Lys Glu Leu His Asn 20
6121PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 61Cys Asp Ser Asp Asn Ser Asp Asn Ile Asn Arg
Ser Val Ser Val Lys 1 5 10 15 Asp Asn Glu Lys Glu 20
6221PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 62Cys Leu Glu Glu Glu Arg Gly Glu His Leu Asp
Lys Ile Asp Glu Leu 1 5 10 15 Lys Glu Glu Leu Lys 20
6322PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 63Cys Asn Ser Lys Asn Pro Ala Pro Ala Pro Ala
Ser Ala Val Pro Val 1 5 10 15 Lys Lys Glu Ala Thr Lys 20
6421PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 64Cys Val Pro Val Lys Lys Glu Ala Thr Lys Leu
Ser Glu Ala Glu Leu 1 5 10 15 Tyr Asn Lys Ile Gln 20
6522PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 65Cys Lys Lys Glu Ala Thr Lys Leu Ser Glu Ala
Glu Leu Tyr Asn Lys 1 5 10 15 Ile Gln Glu Leu Glu Glu 20
6621PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 66Cys Asn Ser Lys Asn Pro Ala Pro Ala Pro Ala
Val Pro Val Lys Lys 1 5 10 15 Glu Ala Thr Lys Leu 20
6721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 67Cys Asn Ser Lys Asn Pro Ala Pro Ala Val Pro
Val Lys Lys Glu Ala 1 5 10 15 Thr
Lys Leu Ser Glu 20 6821PRTArtificial SequenceDescription of
Artificial Sequence; NOTE = Synthetic Construct 68Cys Ala Glu Leu
Tyr Asn Lys Ile Gln Glu Leu Glu Glu Gly Lys Ala 1 5 10 15 Glu Leu
Phe Asp Lys 20 6921PRTArtificial SequenceDescription of Artificial
Sequence; NOTE = Synthetic Construct 69Cys Arg Val Phe Pro Arg Gly
Thr Val Glu Asn Pro Asp Lys Ala Arg 1 5 10 15 Glu Leu Leu Asn Lys
20 7022PRTArtificial SequenceDescription of Artificial Sequence;
NOTE = Synthetic Construct 70Cys Arg Gly Thr Val Glu Asn Pro Asp
Lys Ala Arg Glu Leu Leu Asn 1 5 10 15 Lys Tyr Asp Val Glu Asn 20
7122PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 71Cys Glu Asn Pro Asp Lys Ala Arg Glu Leu Leu
Asn Lys Tyr Asp Val 1 5 10 15 Glu Asn Ser Met Leu Gln 20
7222PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 72Cys Glu Asn Ser Met Leu Gln Ala Asn Asn Asp
Asn Leu Thr Asp Gln 1 5 10 15 Asn Lys Asn Leu Thr Asp 20
7321PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 73Cys Asn Ser Met Leu Gln Ala Asn Asn Asp Lys
Leu Thr Thr Glu Asn 1 5 10 15 Lys Asn Leu Thr Asp 20
7422PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 74Cys Asp Ser Ser Ser Arg Asp Ile Thr Glu Ala
Gly Val Ser Lys Phe 1 5 10 15 Trp Lys Ser Lys Phe Asp 20
7522PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 75Cys Arg Asp Ile Thr Glu Ala Gly Val Ser Lys
Phe Trp Lys Ser Lys 1 5 10 15 Phe Asp Ala Glu Gln Asn 20
7622PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 76Cys Glu Ala Gly Val Ser Lys Phe Trp Lys Ser
Lys Phe Asp Ala Glu 1 5 10 15 Gln Asn Arg Ala Asn Glu 20
7721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 77Cys Asp Ala Glu Gln Asn Arg Ala Asn Glu Leu
Glu Lys Lys Leu Ser 1 5 10 15 Gly Tyr Glu Lys Asp 20
7819PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 78Cys Glu Glu His Pro Asp Val Val Ala Ala Arg
Glu Ser Val Leu Asn 1 5 10 15 Asn Val Arg 7921PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 79Cys His Pro Asp Val Val Ala Ala Arg Glu Ser Val Leu Asn
Asn Val 1 5 10 15 Arg Val Pro Gly Thr 20 8022PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 80Cys Arg Val Pro Gly Thr Leu Trp Leu Arg Gln Lys Glu Glu
Asn Asp 1 5 10 15 Lys Leu Lys Leu Glu Lys 20 8121PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 81Cys Leu Arg Gln Lys Glu Glu Asn Asp Lys Leu Lys Leu Glu
Lys Lys 1 5 10 15 Gly Leu Glu Thr Glu 20 8222PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 82Cys Glu Glu Glu Arg Thr Phe Thr Glu Leu Pro Tyr Glu Ala
Arg Tyr 1 5 10 15 Lys Ala Trp Lys Ser Glu 20 8322PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 83Cys Glu Leu Pro Tyr Glu Ala Arg Tyr Lys Ala Trp Lys Ser
Glu Asn 1 5 10 15 Asp Glu Leu Arg Glu Asn 20 8420PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 84Cys Asn Asp Glu Leu Arg Glu Asn Tyr Arg Arg Thr Leu Asp
Lys Phe 1 5 10 15 Asn Thr Glu Gln 20 8521PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 85Cys Lys Ala Trp Lys Ser Glu Asn Asp Glu Leu Arg Glu Asn
Tyr Arg 1 5 10 15 Lys Thr Leu Asp Lys 20 8621PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 86Cys Arg Glu Asn Tyr Arg Arg Thr Leu Asp Lys Phe Asn Thr
Glu Gln 1 5 10 15 Gly Lys Thr Thr Arg 20 8721PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 87Cys Glu Glu His Glu Lys Val Thr Gln Ala Arg Glu Ala Val
Ile Arg 1 5 10 15 Glu Met Gln Gln Arg 20 8821PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 88Cys His Glu Lys Val Thr Gln Ala Arg Glu Ala Val Ile Arg
Glu Met 1 5 10 15 Gln Gln Arg Gly Thr 20 8922PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 89Cys Glu Met Gln Gln Arg Gly Thr Asn Phe Gly Pro Leu Leu
Ala Ser 1 5 10 15 Thr Met Arg Asp Asn His 20 9023PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 90Cys Asp Asp Arg Ser Val Ser Thr Asn Ser Gly Ser Val Ser
Thr Pro 1 5 10 15 Tyr Asn Asn Leu Leu Asn Glu 20 9121PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 91Cys Arg Ser Val Ser Thr Asn Ser Gly Ser Val Ser Thr Pro
Tyr Asn 1 5 10 15 Asn Leu Leu Asn Glu 20 9221PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 92Cys Glu Tyr Asp Asp Leu Leu Ala Lys His Gly Glu Leu Leu
Ser Glu 1 5 10 15 Tyr Asp Ala Leu Lys 20 9323PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 93Cys Asp Leu Leu Ala Lys His Gly Glu Leu Leu Ser Glu Tyr
Asp Ala 1 5 10 15 Leu Lys Glu Lys Gln Asp Lys 20 9422PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 94Cys Thr Val Thr Arg Gly Thr Ile Asn Asp Pro Gln Arg Ala
Lys Glu 1 5 10 15 Ala Leu Asp Lys Tyr Glu 20 9521PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 95Cys Asp Pro Gln Arg Ala Lys Glu Ala Leu Asp Lys Tyr Glu
Leu Glu 1 5 10 15 Asn His Asp Leu Lys 20 9621PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 96Cys Glu Asn His Asp Leu Lys Thr Lys Asn Glu Gly Leu Lys
Thr Glu 1 5 10 15 Asn Glu Gly Leu Lys 20 9722PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 97Cys Gln Arg Ala Lys Ala Ala Leu Asp Lys Tyr Glu Leu Glu
Asn His 1 5 10 15 Asp Leu Lys Thr Lys Asn 20 9822PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 98Cys Thr Val Thr Arg Gly Thr Val Asn Asp Pro Gln Arg Ala
Lys Glu 1 5 10 15 Ala Leu Asp Lys Tyr Glu 20 9922PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 99Cys Thr Val Thr Arg Gly Thr Val Asn Asp Pro Gln Arg Ala
Lys Glu 1 5 10 15 Thr Leu Asp Lys Tyr Glu 20 10022PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 100Cys Thr Val Thr Arg Gly Thr Ile Asn Asp Pro Gln Arg
Ala Lys Glu 1 5 10 15 Val Ile Asp Lys Tyr Glu 20 10122PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 101Cys Thr Val Thr Arg Ser Thr Ile Asn Asp Pro Gln Arg
Ala Lys Glu 1 5 10 15 Ala Leu Asp Lys Tyr Glu 20 10222PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 102Cys His Asp Leu Lys Thr Lys Asn Glu Gly Leu Lys Thr
Glu Asn Glu 1 5 10 15 Gly Leu Lys Thr Glu Asn 20 10322PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 103Cys Glu Glu Ala Ser Asn Asn Gly Gln Leu Thr Leu Gln
His Lys Asn 1 5 10 15 Asn Ala Leu Thr Ser Glu 20 10421PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 104Cys Gln His Lys Asn Asn Ala Leu Thr Ser Glu Asn Glu
Ser Leu Arg 1 5 10 15 Arg Glu Lys Asp Arg 20 10521PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 105Cys Glu Ser Leu Arg Arg Glu Lys Asp Arg Tyr Leu Tyr
Glu Lys Glu 1 5 10 15 Glu Leu Glu Lys Lys 20 10621PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 106Cys Arg Arg Glu Glu Lys Asp Arg Tyr Leu Tyr Glu Lys
Glu Glu Leu 1 5 10 15 Glu Lys Lys Asn Lys 20 10720PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 107Cys Asp Asn Gln Ser Pro Ala Pro Val Lys Lys Glu Ala
Lys Lys Leu 1 5 10 15 Asn Glu Ala Glu 20 10821PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 108Cys Lys Lys Glu Ala Lys Lys Leu Asn Glu Ala Glu Leu
Tyr Asn Lys 1 5 10 15 Ile Gln Glu Leu Glu 20 10923PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 109Cys Glu Leu Tyr Asn Lys Ile Gln Glu Leu Glu Glu Gly
Lys Ala Glu 1 5 10 15 Leu Phe Asp Lys Leu Glu Lys 20
11021PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 110Cys Asp Asn Gln Ser Pro Ala Leu Val Lys
Lys Glu Ala Lys Lys Leu 1 5 10 15 Asn Glu Ala Glu Leu 20
11122PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 111Cys Asp Asn Gln Ser Pro Ala Pro Ala Pro
Val Lys Lys Glu Ala Lys 1 5 10 15 Lys Leu Asn Glu Ala Glu 20
11221PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 112Cys Gln Glu Leu Glu Glu Gly Lys Ala Glu
Leu Phe Asp Lys Leu Glu 1 5 10 15 Lys Val Glu Glu Glu 20
11322PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 113Cys Ala Ala Pro Leu Thr Arg Ala Thr Ala
Asp Asn Lys Asp Glu Leu 1 5 10 15 Ile Lys Arg Ala Asn Asp 20
11421PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 114Cys Arg Ala Thr Ala Asp Asn Lys Asp Glu
Leu Ile Lys Arg Ala Asn 1 5 10 15 Asp Tyr Glu Ile Gln 20
11521PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 115Cys Glu Ile Gln Asn His Gln Leu Thr Val
Glu Asn Lys Lys Leu Lys 1 5 10 15 Thr Asp Lys Glu Gln 20
11621PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 116Cys Arg Ala Thr Ala Asp Asn Lys Asp Glu
Leu Ile Lys Arg Ala Asn 1 5 10 15 Gly Tyr Glu Ile Gln 20
11721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 117Cys Lys Asp Glu Leu Ile Lys Arg Lys Glu
Leu Thr Ile Ile Glu Ile 1 5 10 15 Gln Asn His Gln Leu 20
11822PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 118Cys Asn His Gln Leu Thr Val Glu Asn Lys
Lys Leu Lys Thr Asp Lys 1 5 10 15 Glu Gln Leu Thr Lys Glu 20
11921PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 119Cys Asp Ser Ser Arg Glu Val Thr Asn Glu
Leu Thr Ala Ser Met Trp 1 5 10 15 Lys Ala Gln Ala Asp 20
12021PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 120Cys Arg Glu Val Thr Asn Glu Leu Thr Ala
Ser Met Trp Lys Ala Gln 1 5 10 15 Ala Asp Ser Ala Lys 20
12122PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 121Cys Lys Ala Lys Glu Leu Glu Lys Gln Val
Glu Glu Tyr Lys Lys Asn 1 5 10 15 Tyr Glu Thr Leu Glu Lys 20
12221PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 122Cys Asp Ser Ser Arg Glu Val Thr Asn Glu
Leu Ala Ala Ser Met Trp 1 5 10 15 Lys Ala Gln Ala Asp 20
12321PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 123Cys Asp Ser Ser Arg Asp Ser Ser Arg Glu
Val Thr Asn Glu Leu Thr 1 5 10 15 Ala Ser Met Trp Lys 20
12422PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 124Cys Lys Ala Lys Glu Leu Glu Lys Gln Val
Glu Glu Tyr Lys Lys Asn 1 5 10 15 Tyr Glu Thr Leu Glu Lys 20
12521PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 125Cys Glu Gln Ala Lys Asn Asn Asn Gly Glu
Leu Thr Leu Gln Gln Lys 1 5 10 15 Tyr Asp Ala Leu Thr 20
12622PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 126Cys Glu Leu Thr Leu Gln Gln Lys Tyr Asp
Ala Leu Thr Asn Glu Asn 1 5 10 15 Lys Ser Leu Arg Arg Glu 20
12721PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 127Cys Asn Glu Asn Lys Ser Leu Arg Arg Glu
Arg Asp Asn Tyr Leu Asn 1 5 10 15 Tyr Leu Tyr Glu Lys 20
12821PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 128Cys Arg Arg Glu Arg Asp Asn Tyr Leu Asn
Tyr Leu Tyr Glu Lys Glu 1 5 10 15 Glu Leu Glu Lys Lys 20
12923PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 129Cys Ala Asp His Pro Ser Tyr Thr Ala Ala
Lys Asp Glu Val Leu Ser 1 5 10 15 Lys Phe Ser Val Pro Gly His 20
13021PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 130Cys Lys Asp Glu Val Leu Ser Lys Phe Ser
Val Pro Gly His Val Trp 1 5 10 15 Ala His Glu Arg Glu 20
13118PRTArtificial SequenceDescription of Artificial Sequence; NOTE
= Synthetic Construct 131Cys His Glu Arg Glu Lys Asn Asp Lys Leu
Ser Ser Glu Asn Glu Gly 1 5 10 15 Leu Lys 13221PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 132Cys Asp Lys Leu Arg Leu Glu Lys Glu Glu Leu Lys Thr
Asp Leu Gln 1 5 10 15 Lys Lys Glu Arg Glu 20 13321PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 133Cys Lys Asn Asp Lys Leu Ser Ser Glu Asn Glu Gly Leu
Lys Ala Gly 1 5 10 15 Leu Gln Glu Lys Glu 20 13421PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 134Cys Glu Gly Asn Ala Arg Leu Ala Gln Ala Gln Glu Glu
Ala Leu Arg 1 5 10 15 Asp Val Leu Asn Asn 20 13521PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 135Cys Arg Leu Ala Gln Ala Gln Glu Glu Ala Leu Arg Asp
Val Leu Asn 1 5 10 15 Asn Thr Pro His Asn 20 13622PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 136Cys Gln Ala Gln Glu Glu Ala Leu Arg Asp Val Leu Asn
Asn Thr Pro 1 5 10 15 His Asn Gln Leu Arg Asp 20 13722PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 137Cys Asp Val Leu Asn Asn Thr Pro His Asn Gln Leu Arg
Asp Ala Tyr 1 5 10 15 Ala Gly Ala Phe Arg Arg 20 13823PRTArtificial
SequenceDescription of Artificial Sequence; NOTE = Synthetic
Construct 138Cys Gln Leu Arg Asp Pro Tyr Ala Gly Ala Phe Arg Arg
Asn Asn Glu 1 5 10 15 Leu Glu Lys Ile Ile Gln Glu 20
* * * * *