NZ618243B2 - Group a streptococcus multivalent vaccine - Google Patents
Group a streptococcus multivalent vaccine Download PDFInfo
- Publication number
- NZ618243B2 NZ618243B2 NZ618243A NZ61824312A NZ618243B2 NZ 618243 B2 NZ618243 B2 NZ 618243B2 NZ 618243 A NZ618243 A NZ 618243A NZ 61824312 A NZ61824312 A NZ 61824312A NZ 618243 B2 NZ618243 B2 NZ 618243B2
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- amino acid
- acid sequence
- protein
- immunogenic
- fusion polypeptide
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- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/315—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Streptococcus (G), e.g. Enterococci
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56944—Streptococcus
Abstract
Disclosed is an immunogenic composition comprising at least 31 immunogenic peptides, wherein each immunogenic peptide is different and comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein or a Spa protein, wherein each different M protein is independently selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18, and wherein the immunogenic composition induces an immune response against at least each of GAS 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118 serotypes. Also disclosed is the use of such an immunogenic composition for preventing or treating a GAS infection in a subject. ntly selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18, and wherein the immunogenic composition induces an immune response against at least each of GAS 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118 serotypes. Also disclosed is the use of such an immunogenic composition for preventing or treating a GAS infection in a subject.
Description
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GROUP A STREPTOCOCCUS MULTIVALENT VACCINE
CROSS-REFERENCE TO RELATED APPLICATION
This application claims benefit under 35 U.S.C. § 119(e) of U.S.
Provisional Patent Application No. 61/498,397 filed June 17, 2011, and U.S.
Provisional Patent Application No. 61/641,448 filed May 2, 2012, which are each
incorporated herein by reference in their entirety.
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under Grant Numbers
AI-010085, AI-060592, and 5T35DK007405 awarded by the National Institutes of
Health. The government has certain rights in the invention.
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text
format in lieu of a paper copy, and is hereby incorporated by reference into the
specification. The name of the text file containing the Sequence Listing is
920098_414PC_SEQUENCE_LISTING.txt. The text file is 93 KB, was created on
June 14, 2012 and is being submitted electronically via EFS-Web.
BACKGROUND
Technical Field
Safe and effective vaccines to prevent group A streptococcal (GAS)
infections are needed. A multivalent vaccine is described herein that induces an
immune response against multiple GAS serotypes and that may be useful for
immunizing subjects in need thereof.
Description of the Related Art
Efforts to develop safe and effective vaccines to prevent group A
streptococcal (GAS) infections have been ongoing for decades. Although a number of
GAS antigens have been identified as potential vaccine components (see, e.g., Steer et
al., Curr. Opin. Infect. Dis. 22:544-52(2009)), the lead candidates are the type-specific
peptides representing the amino-terminal regions of the surface M proteins (see, e.g.,
Kotloff et al., JAMA 292:709-15 (2004); McNeil et al., Clin. Infect. Dis. 41:1114-22
(2005)).
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The major burden of disease in North America, Europe, and other
economically developed regions and countries is uncomplicated pharyngitis and
serious, invasive infections (see, e.g., Carapetis et al., The Lancet Infectious Diseases
:685-94 (2005)). The global burden of GAS infections is most significant in poor
countries where acute rheumatic fever (ARF) and rheumatic heart disease (RHD) are
rampant (see, e.g., Carapetis et al., supra). Vaccine prevention of the infections that
trigger ARF using M protein-based vaccines has been considered a challenge because
the GAS emm types present in developing countries differ compared to economically
developed areas of the world (see, e.g., Steer et al., The Lancet Infectious Diseases
9:611-16 (2009)). Immunogenicity of a 26-valent M protein-based vaccine in pre-
clinical (see, e.g., Hu et al., supra) and clinical studies (see, e.g., McNeil et al., supra)
has been reported. However, the 26-valent vaccine does not provide protection against
infection by a sufficient number of different GAS serotypes for optimum benefit and
use in both developing and developed countries. Accordingly, a need exists for
development of improved therapeutics and vaccines, which can be economically
produced, for treating and preventing GAS infections.
It is an object of the present invention to address the above need or to
provide one or more of an immunogenic composition, use of an immunogenic
composition, a fusion polypeptide, an isolated polynucleotide, a recombinant expression
vector, an isolated host cell, a process for producing a fusion polypeptide, and an in
vitro method for detecting an antibody which overcome or ameliorate at least one of the
disadvantages of the prior art. These objects are to be read disjunctively with the
alternative object of to at least provide the public with a useful choice.
BRIEF SUMMARY
Briefly, provided herein are immunogenic peptides, fusion polypeptides,
and immunogenic compositions comprising these immunogenic peptides and fusion
polypeptides, capable of inducing an immune response against multiple strains of GAS.
The immunogenic compositions described herein are a significant improvement to
immunogenic compositions previously described in the art for preventing or treating
GAS infections. Methods for using the immunogenic compositions are also provided.
The various embodiments of the immunogenic peptides, fusion polypeptides, and
immunogenic compositions and methods are summarized below.
Embodiment 1. An immunogenic composition comprising at least 31
immunogenic peptides, wherein each immunogenic peptide is different and comprises
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at least 25 contiguous amino acids from the amino terminal portion of a different M
protein or a Spa protein, wherein each different M protein is independently selected
from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14,
18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118,
and the Spa protein is from GAS serotype 18, and wherein the immunogenic
composition induces an immune response against at least each of GAS 1, 2, 3, 4, 5, 6,
11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114,
and 118 serotypes.
Embodiment 2. The immunogenic composition of Embodiment 1,
wherein at least four of the different immunogenic peptides are linked in tandem to
form a fusion polypeptide.
Embodiment 3. The immunogenic composition of Embodiment 1 or
Embodiment 2 comprising a first fusion polypeptide, a second fusion polypeptide, a
third fusion polypeptide, and a fourth fusion polypeptide that each comprises at least six
of the different immunogenic peptides linked in tandem.
Embodiment 4. The immunogenic composition of Embodiment 3,
wherein the first fusion polypeptide comprises eight of the different immunogenic
peptides linked in tandem, wherein each of the eight immunogenic peptides comprises
at least 25 contiguous amino acids from the amino terminal portion of a different M
protein or the Spa protein, wherein each different M protein is independently selected
from the M protein of GAS serotype 1, 2, 3, 6, 12, 18, and 28, and the Spa protein is
from GAS serotype 18.
Embodiment 5. The immunogenic composition of Embodiment 4,
wherein the immunogenic peptide located at the carboxy terminal end of the first fusion
polypeptide is a duplicate of the immunogenic peptide located at the amino terminal
end of the first fusion polypeptide.
Embodiment 6. The immunogenic composition of Embodiment 5,
wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein of GAS serotype 1.
Embodiment 7. The immunogenic composition of any one of
Embodiments 3-6, wherein the second fusion polypeptide comprises eight of the
different immunogenic peptides linked in tandem, and wherein each of the eight
immunogenic peptides comprises at least 25 contiguous amino acids from the amino
terminal portion of a different M protein independently selected from the M protein of
GAS serotype 4, 5, 11, 14, 19, 24, 29, and 75.
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Embodiment 8. The immunogenic composition of Embodiment 7,
wherein the immunogenic peptide located at the carboxy terminal end of the second
fusion polypeptide is a duplicate of the immunogenic peptide located at the amino
terminal end of the second fusion polypeptide.
Embodiment 9. The immunogenic composition of Embodiment 8,
wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein of GAS serotype 4.
Embodiment 10. The immunogenic composition of any one of
Embodiments 3-9, wherein the third fusion polypeptide comprises eight of the different
immunogenic peptides linked in tandem, and wherein each of the eight immunogenic
peptides comprises at least 25 contiguous amino acids from the amino terminal portion
of a different M protein independently selected from the M protein of GAS serotype 22,
44, 58, 73, 77, 78, 89, and 118.
Embodiment 11. The immunogenic composition of Embodiment 10,
wherein the immunogenic peptide located at the carboxy terminal end of the third
fusion polypeptide is a duplicate of the immunogenic peptide located at the amino
terminal end of the third fusion polypeptide.
Embodiment 12. The immunogenic composition of Embodiment 11,
wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein of GAS serotype 77.
Embodiment 13. The immunogenic composition of any one of
Embodiments 3-12, wherein the fourth fusion polypeptide comprises seven of the
different immunogenic peptides linked in tandem, and wherein each of the seven
immunogenic peptides comprises at least 25 contiguous amino acids from the amino
terminal portion of a different M protein independently selected from the M protein of
GAS serotype 49, 81, 82, 83, 87, 92, and 114.
Embodiment 14. The immunogenic composition of Embodiment 13,
wherein the immunogenic peptide located at the carboxy terminal end of the fourth
fusion polypeptide is a duplicate of the immunogenic peptide located at the amino
terminal end of the fourth fusion polypeptide.
Embodiment 15. The immunogenic composition of Embodiment 14,
wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein of GAS serotype 83.
Embodiment 16. The immunogenic composition of any one of
Embodiments 1-15, wherein each of the immunogenic peptides comprises (a) the at
least 25 contiguous amino acids from the amino terminal portion of the different M
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protein or the Spa protein in duplicate; (b) at least 40 contiguous amino acids from the
amino terminal portion of the different M protein or the Spa protein; (b) at least 45
contiguous amino acids from the amino terminal portion of the different M protein or
the Spa protein; or (d) at least 50 contiguous amino acids from the amino terminal
portion of the different M protein or the Spa protein.
Embodiment 17. An immunogenic composition comprising:
(a) a first fusion polypeptide comprising an amino acid sequence at least
85% identical to the amino acid sequence set forth in SEQ ID NO:1;
(b) a second fusion polypeptide comprising an amino acid sequence at
least 85% identical to the amino acid sequence set forth in SEQ ID NO:2;
(c) a third fusion polypeptide comprising an amino acid sequence at least
85% identical to the amino acid sequence set forth in SEQ ID NO:3; and
(d) a fourth fusion polypeptide comprising an amino acid sequence at
least 85% identical to the amino acid sequence set forth in SEQ ID NO:4,
wherein the immunogenic composition induces an immune response
against at least each of GAS 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58,
73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118 serotypes.
Embodiment 18. The immunogenic composition of Embodiment 17
wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 90%
identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion
polypeptide comprises an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an
amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ
ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at
least 90% identical to the amino acid sequence set forth in SEQ ID NO:4.
Embodiment 19. The immunogenic composition of Embodiment 17,
wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 95%
identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion
polypeptide comprises an amino acid sequence at least 95% identical to the amino acid
sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an
amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ
ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at
least 95% identical to the amino acid sequence set forth in SEQ ID NO:4.
Embodiment 20. The immunogenic composition of Embodiment 17,
wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 97%
identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion
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polypeptide comprises an amino acid sequence at least 97% identical to the amino acid
sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an
amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ
ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at
least 97% identical to the amino acid sequence set forth in SEQ ID NO:4.
Embodiment 21. The immunogenic composition of Embodiment 17,
wherein (a) the first fusion polypeptide comprises the amino acid sequence set forth in
SEQ ID NO:1; (b) the second fusion polypeptide comprises the amino acid sequence set
forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises the amino acid
sequence set forth in SEQ ID NO:3; and (d) the fourth fusion polypeptide comprises the
amino acid sequence set forth in SEQ ID NO:4.
Embodiment 22. The immunogenic composition of any one of
Embodiments 1-21, further comprising a pharmaceutically acceptable excipient.
Embodiment 23. The immunogenic composition of any one of
Embodiments 1-22, further comprising a pharmaceutically acceptable adjuvant.
Embodiment 24. The immunogenic composition of any one of
Embodiments 1-23, wherein the immune response against group A streptococcus
comprises an immune response against at least each of GAS 8, 9, 15, 25, 30, 33, 36, 40,
48, 51, 52, 53, 59, 60, 65, 66, 68, 76, 79, 85, 94, 95, 97, 102, 105, 109, 111, and 122
serotypes.
Embodiment 25. A method for inducing an immune response against
group A streptococcus in a subject, comprising administering to the subject the
immunogenic composition of any one of Embodiments 1-24.
Embodiment 26. A method for reducing the likelihood of occurrence of a
group A streptococcus infection in a subject, comprising administering to the subject
the immunogenic composition of any one of Embodiments 1-24.
Embodiment 27. A method for preventing or treating a group A
streptococcus infection in a subject, comprising administering to the subject the
immunogenic composition of any one of Embodiments 1-24.
Embodiment 28. The immunogenic composition of any one of
Embodiments 1-24 for use in preventing or treating a group A streptococcus infection.
Embodiment 29. Use of the immunogenic composition of any one of
Embodiments 1-24 for the manufacture of a vaccine to prevent or treat a group A
streptococcus infection.
Embodiment 30. The immunogenic composition of any one of
Embodiments 1-24 for preventing or treating a group A streptococcus infection.
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Embodiment 31. A fusion polypeptide comprising:
(a) an amino acid sequence at least 85% identical to the amino acid
sequence set forth in SEQ ID NO:1;
(b) an amino acid sequence at least 85% identical to the amino acid
sequence set forth in SEQ ID NO:2;
(c) an amino acid sequence at least 85% identical to the amino acid
sequence set forth in SEQ ID NO:3; or
(d) an amino acid sequence at least 85% identical to the amino acid
sequence set forth in SEQ ID NO:4,wherein the fusion polypeptide induces an immune
response against group A streptococcus.
Embodiment 32. The fusion polypeptide of Embodiment 31 comprising:
(a) an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO:1;
(b) an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO:2;
(c) an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO:3; or
(d) an amino acid sequence at least 90% identical to the amino acid
sequence set forth in SEQ ID NO:4.
Embodiment 33. The fusion polypeptide of Embodiment 31 comprising:
(a) an amino acid sequence at least 95% identical to the amino acid
sequence set forth in SEQ ID NO:1;
(b) an amino acid sequence at least 95% identical to the amino acid
sequence set forth in SEQ ID NO:2;
(c) an amino acid sequence at least 95% identical to the amino acid
sequence set forth in SEQ ID NO:3; or
(d) an amino acid sequence at least 95% identical to the amino acid
sequence set forth in SEQ ID NO:4.
Embodiment 34. The fusion polypeptide of Embodiment 31 comprising:
(a) an amino acid sequence at least 97% identical to the amino acid
sequence set forth in SEQ ID NO:1;
(b) an amino acid sequence at least 97% identical to the amino acid
sequence set forth in SEQ ID NO:2;
(c) an amino acid sequence at least 97% identical to the amino acid
sequence set forth in SEQ ID NO:3; or
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(d) an amino acid sequence at least 97% identical to the amino acid
sequence set forth in SEQ ID NO:4.
Embodiment 35. The fusion polypeptide of Embodiment 31 comprising:
(a) the amino acid sequence set forth in SEQ ID NO:1;
(b) the amino acid sequence set forth in SEQ ID NO:2;
(c) the amino acid sequence set forth in SEQ ID NO:3; or
(d) the amino acid sequence set forth in SEQ ID NO:4.
Embodiment 36. An isolated polynucleotide encoding the fusion
polypeptide of any one of Embodiments 31-35.
Embodiment 37. A recombinant expression vector comprising the
isolated polynucleotide of Embodiment 36 operatively linked to at least one expression
control region.
Embodiment 38. An isolated host cell transfected, transduced, or
transformed with the recombinant expression vector of Embodiment 37.
Embodiment 39. A process for producing the fusion polypeptide of any
one of Embodiments 31-35, the method comprising:
(a) culturing the isolated host cell of Embodiment 38; and
(b) isolating the fusion polypeptide from the host cell culture.
Embodiment 40. A method for detecting an antibody that specifically
binds to the fusion polypeptide of any one of Embodiments 31-35 in a biological
sample suspected of containing the antibody, the method comprising:
(a) contacting the biological sample with
(i) an immunogenic peptide comprising at least 25 contiguous
amino acids of the amino terminal portion of an M protein or a Spa protein, wherein the
M protein is selected from the M protein of group A streptococcus (GAS) serotype 1, 2,
3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89,
92, 114, and 118, and the Spa protein is from GAS serotype 18;
(ii) a dimeric peptide, wherein each peptide of the dimeric
peptide is different and comprises at least 25 contiguous amino acids of the amino
terminal portion of a different M protein or a Spa protein, wherein each different M
protein is independently selected from the M protein of group A streptococcus (GAS)
serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81,
82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18; or
(iii) the fusion polypeptide of any one of Embodiment s 31-35;
and
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(b) detecting specific binding of the immunogenic peptide, dimeric
peptide, or fusion polypeptide with the biological sample, thereby indicating that the
biological sample contains the antibody.
Embodiment 41. Use of the immunogenic composition of any one of
embodiments 1-24 in the manufacture of a vaccine for inducing an immune in response
against group A streptococcus in a subject.
Embodiment 42. Use of the immunogenic composition of any one of
embodiments 1-24 in the manufacture of a vaccine for reducing the likelihood of
occurrence of a group A streptococcus infection in a subject.
As used herein and in the appended claims, the singular forms “a,”
“and,” and “the” include plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to “a polypeptide” may refer to one or more polypeptides,
or a plurality of such polypeptides, and reference to “a cell” or “the cell” includes
reference to one or more cells and equivalents thereof (e.g., plurality of cells) known to
those skilled in the art, and so forth. Similarly, reference to “a composition” includes a
plurality of such compositions, and refers to one or more compositions unless the
context clearly dictates otherwise. When steps of a method are described or claimed,
and the steps are described as occurring in a particular order, the description of a first
step occurring (or being performed) “prior to” (i.e., before) a second step has the same
meaning if rewritten to state that the second step occurs (or is performed) “subsequent”
to the first step. The term “about” when referring to a number or a numerical range
means that the number or numerical range referred to is an approximation within
experimental variability (or within statistical experimental error), and thus the number
or numerical range may vary between 1% and 15% of the stated number or numerical
range. The term “comprising” (and related terms such as “comprise” or “comprises” or
“having” or “including”) is not intended to exclude that in other certain embodiments,
for example, an embodiment of any composition of matter, composition, method, or
process, or the like, described herein, may “consist of” or “consist essentially of” the
described features.
As used herein, the term “isolated” means that a material is removed
from its original environment (e.g., the natural environment if it is naturally occurring).
For example, a naturally occurring nucleic acid or polypeptide present in a living
animal is not isolated, but the same nucleic acid or polypeptide, separated from some or
all of the co-existing materials in the natural system, is isolated. Such a nucleic acid
could be part of a vector and/or such nucleic acid or polypeptide could be part of a
composition, and still be isolated in that the vector or composition is not part of the
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natural environment for the nucleic acid or polypeptide. The term “gene” means the
segment of DNA involved in producing a polypeptide chain; it includes regions
preceding and following the coding region “leader and trailer” as well as intervening
sequences (introns) between individual coding segments (exons). Amino acids may be
referred to herein according to the single letter and three letter codes, which are
understood according to common textbook knowledge in the art, and therefore with
which a person skilled in the art is familiar. The term “fusion polypeptide” used herein
may also be used interchangeably with “fusion protein,” and unless specifically
indicated otherwise, the two terms are not meant to indicate molecules that have
distinguishable properties or characteristics.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 presents a schematic diagram of four proteins comprising a 30-
valent M protein-based GAS immunogenic composition.
Figure 2A-2H provides the nucleotide sequences of four synthetic
polynucleotides (Figure 2A (SEQ ID NO:17); 2C (SEQ ID NO:18); 2E (SEQ ID
NO:19); and 2G (SEQ ID NO:20)) and the respective amino acid sequence of the
encoded constituent recombinant polypeptides (Figure 2B (SEQ ID NO:9); 2D (SEQ ID
NO:10); 2F (SEQ ID NO:11); and 2H (SEQ ID NO:12)). Each polynucleotide was
synthesized to contain an upstream T7 promoter, ribosome binding site, start codon
(ATG in bold, beginning at position 108 of each of Figure 2A, 2C, 2E, 2G), 3'
polyhistidine motif, and a termination codon.
Figure 3 depicts dose response in rabbits of the level of the antibody titer
in animals immunized with the 30-valent immunogenic composition described herein.
Figure 4A-B illustrates the geometric mean antibody levels (Log )
evoked in three rabbits after immunization with three 800 μg doses of the 30-valent
GAS immunogenic composition. Solid-phase bound antigens were recombinant dimeric
peptides or synthetic peptides (M44, M78, M118, M83, M81, M87, M49) that were
used and that had the same amino acid sequence as the respective peptide used in the
-valent GAS immunogenic composition. Figure 4B provides a graphic of the level of
bactericidal antibodies evoked by the 30-valent M protein-based GAS immunogenic
composition. Immune serum from one of the rabbits immunized with 800 μg doses of
the immunogenic composition was used in bactericidal assays against all serotypes of
GAS represented in the immunogenic composition. Percent killing was calculated as
described in the Examples.
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Figure 5A-B illustrates the level of bactericidal antibodies evoked by a
-valent immunogenic composition against serotypes of GAS not represented by a
GAS peptide in the 30-valent immunogenic composition.
Figure 6 presents a comparison of cross-opsonic bactericidal antibodies
evoked by the 30-valent immunogenic composition vs. a 26-valent vaccine (see, e.g.,
Hu et al., supra; U.S. Patent No. 7,270,827). The level of bactericidal killing promoted
by the 30-valent immunogenic composition against 10 serotypes that were not
represented in any of the fusion polypeptide constructs was significantly greater than
bactericidal killing observed with the 26-valent antiserum (average 64.9% vs. 23.6%,
p=0.0008, Student's t-test, paired, two-tailed).
DETAILED DESCRIPTION
Provided herein are immunogenic compositions that are capable of
evoking (i.e., inducing) an immune response against multiple strains of group A
streptococcus (GAS) and that represent a significant improvement to immunogenic
compositions previously described in the art. As described herein, the combination of
immunogenic peptides derived from GAS strains in the multivalent immunogenic
compositions evoke immune responses not only against the GAS strains from which the
immunogenic peptides were derived but also against multiple additional strains of GAS
not represented by immunogenic peptides in the composition.
Group A streptococcus (Streptococcus pyogenes) has two major,
surface-exposed anti-phagocytic factors, capsule and M protein, which allow these
organisms to colonize and survive in a host. The cell surface M protein of group A
streptococci (GAS) is one of the major virulence factors for this pathogen. The M
protein, which is encoded by the emm gene, extends from the cell surface as an -
helical coiled-coil dimer that appears as a fibril on the surface of a GAS bacterium. The
M proteins form an antigenically diverse group and GAS strains have been
serologically categorized according to M protein serotypes. More than 120 M protein
serotypes have been identified, and within some serotypes, subtypes have also been
identified. By way of example, the M proteins of GAS serotype 3 include related
subtypes, which are designated as 3.0, 3.1, 3.2, etc. Antibodies to the M protein can
facilitate opsonophagocytosis by phagocytic cells present in blood.
As described herein and by way of example, a 30-valent immunogenic
composition (which may also be referred to herein and in the art as a vaccine) was
constructed that contains immunogenic peptides derived from the M protein of GAS
serotypes prevalent in North America, Europe, and developing countries. Selection of
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serotypes represented in the composition was based on available epidemiology and
serotype prevalence of GAS infections in North America and Europe (see, e.g.,
Shulman et al., Clin. Infect. Dis. 39:325-32 (2004)); Shulman et al., Clin. Infect. Dis.
49:78-84 (2009); O'Loughlin et al., Clin. Infect. Dis. 45:853-62 (2007); Luca-Harari et
al., J. Clin. Microbiol. 47:1155-65 (2009)), and in some developing countries (see, e.g.,
Steer et al., supra). These immunogenic compositions also contain an immunogenic
peptide from a protein referred to in the art as Spa, which evokes antibodies that cross
react with more than one GAS serotype. The immunogenic compositions described
herein, such as the composition comprising thirty different M protein peptide
immunogens and a Spa peptide immunogen, more adequately represents the
epidemiology of pharyngitis and invasive GAS infections.
In addition, the immunogenic compositions described herein include an
immunogenic peptide derived from the M protein of the GAS serotype 4. GAS
serotype 4 causes a significant portion of GAS disease, approximately 9% of
uncomplicated pharyngitis in North America and 5% of invasive GAS infections in
Europe (see, e.g., Shulman et al., Clin. Infect. Dis. 2009, supra; Luca-Harari et al.,
supra). A member of the M protein family called Arp4 (see, e.g., Johnsson et al., J.
Immunol. 153:3557-64 (1994)) present on the cell surface of GAS serotype 4 bacteria
was initially believed not to be required for virulence or for resistance to phagocytosis,
unlike other members of the M protein family (see, e.g., Husmann et al., Infect. Immun.
63:345-48 (1995)). Upon further experimentation, an immunogenic peptide from the
amino terminal end of the Arp4 (M4) protein that evokes opsonic antibodies in an
immunized host was identified (see, e.g., Courtney et al., supra). The addition of an
M4 immunogenic peptide to a GAS immunogenic composition improves the efficacy of
multivalent vaccines with respect to both uncomplicated and complicated infections by
increasing, from approximately 78% to 90%, the number of GAS strains to which such
a multivalent immunogenic composition induces an immune response.
Distinct from M protein, a polypeptide designated Spa (Streptococcal
protective antigen) contains epitopes that induce production of opsonic, protective
antibodies. Spa polypeptides may be serotype specific, such as the Spa polypeptide
from GAS serotype 36, whereas other Spa polypeptides, such as Spa polypeptide
isolated from GAS serotype 18, evoke antibodies that bind to multiple GAS serotypes.
The Spa polypeptide of GAS serotype 18 induces antibodies that bind to and are
protective against serotypes 3 and 28 as well as serotype 18 (see, e.g., U.S. Patent No.
7,063,850; Ahmed et al., Eur. J. Clin. Microbiol. Infect. Dis. 29:51-57 (2010) Epub
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2009 Oct 29; McLellan et al., Infect. Immun. 69:2943-49 (2001); Dale et al., J. Clin.
Invest. 103:1261-68 (1999)).
In one embodiment, an immunogenic composition is provided that
comprises four different fusion proteins that each comprise seven or eight GAS M
protein or GAS Spa protein immunogenic fragments linked in tandem. An exemplary
composition was immunogenic in rabbits and evoked bactericidal antibodies against all
serotypes of GAS represented by immunogenic peptides included in the composition. In
addition, unexpectedly and in contrast to a 26-valent immunogenic composition
previously described in the art, antisera obtained from animals immunized with the
exemplary 30-valent composition described herein also contained significant levels of
bactericidal antibodies against GAS serotypes from which an immunogenic peptide
fragment was not included in the composition (i.e., a non-vaccine GAS serotype or non-
represented GAS serotype). Therefore, the potential efficacy of the immunogenic
compositions described herein, such as the 30-valent immunogenic composition, may
extend well beyond the serotypes represented by the subunit M peptides and the Spa
peptide. This and other embodiments and uses therefor are described in greater detail
herein.
Immunogenic Compositions
In one embodiment, an immunogenic composition is provided that
comprises a plurality of GAS immunogenic peptide. In a particular embodiment, the
immunogenic composition comprises at least 31 different immunogenic peptides. Each
immunogenic peptide comprises an amino acid sequence that is located at the amino
(also called herein and in the art, NH ) terminal portion of one of 31 different GAS
polypeptides, respectively. In a particular embodiment, each of at least 30 different
immunogenic peptides is derived from the amino acid sequence of at least 30 M
proteins from 30 different GAS serotypes, respectively. Each immunogenic peptide of
an M protein comprises at least 25 contiguous amino acids from the amino terminal
portion of the M protein. The compositions described herein also comprise an
immunogenic peptide that comprises at least 25 contiguous amino acids from the amino
terminal portion of a Spa polypeptide, which in particular embodiments is the Spa
polypeptide expressed and present on the cell surface of GAS serotype M18 strains. In
another particular embodiment, an immunogenic composition is provided that
comprises at least four fusion polypeptides that comprise the plurality of immunogenic
peptides (e.g., at least 31 different immunogenic peptides derived from GAS M proteins
or GAS Spa proteins).
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The amino terminal regions of M proteins have been shown to evoke
antibodies with the greatest bactericidal (protective) activity and are least likely to
cross-react with human tissues (see, e.g., Dale, Adv. Exp. Med. Biol. 609:53-63 (2008)).
One approach has been to construct recombinant hybrid proteins containing M protein
peptides combined into multivalent vaccines designed to elicit opsonic antibodies
against epidemiologically important serotypes of group A streptococci (see, e.g., Dale,
Inf. Dis. Clin. N. Amer. 13:227-43 (1999); Hu et al., Infect. Immun. 70:2171-77 (2002)).
The anti-GAS immunogenic compositions described herein provide a
significant improvement compared with previously described vaccine compositions. In
one embodiment, a 30-valent immunogenic composition is provided that includes
immunogenic peptides of M proteins and a Spa protein that evoked an immune
response not only against each GAS serotype represented by an M protein
immunogenic peptide but also, significantly, to more non-vaccine serotypes (i.e., GAS
serotypes not represented in the immunogenic composition by an M protein
immunogenic peptide or a Spa immunogenic peptide). Importantly, in addition, the
immunogenic compositions described herein include an immunogenic peptide from the
M protein of GAS serotype 4 (see, e.g., Courtney et al., Molec. Microbiol. 59:936-47
(2006)). GAS serotype 4 causes approximately 9% of uncomplicated pharyngitis in
North America and 5% of invasive GAS disease in Europe (see, e.g., Shulman et al.,
Clin. Infect. Dis. 49:78-84 (2009); Luca-Harari et al., J. Clin. Microbiol. 47:1155-65
(2009))); therefore, inclusion of an M4 immunogenic peptide greatly improves the
efficacy of a multivalent GAS vaccine.
The immunogenic compositions comprising GAS immunogenic peptides
described herein evoke an immune response against GAS and more particularly against
each of the GAS serotypes that are represented in the vaccine and from which the
immunogenic peptides were derived. In addition, the immunogenic compositions
described herein evoke an immune response against GAS serotypes from which an
immunogenic peptide was not obtained and therefore not included in the immunogenic
composition (also called herein non-vaccine GAS serotypes or non-represented GAS
serotypes). Terms, such as “30-valent” or “26-valent,” used herein refer to the number
of GAS serotypes from which immunogenic peptides were obtained or derived. The
number of immunogenic peptides included in an immunogenic composition may be
greater than the indicated valency. For example, the immunogenic compositions
comprising 31 immunogenic peptides described herein may be referred to as a 30-valent
composition because the Spa immunogenic peptide (described herein and in the art)
included in the composition can be obtained from GAS serotype M18, and an
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immunogenic peptide from the M protein of serotype M18 is also included in the
vaccine composition.
Immunogenic compositions were designed and constructed to provide
compositions that evoke an immune response against the greatest number of clinically
relevant GAS serotypes regardless of geographic location. Accordingly, to maximize
clinical use and efficacy, GAS serotypes prevalent in one or more geographic areas,
which may be determined from epidemiological data available in the art, may serve as
the source of M protein immunogenic fragments or other GAS polypeptide
immunogenic fragments (e.g., a Spa immunogenic fragment). GAS serotypes
represented by immunogenic fragments in immunogenic compositions described herein
include those that cause non-invasive infections (e.g., pharyngitis, impetigo, erysipelas,
and cellulitis) and GAS serotypes that cause invasive infections (e.g., GAS infections of
the blood (bacteremia), muscle, and lung (pneumonia), necrotizing fasciitis, and
streptococcal toxic shock syndrome), and nonsuppurative sequelae such as acute
rheumatic fever, reactive arthritis, and glomerulonephritis (see, e.g., Cunningham, Clin.
Microbiol. Rev. 13:470 (2000)).
Epidemiology data may be obtained from public and/or private local,
national, and international organizations and investigators. By way of example,
serotypes of GAS from which immunogenic peptides of M proteins may be selected for
use in immunogenic compositions based on the epidemiology of non-invasive
infections, for example, pharyngitis in pediatric subjects in North America (see, e.g.,
Shulman et al., Clin. Infect. Dis. 2009, supra); (2) invasive GAS infections (see, e.g.,
Active Bacterial Core Surveillance of the Emerging Infections Program Network,
supported by the Centers for Disease Control and Prevention) (see, e.g., O’Loughlin et
al., supra); (3) invasive infections in Europe (for example, as reported by the StrepEuro
consortium, see Luca-Harari et al., supra); and/or (4) GAS serotypes prevalent in
developing countries (see, e.g., Steer et al., The Lancet Infectious Diseases 9:611-16
(2006)). Also included in the immunogenic compositions described herein (e.g., the 30-
valent vaccine composition) are M peptides from serotypes of GAS that are currently or
have been historically considered “rheumatogenic” (see, e.g., Shulman et al., Clin.
Infect. Dis. 42:441-47 (2006)). In addition, the amino-terminal peptide fragment of
Spa18, a protective antigen that is expressed by at least several serotypes of group A
streptococci, such as but not limited to, GAS serotype 18, GAS serotype 3, and GAS
serotype 28, may be included in the multivalent immunogenic compositions described
herein (see, e.g., Dale et al., J. Clin. Invest. 103:1261-68 (1999); U.S. Patent No.
7,063,850; International Patent Application Publication No. WO 00/37648).
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The amino acid sequences of M proteins and subtypes of M proteins are
available in protein databases, such as GenBank, GenEMBL, and Swiss Prot databases.
M protein sequences are also available at the Center for Disease Control (CDC) emm
typing center website that may be found by accessing the Internet at
cdc.gov/ncidod/biotech/strep/emmtypes. The amino acid sequence of Spa polypeptides
are also available in public databases and described in Dale et al., J. Clin. Invest.
103:1261-68 (1999); U.S. Patent No. 7,063,850; and International Patent Application
Publication No. WO 00/37648. Criteria for subtyping of GAS M serotypes as currently
used in the art is described by Facklam et al., Emerg. Infect. Dis. 5:247-53 (1999).
Also long known in the art, region(s) of M proteins comprise amino acid
sequences that induce antibodies that cross-react with host tissue, for example, human
heart (see, e.g., Dale et al., J. Exp. Med. 156:1165-76 (1982); Dale et al., J. Exp. Med.
161:113-22 (1985)), brain (see, e.g., Bronze et al., J. Immunol. 151:2820-28 (1993)),
muscle, kidney, and cartilage. Accordingly, the design of the multivalent immunogenic
compositions described herein includes analysis of amino acid sequences in M proteins
to determine if each M protein has five or more contiguous amino acids in common
with one or more human proteins and which therefore could potentially induce
antibodies that might bind to human tissue. Immunogenic peptides of M proteins and
fusion polypeptides comprising the immunogenic peptides included in the
immunogenic compositions described herein lack five or more contiguous amino acids
that are identical to five or more contiguous amino acids of a known human protein.
Comparison of the amino acid sequences of M proteins with human
proteins can be accomplished using one or more of the several alignment programs
available to the person skilled in the art. Such alignment tools (which may also be
useful for comparing nucleotide sequences, such as those encoding an M protein)
include, without limitation, software programs such as BlastP, BLAST, tBLAST, and
MegAlign. Other software programs are provided in the Lasergene bioinformatics
computing suite (DNASTAR Madison, Wisconsin); CLUSTALW program (see, e. g.,
Thompson et al., Nucleic Acids Res. 22:4673-80 (1991)); and “GeneDoc” (Nicholas et
al., EMBNEW News 4:14 (1991)). References for algorithms such as ALIGN or
BLAST may be found in, for example, Altschul, J. Mol. Biol. 219:555-565, 1991; and
Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992. Algorithms
including the Align and BLAST algorithms are available at the NCBI website (see
[online] Internet at ncbi.nlm.nih.gov/cgi-bin/BLAST). Default parameters may be used.
Additional methods available in the art for comparing nucleotide or amino acid
sequences and determining optimal alignment include methods described, for example,
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in Peruski and Peruski, The Internet and the New Biology: Tools for Genomic and
Molecular Research (ASM Press, Inc. 1997); Wu et al. (eds.), “Information
Superhighway and Computer Databases of Nucleic Acids and Proteins,” in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997); and Bishop (ed.), Guide to
Human Genome Computing, 2nd Ed. (Academic Press, Inc. 1998).
A GAS immunogenic peptide that may be used in the multivalent
vaccine compositions (i.e., multivalent immunogenic compositions) described herein
comprises an immunogenic portion of a full-length GAS polypeptide and may comprise
at least 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids (or any number
of amino acids between 25-30, 30-35, 35-40, 40-45, 45-50, 40-55, or 55-60, or more
than 60 contiguous amino acids) of the amino terminal portion of the mature
polypeptide (i.e., the polypeptide from which the signal peptide sequence has been
removed). In certain particular embodiments, the immunogenic peptide may comprise
at least 25, 30, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids from the amino
terminal portion of an M protein or a Spa protein (or any number of amino acids
between 25-30, 30-35, 35-40, 40-45, 45-50, 40-55, or 55-60, or more than 60
contiguous amino acids from the amino terminal portion of an M protein or a Spa
protein). An immunogenic portion of a mature or full-length GAS polypeptide has one
or more epitopes that induces an immune response, which includes production of
antibodies that specifically bind to the immunogenic peptide, and to the immunogenic
portion within the mature and full-length polypeptide, and to GAS bacteria that express
the polypeptide. In more particular embodiments, an immunogenic peptide comprises
at least 25 or at least 50 contiguous amino acids from the amino terminal portion of an
M protein or a Spa protein. In certain specific embodiments, one or more immunogenic
peptides comprise at least 25 contiguous amino acids from the amino terminal portion
of an M protein or a Spa protein, which immunogenic peptide is present in duplicate
and the duplicates are directly linked in tandem. In other words and by way of
illustration, if the immunogenic region selected includes amino acid residues 1 - 25 of
the amino terminal portion of an M protein or Spa protein, the immunogenic peptide in
duplicate includes amino acid residues 1-25 linked in tandem to a second repeat of
amino acids 1-25 (i.e., amino acid residue 25 of the first duplicate bonds directly to
amino acid residue 1 of the second duplicate).
In one embodiment, the multivalent immunogenic compositions
comprise immunogenic peptides that are representative (or obtained from, derived
from) at least 30 different GAS serotypes. In more specific embodiments, one of the
GAS serotypes represented in the immunogenic composition described herein is M4,
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and an immunogenic peptide comprises an amino acid sequence that includes at least 25
contiguous amino acids from the amino terminal portion of the M4 protein. In other
certain embodiments, an immunogenic composition comprises at least 31 immunogenic
peptides that are representative of at least 30 different GAS serotypes. In more
particular embodiments, the amino acid sequence of each of the 31 immunogenic
peptides is different and each comprises at least 25, 30, 35, 40, 45, 50, 55, or 60 or more
contiguous amino acids (or any number of amino acids between 25-30, 30-35, 35-40,
40-45, 45-50, 40-55, or 55-60, or more than 60 contiguous amino acids) from the amino
terminal portion of the M protein from one of GAS serotypes (1) M1; (2) M2; (3) M3;
(4) M4; (5) M5; (6) M6; (7) M11; (8) M12; (9) M14; (10) M18; (11) M19; (12) M22;
(13) M24; (14) M28; (15) M29; (16) M44; (17) M49; (18) M58; (19) M73; (20) M75;
(21) M77; (22) M78; (23) M81; (24) M82; (25) M83; (26) M87; (27) M89; (28) M92;
(29) M114; (30) M118, and (31) from the amino terminal portion of the Spa protein
from GAS serotype, for example, such as GAS serotype M18. In another more specific
embodiment, one or more of the immunogenic peptides comprises at least 25
contiguous amino acids in duplicate. In another specific embodiment one or more of
the immunogenic peptides comprises at least 40 contiguous amino acids or at least 45
contiguous amino acids. In still another specific embodiment, one or more of the
immunogenic peptides comprises at least at least 50 contiguous amino acids. As used
herein, when referring to GAS bacteria according to serotype, the bacteria may be
called, for example, GAS serotype 3, GAS M serotype 3, or GAS serotype M3. When
referring to the M protein of a particular serotype, the designation of the GAS serotype
from which the M protein is derived, such as M3, is typically followed by the word
protein or immunogenic peptide depending on the context (i.e., M3 protein or M3
immunogenic peptide).
Accordingly, in other words, the embodiment of an immunogenic
composition described above comprises at least the following immunogenic peptides:
an immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 1; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 2; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 3; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 4; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 5; an immunogenic peptide
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comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 6; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 11; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 12; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 14; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 18; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 19; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 22; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 24; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 28; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 29; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 44; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 49; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 58; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 73; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 75; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 77; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 78; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 81; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 82; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 83; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
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protein from GAS serotype 87; an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 89; an immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 92; an
immunogenic peptide comprising at least 25 contiguous amino acids from the amino
terminal portion of the M protein from GAS serotype 114; an immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 118; and an immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the Spa protein from a GAS
serotype, for example, such as GAS serotype 18. In certain particular embodiments as
described herein, each aforementioned immunogenic peptide may comprise at least 25,
, 35, 40, 45, 50, 55, or 60 or more contiguous amino acids from the amino terminal
portion of the respective M protein or Spa protein (or any number of amino acids
between 25-30, 30-35, 35-40, 40-45, 45-50, 40-55, or 55-60, or more than 60
contiguous amino acids from the amino terminal portion of the respective M protein or
Spa protein). In another more specific embodiment, one or more of the immunogenic
peptides comprises at least 25 contiguous amino acids in duplicate. In another specific
embodiment one or more of the immunogenic peptides comprises at least 40 contiguous
amino acids or at least 45 contiguous amino acids. In still another specific embodiment,
one or more of the immunogenic peptides comprises at least at least 50 contiguous
amino acids.
In other certain embodiments, an immunogenic composition may
comprise 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more different
immunogenic peptides. The different immunogenic peptides may include the 31
immunogenic peptides from the amino terminal portions of the M proteins from GAS
M serotypes 1; 2; 3; 4; 5; 6; 11; 12; 14; 18; 19; 22; 24; 28; 29; 44; 49; 58; 73; 75; 77;
78; 81; 82; 83; 87; 89; 92; 114; 118, and Spa protein from the GAS serotype 18 (as
described above) and further comprise immunogenic peptides from the amino terminal
portions of M proteins from at least one additional, different GAS serotype or subtype
and/or one or more additional, different immunogenic peptide from the amino terminal
portion of a Spa protein. Selection of additional immunogenic peptides may be
informed by epidemiology data; lack of shared identity of 5 or more contiguous amino
acids of the GAS protein with a human protein; immunogenicity, bactericidal, and/or
protection data, including the capability of the one or more additional immunogenic
peptides to increase the number of non-represented GAS serotypes recognized by
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antibodies produced in response to immunization with such an immunogenic
composition.
As described herein and known to a person skilled in the art, the amino
terminal portion of M proteins and Spa proteins comprise the regions of M proteins and
Spa proteins that have one or more immunogenic epitopes which evoke a protective
immune response but do not induce antibodies that cross-react with proteins expressed
by human cells and tissues. As used herein the amino terminal portion of an M protein
or a Spa protein refers to the mature protein, which is the expressed protein lacking the
signal peptide sequence. As is well understood in the art, polypeptides that are secreted
or are membrane bound proteins are translocated through or to the membrane,
respectively, by a translocation apparatus that interacts with a signal peptide at the
amino terminal end of a nascent polypeptide. In bacteria, a signal peptide sequence is
typically cleaved from a nascently translated polypeptide in vivo by a bacterial
protease to form the mature polypeptide. Accordingly, unless specified otherwise, a
description herein of an immunogenic peptide as an immunogenic peptide comprising
the amino acid sequence of residues 1-50 of an M protein (for example), residue 1 is
at the amino terminus of the M protein in the absence of the signal peptide sequence.
Exemplary amino acid sequences for immunogenic peptides are provided in SEQ ID
NOS:29-59 (see Sequence Listing and Table 1).
The term “amino terminal portion” of an M protein or a Spa protein is
readily understood by a person having ordinary skill in the art as the portion or region
of an polypeptide located in the amino terminal half of the polypeptide. The
immunogenic peptides described herein that comprise amino acids from the amino
terminal portion of an M protein or a Spa protein may, but not necessarily, comprise
at least 25 contiguous amino acids from the amino terminal end of the respective M
protein or Spa protein (i.e., amino acids 1-25 of the mature polypeptide). In other
instances, an immunogenic peptide may comprise at least 25 contiguous amino acids
derived from the amino terminal portion of an M protein or Spa protein that lack one
or more amino acids located at the amino terminus of the mature protein (e.g., amino
acids 2-26, 3-27, 4-28 and the like of the mature polypeptide). In other embodiments,
and by way of example, the at least 25 contiguous amino acids may be derived from
the amino terminal portion of the M protein or Spa protein that begins at amino acid
position 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.
As described herein, the amino acid sequence of each immunogenic peptide is
selected, at least in part, on the basis of the presence of at least one immunogenic
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epitope and the lack of at least five contiguous amino acids that are identical to at
least five contiguous amino acids present in a known human protein.
In one embodiment, the immunogenic composition comprising at least
31 different immunogenic peptides contains each of the individual immunogenic
peptides as separate peptides. In certain embodiments, the immunogenic composition
may comprise 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more different
immunogenic peptides and contains each of the individual immunogenic peptides as a
separate peptide. Each of these immunogenic peptides may each be chemically
synthesized or may be recombinantly produced according to techniques and methods
described in greater detail herein and in the art. The individual peptides are then
combined together and formulated in an immunogenic composition. Also as described
in greater detail herein, the immunogenic compositions may comprise one or more
pharmaceutically suitable excipients, and may further comprise one or more
pharmaceutically suitable adjuvants. In still another embodiment, the immunogenic
compositions comprise the at least 31 different immunogenic peptides as a combination
of individual immunogenic peptides, dimeric peptides, and/or trimeric peptides.
In still other embodiments, at least four different immunogenic peptides
may be linked together in tandem to form a fusion polypeptide for inclusion in an
immunogenic composition. In certain embodiments, the immunogenic composition
comprising at least 31 different immunogenic peptides (for example, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or more different immunogenic peptides) may
include at least one, two, three, four, five, six, seven, or more fusion polypeptides,
wherein each fusion polypeptide comprises at least four different immunogenic peptides
linked together in tandem. In more particular embodiments, one or more fusion
polypeptides included in an immunogenic composition may comprise 4, 5, 6, 7, 8, 9,
, 11, 12, 13, 14, or 15, or more individual immunogenic peptides linked in tandem.
In more specific embodiments, each fusion polypeptide comprising different
immunogenic peptides linked in tandem and that is used in the immunogenic
compositions described herein comprises at least 6, 7, 8, 9, or 10 different immunogenic
peptides. In even more specific embodiments described herein, an immunogenic
composition comprises at least 31 different immunogenic peptides, each from the amino
terminal portion of a GAS M protein or Spa protein, and at least six, seven, eight or
nine different immunogenic peptides are linked together in a manner that provides four
fusion polypeptides. In still another specific embodiment, an immunogenic
composition comprises at least 31 different immunogenic peptides, each from the amino
terminal portion of a GAS M protein or Spa protein, and seven or eight different
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immunogenic peptides are linked together in tandem per fusion polypeptide in a manner
that provides four fusion polypeptides. Stated another way, each of the at least 31
different immunogenic peptides is included in one of these four fusion polypeptides.
In more specific embodiments, an immunogenic composition comprises
at least 31 different immunogenic peptides (e.g., immunogenic peptides from the amino
terminal portions of the M proteins from GAS M serotypes 1; 2; 3; 4; 5; 6; 11; 12; 14;
18; 19; 22; 24; 28; 29; 44; 49; 58; 73; 75; 77; 78; 81; 82; 83; 87; 89; 92; 114; 118, and
Spa protein from GAS serotype 18), wherein at least six, seven, eight, or nine different
immunogenic peptides are linked together in tandem to form at least four fusion
polypeptides. In one particular embodiment, each of the four fusion polypeptides
comprises at least 7 or at least 8 different immunogenic peptides. In one embodiment,
the immunogenic composition comprises four fusion polypeptides (which for
convenience may be called a first fusion polypeptide, a second fusion polypeptide, a
third fusion polypeptide, and a fourth fusion polypeptide) and each fusion polypeptide
comprises 7 or 8 different immunogenic peptides independently selected from the at
least 31 different immunogenic peptides. Which immunogenic peptides are included on
each of the first, second, third, and fourth fusion polypeptides and the order with which
the immunogenic peptides are linked may be readily determined by a person skilled in
the art using methods and techniques described herein and routinely practiced in the art
and does not require undue empirical, trial and error analysis to ensure optimization of
the immunogenicity of each polypeptide. To determine that the amino acids located at
the junction where two different immunogenic peptides are tandemly linked does not
introduce a contiguous 5-amino acid sequence that shares identity with a human
protein, the amino acid sequence of each fusion polypeptide is compared with the
sequences of human proteins available in databases described herein and known to the
person skilled in the art.
Fusion polypeptides comprising three or more different immunogenic
peptides from the amino terminal portions of the M proteins and Spa proteins from
GAS serotypes are constructed so that the immunogenic peptide located at the amino
terminal end is repeated (i.e., duplicated) at the carboxy terminal end of the fusion
polypeptide. In the absence of a duplicate immunogenic peptide at the carboxy terminal
end of the fusion polypeptide, the immunogenicity of the penultimate and carboxy
terminal peptide may be reduced or compromised. Without wishing to be bound by
theory, repetition of the amino terminal peptide at the carboxy terminal end preserves
the immunogenicity of all peptides included in the fusion protein and reduces or
abrogates the need for inclusion of a non-GAS carrier protein, such as tetanus toxoid,
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keyhole limpet hemocyanin, or other protein or protein fragment, used in the art to
enhance an immune response to an antigen of interest. See U.S. Patent Nos. 6,716,433
and 7,402,316.
As described herein, the amino acid sequences of M proteins and Spa
proteins are readily available in the art. Databases, such as provided at the CDC emm
typing center website, may be accessed to determine the variability in the amino acid
sequences within an M protein from a GAS serotype (referred to herein and in the art as
GAS subtypes) or within a Spa protein from a GAS serotype. Alignment tools for
comparison of the amino terminal portions of M proteins (or Spa proteins) can be used
to identify amino acid residues within the amino terminal portion that may be variable.
The amino acid sequences of the immunogenic peptides and fusion polypeptides
described herein are exemplary, and immunogenic peptides and fusion polypeptides
useful for inducing an immune response against GAS may include substitutions,
insertions, and deletions of one or more amino acids that do not adversely affect
(decrease or reduce in a statistically significant manner or biologically or clinical
significant manner) the immunogenicity of the immunogenic peptide or fusion
polypeptide. Immunogenicity of a variant peptide or variant fusion polypeptide that is
substantially similar in a statistically, biologically, or clinically significant manner to
the immunogenicity of an immunogenic peptide or fusion polypeptide, respectively,
described herein is desired. Exemplary amino acid sequences for immunogenic
peptides from the amino terminal portions of the M proteins from GAS M serotypes 1;
2; 3; 4; 5; 6; 11; 12; 14; 18; 19; 22; 24; 28; 29; 44; 49; 58; 73; 75; 77; 78; 81; 82; 83;
87; 89; 92; 114; 118, and Spa protein from GAS serotype 18 are provided in Table 1.
Also provided herein in more specific embodiments are immunogenic
compositions that may be used in particular geographic regions. By way of example,
provided herein in certain embodiments are immunogenic compositions that comprise
at least 23 different immunogenic peptides (e.g., immunogenic peptides from the amino
terminal portions of the M proteins from GAS M serotypes 1; 2; 3; 4; 5; 6; 11; 12; 14;
18; 19; 22; 24; 28; 29; 44; 58; 73; 75; 77; 78; 89; 118, and Spa protein from GAS
serotype 18), wherein at least six, seven, eight, or nine different immunogenic peptides
are linked together in tandem to form at least three or at least four fusion polypeptides.
In one particular embodiment, an immunogenic composition comprises each of three
fusion polypeptides that each comprises at least 7 or at least 8 different immunogenic
peptides. In one embodiment, the immunogenic composition comprises three fusion
polypeptides (which for convenience may be called a first fusion polypeptide, a second
fusion polypeptide, and a third fusion polypeptide) and each fusion polypeptide
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comprises 7 or 8 different immunogenic peptides. This exemplary immunogenic
composition comprises GAS serotypes that are more prevalent in GAS infections in
North America than in other global regions, such as Europe. GAS M serotypes, such as
49, 81, 82, 83, 87, 92 and 114, are identified in Europe more frequently than in North
America as the cause of a GAS infection.
In certain embodiments, an immunogenic peptide or fusion polypeptide
described herein includes immunogenic peptide or fusion polypeptide species,
respectively, that have one or more amino acid substitutions, insertions, or deletions
(also called herein a variants). Immunogenic peptide species or a fusion polypeptide
species comprising the immunogenic peptides described herein includes immunogenic
peptide species and fusion polypeptide species, respectively, that have one or more
amino acid substitutions, insertions, or deletions (also called herein a variant).
Immunogenic peptide variants include those that comprise an amino acid sequence
different from an exemplary amino acid sequence described herein of the immunogenic
peptide and that comprise the amino acid sequence (at least 25 contiguous amino acids
of the amino terminal portion of a Spa protein or M protein) of a different subtype of
the same respective GAS serotype (see, e.g., Dale et al., Clin. Diagn. Lab. Immunol.
12:833-36 (2005); Facklam et al., Emerg. Infect. Dis. 5:247-53 (1999)). Fusion
polypeptide variants include species comprising at least one immunogenic peptide
variant. Amino acid sequences of M protein subtypes are available in public databases,
including the CDC emm typing center website (supra).
Immunogenic peptide variants and fusion polypeptide variants also
include those that have amino acid substitutions, deletions or insertions, which may be
introduced during chemical synthesis or recombinant production, whichever method is
used to produce the particular immunogenic peptide or fusion polypeptide.
Immunogenic peptide variants and fusion polypeptide variants useful for inducing an
immune response against GAS may also include substitutions, insertions, and deletions
of one or more amino acids of the amino acid sequences described herein and that do
not adversely affect or alter (decrease or reduce) the immunogenicity of the
immunogenic peptide or fusion polypeptide in a statistically, biologically, or clinically
significant manner. Stated another way, a variant of an immunogenic peptide or of a
fusion polypeptide described herein retains immunogenicity exhibited by the
immunogenic peptide or fusion polypeptide, respectively. As described herein,
retention of immunogenicity includes the capability to evoke an immune response
against GAS, such as production of antibodies that specifically bind to the cognate
immunogenic peptide and/or fusion polypeptide, cognate full-length or mature M
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protein or Spa protein (either isolated or present on the cell surface of GAS bacteria),
and which antibodies include those with bactericidal and phagocytic activity.
In general, an amino acid substitution that may be included in an
immunogenic peptide or a fusion polypeptide is a conservative substitution.
Conservative substitutions of amino acids are well known and may occur naturally in an
M protein or Spa polypeptide or may be introduced when the peptide or fusion
polypeptide is recombinantly produced. A variety of criteria understood by a person
skilled in the art indicate whether an amino acid that is substituted at a particular
position in an immunogenic peptide or fusion polypeptide is conservative (or similar).
For example, a similar amino acid or a conservative amino acid substitution is one in
which an amino acid residue is replaced with an amino acid residue having a similar
side chain. Similar amino acids may be included in the following categories: amino
acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic
side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,
histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-
branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic
side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered
more difficult to classify, shares properties with amino acids that have aliphatic side
chains (e.g., leucine, valine, isoleucine, and alanine). In certain circumstances,
substitution of glutamine for glutamic acid or asparagine for aspartic acid may be
considered a similar substitution in that glutamine and asparagine are amide derivatives
of glutamic acid and aspartic acid, respectively. As understood in the art “similarity”
between two polypeptides is determined by comparing the amino acid sequence and
conserved amino acid substitutes thereto of the peptide or polypeptide to the sequence
of a second peptide or polypeptide, respectively, using any one of the algorithms, such
as Align or the BLAST algorithm, or other algorithms described herein and practiced in
the art.
Amino acid substitutions, deletions, and additions may be introduced
into an immunogenic peptide or fusion polypeptide during chemical synthesis of the
polynucleotide that encodes the peptide or fusion polypeptide. Alternatively, amino
acid substitutions, deletions, and additions may be introduced into an immunogenic
peptide or fusion polypeptide recombinantly using well-known and routinely practiced
mutagenesis methods (see, e.g., Sambrook et al. Molecular Cloning: A Laboratory
Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY 2001)). Oligonucleotide-
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directed site-specific (or segment specific) mutagenesis procedures may be employed to
provide an altered polynucleotide that has particular codons altered according to the
substitution, deletion, or insertion desired. Alternatively, random mutagenesis
techniques, such as alanine scanning mutagenesis, error prone polymerase chain
reaction mutagenesis, and oligonucleotide-directed mutagenesis may be used to prepare
immunogenic peptide and fusion polypeptide variants (see, e.g., Sambrook et al.,
supra). An immunogenic peptide variant or a fusion polypeptide variant includes an
immunogenic peptide or fusion polypeptide, respectively, that has at least 85%, 90%,
92%, 95%, 97%, 98%, or 99% amino acid sequence identity to any of the exemplary
immunogenic peptide and fusion polypeptide amino acid sequences provided herein,
including in the sequence listing (for example, fusion polypeptides represented by SEQ
ID NOS:1-12, and immunogenic peptides represented by SEQ ID NOS:29-59). Percent
identity of one amino acid sequence to one or more additional sequences may be
determined using any one of the alignment tools described herein and used in the art.
Given the description in the art and herein regarding regions of M
proteins and Spa proteins that exhibit immunogenic activity and lack undesirable
epitopes that induce antibodies that cross-react with proteins expressed on human cells,
persons skilled in the art can readily determine which amino acids in the amino terminal
portions of M proteins and Spa proteins may be more amenable to alteration (i.e.,
substitution, deletion, or addition of one or more amino acids) and which amino acids
may not be amenable to change. Also given the description herein and given the many
molecular biology, protein expression, and protein isolation techniques and methods
routinely practiced in the art for introducing mutations in a peptide or polypeptide,
isolating these peptides and polypeptides and variants thereof, and analyzing same, the
desired immunogenicity can be made readily and without undue experimentation.
Retention of immunogenicity includes the capability to evoke an immune response
against GAS, such as production of antibodies that specifically bind to the cognate
immunogenic peptide and/or fusion polypeptide, cognate mature M protein or Spa
protein (either isolated or present on the cell surface of GAS bacteria), or cognate full-
length M protein or Spa protein, and which antibodies include those with bactericidal
and phagocytic activity.
Assays for assessing whether a variant of an immunogenic peptide of an
M protein or of a Spa protein, or a fusion polypeptide comprising the immunogenic
peptides, folds into a conformation comparable to the non-variant peptide or fusion
polypeptide include, for example, the ability of the peptide or fusion polypeptide to
react with mono- or polyclonal antibodies that are specific for native or unfolded
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epitopes, the retention of ligand-binding functions, and the sensitivity or resistance of
the mutant peptide or fusion polypeptide to digestion with proteases (see Sambrook et
al., supra). Immunogenic peptide variants and fusion polypeptide variants as described
herein can be identified, characterized, and/or made according to these methods
described herein or other methods known in the art, which are routinely practiced by
persons skilled in the art.
Variants of individual immunogenic peptides and of fusion polypeptides
comprising immunogenic peptides, can be prepared without altering a biological
activity of the resulting molecule (i.e., without altering one or more immunogenic
activities in a statistically significant, clinically significant, or biologically significant
manner). As described in greater detail herein, for example, such substitutions are
generally made by interchanging an amino acid with another amino acid that is included
within the same group, such as the group of polar residues, charged residues,
hydrophobic residues, and/or small residues, and the like. The effect of any amino acid
substitution may be determined empirically merely by testing the resulting modified
peptide or fusion polypeptide for the ability to function in a biological assay, or to bind
to a cognate ligand or target molecule, such as a monoclonal or polyclonal antibody.
Exemplary Anti-GAS Immunogenic Composition Comprising Four Fusion
Polypeptides
In one particular embodiment, an immunogenic composition useful for
inducing an immune response against GAS comprises 31 different immunogenic
peptides, and each immunogenic peptide is incorporated into one of four fusion
polypeptides. Each immunogenic peptide is from the amino terminal portion of the M
protein from one of GAS M serotypes 1; 2; 3; 4; 5; 6; 11; 12; 14; 18; 19; 22; 24; 28; 29;
44; 49; 58; 73; 75; 77; 78; 81; 82; 83; 87; 89; 92; 114; 118, or from the amino terminal
portion of Spa protein from GAS serotype 18. Three fusion polypeptides (for
convenience, called a first, second, and third fusion polypeptide, respectively) each
comprise at least 8 different immunogenic peptides linked in tandem, and the fourth
fusion polypeptide comprises at least 7 different immunogenic peptides linked in
tandem. In a particular embodiment, the first fusion polypeptide comprises 8
immunogenic peptides, which are different, and each immunogenic peptide comprises
at least 25 contiguous amino acids from the amino terminal portion of the M protein
from one of GAS M serotypes 1, 2, 3, 6, 12, 18, 28, and from the amino terminal
portion of the Spa protein from GAS serotype 18. Stated another way, a first fusion
polypeptide comprises at least 8 different immunogenic peptides linked in tandem, and
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each immunogenic peptide comprises at least 25 contiguous amino acids from the
amino terminal portion of one of M1 protein, M2 protein, M3 protein, M6 protein, M12
protein, M18 protein, M28 protein, and Spa 18 protein (i.e., the fusion polypeptide
therefore comprises an immunogenic peptide from the amino terminal portion of M1
protein, an immunogenic peptide from the amino terminal portion of M2 protein, an
immunogenic peptide from the amino terminal portion of M3 protein etc.). One of the
immunogenic peptides is repeated at each of the amino and carboxy terminal ends of
the fusion polypeptide. In a more particular embodiment, the first fusion polypeptide
comprises duplicates (repeats) of the immunogenic peptide comprising at least 25
contiguous amino acids from the amino terminal portion of the M protein from GAS
serotype 1; one duplicate is located at the amino terminal end of the fusion polypeptide
and the second duplicate is located at the carboxy terminal end of the fusion
polypeptide.
The second fusion polypeptide comprises 8 immunogenic peptides,
which are different, and each immunogenic peptide comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein from one of GAS M
serotypes 4, 5, 11, 14, 19, 24, 29, and 75. Stated another way and similar to the
description of the first fusion polypeptide, the second fusion polypeptide comprises at
least 8 different immunogenic peptides linked in tandem, and each immunogenic
peptide comprises at least 25 contiguous amino acids from the amino terminal portion
of one of M4 protein, M5 protein, M11 protein, M14 protein, M19 protein, M24
protein, M29 protein, and M75 protein. One of the immunogenic peptides is repeated at
each of the amino and carboxy terminal ends of the second fusion polypeptide. In a
more particular embodiment, the second fusion polypeptide comprises duplicates
(repeats) of the immunogenic peptide comprising at least 25 contiguous amino acids
from the amino terminal portion of the M protein from GAS serotype 4; one duplicate is
located at the amino terminal end of the fusion polypeptide and the second duplicate is
located at the carboxy terminal end of the fusion polypeptide.
In certain embodiments, the third fusion polypeptide comprises 8
immunogenic peptides, which are different, and each immunogenic peptide comprises
at least 25 contiguous amino acids from the amino terminal portion of the M protein
from one of GAS M serotypes 22, 44, 58, 73, 77, 78, 89, and 118. Stated another way
and as above, a third fusion polypeptide comprises at least 8 different immunogenic
peptides linked in tandem, and each immunogenic peptide comprises at least 25
contiguous amino acids from the amino terminal portion of one of M22 protein, M44
protein, M58 protein, M73 protein, M77 protein, M78 protein, M89 protein, and M118
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protein. One of the immunogenic peptides is repeated at each of the amino and carboxy
terminal ends of the third fusion polypeptide. In a more particular embodiment, the
third fusion polypeptide comprises duplicates (repeats) of the immunogenic peptide
comprising at least 25 contiguous amino acids from the amino terminal portion of the M
protein from GAS serotype 77; one duplicate is located at the amino terminal end of the
fusion polypeptide and the second duplicate is located at the carboxy terminal end of
the fusion polypeptide.
The fourth fusion polypeptide comprises 7 immunogenic peptides, which
are each different, and each immunogenic peptide comprises at least 25 contiguous
amino acids from the amino terminal portion of the M protein from one of GAS M
serotypes 49, 81, 82, 83, 87, 92, and 114. Stated another way and as above, a fourth
fusion polypeptide comprises at least 8 different immunogenic peptides linked in
tandem, and each immunogenic peptide comprises at least 25 contiguous amino acids
from the amino terminal portion of one of M49 protein, M81 protein, M82 protein, M83
protein, M87 protein, M92 protein, and M114 protein. One of the immunogenic
peptides is repeated at each of the amino and carboxy terminal ends of the fourth fusion
polypeptide. In a more particular embodiment, the fourth fusion polypeptide comprises
duplicates (repeats) of the immunogenic peptide comprising at least 25 contiguous
amino acids from the amino terminal portion of the M protein from GAS serotype 83;
one duplicate is located at the amino terminal end of the fusion polypeptide and the
second duplicate is located at the carboxy terminal end of the fusion polypeptide.
Each immunogenic peptide in each of the first, second, third, and fourth
fusion polypeptides may comprise independently at least 25, 30, 35, 40, 45, 50, 55, or
60 or more contiguous amino acids from the amino terminal portion of the respective M
protein or Spa protein (or any number of amino acids between 25-30, 30-35, 35-40, 40-
45, 45-50, 40-55, or 55-60, or more than 60 contiguous amino acids from the amino
terminal portion of the respective M protein or Spa protein). In another more specific
embodiment, one or more of the immunogenic peptides comprises at least 25
contiguous amino acids in duplicate and each duplicate is in tandem (i.e., not separated
by one or more immunogenic peptides from a different M or Spa protein of the fusion
protein). In another specific embodiment one or more of the immunogenic peptides
comprises at least 40 contiguous amino acids or at least 45 contiguous amino acids from
the amino terminal portion of the respective M protein or Spa protein. In still another
specific embodiment, one or more of the immunogenic peptides comprises at least 50
contiguous amino acids from the amino terminal portion of the respective M protein or
Spa protein.
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In certain specific embodiments, as discussed herein, the immunogenic
peptides may be derived from subtypes of the respective M protein. For example, in
certain specific embodiments, the immunogenic peptide from the M protein of GAS
serotype 3 is from GAS serotype 3.1. In other specific embodiments, the immunogenic
peptide from the M protein of GAS serotype 6 is from GAS serotype 6.4. In still
another specific embodiment, the immunogenic peptide from the M protein of GAS
serotype 5 is from GAS serotype 5.0 (parent subtype). In a certain particular
embodiment, the immunogenic peptide from the M protein of GAS serotype 14 is from
GAS serotype 14.3. In yet another specific embodiment, the immunogenic peptide
from the M protein of GAS serotype 29 is from GAS serotype 29.2. In still yet another
specific embodiment, the immunogenic peptide from the M protein of GAS serotype 49
is from GAS serotype 49.1. In still another specific embodiment, the immunogenic
peptide from the M protein of GAS serotype 83 is from GAS serotype 83.1. If no
subtype is designated, the M protein is derived from the subtype considered in the art as
the “parent” subtype, such as, by way of illustration, M12.0, M18.0, M28.0, and the
like.
In a more specific embodiment, the first fusion polypeptide comprising
eight immunogenic peptides, which immunogenic peptides are independently from the
amino terminal portion of the M protein from one of GAS M serotypes 1, 2, 3, 6, 12,
18, 28, and from the amino terminal portion of the Spa protein from GAS serotype 18
as described above, are linked in tandem in the following order from amino terminal to
carboxy terminal: M1, M3.1, M6.4, M2, M18, M28, M12, Spa, and M1. Each of the
M1, M18, M28, M12, and Spa immunogenic peptides comprises amino acid residues 1-
50 from each of the respective mature M proteins or Spa protein. The immunogenic
peptide representing the GAS serotype 3.1 comprises amino acid residues at positions
22-71 of the mature M3.1 protein. Each of the immunogenic peptides of the M3.1
protein and the M6.4 protein include amino acid residues 1-25 of the respective mature
M protein linked directly in tandem to a duplicate (i.e., repeat) of residues 1-25. The
immunogenic peptide of the M2 protein include amino acid residues 2-26 of the
respective mature M protein linked directly in tandem to a duplicate (i.e., repeat) of
residues 2-26. The second fusion polypeptide comprising eight immunogenic peptides,
which immunogenic peptides are independently from the amino terminal portion of the
M protein from one of GAS M serotypes 4, 5, 11, 14, 19, 24, 29, and 75, are linked in
tandem in the following order from amino terminal to carboxy terminal: M4, M5.0,
M11, M75, M19, M29.2, M14.3, M24, and M4. Each of the M4, M11, M75, M29.2,
M14.3, and M24 immunogenic peptides comprises amino acid residues 1-50 from the
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amino terminal portion of the respective mature M protein. Each of the immunogenic
peptides of the M5.0 protein and the M19 protein includes amino acid residues 1-25 of
the respective mature M protein linked directly in tandem to a duplicate (i.e., repeat) of
residues 1-25. The third fusion polypeptide comprising eight immunogenic peptides,
which immunogenic peptides are independently from the amino terminal portion of the
M protein from one of GAS M serotypes 22, 44, 58, 73, 77, 78, 89, and 118, are linked
in tandem in the following order from amino terminal to carboxy terminal: M77, M22,
M73, M89, M58, M44, M78, M118, and M77. Each of these M protein immunogenic
peptides comprises amino acid residues 1-50 at the amino terminal portion of the
respective mature M proteins. The fourth fusion polypeptide comprising seven
immunogenic peptides, which immunogenic peptides are independently from the amino
terminal portion of the M protein from one of GAS M serotypes 49, 81, 82, 83, 87, 92,
and 114, are linked in tandem in the following order from amino terminal to carboxy
terminal: M83.1, M82, M81, M87, M49.1, M92, M114, and M83.1. Each of these M
protein immunogenic peptides comprises amino acid residues 1-50 at the amino
terminal portion of the respective mature M proteins. See also Figure 1.
Exemplary amino acid sequences for each of the specific embodiments
of a first, second, third, and fourth fusion polypeptides are provided in SEQ ID NOS: 1,
2, 3, and 4, respectively. Any of the immunogenic peptides, dimeric peptides, and
fusion polypeptides, including the exemplary fusion polypeptides described herein may
further comprise a heterologous peptide or polypeptide at either the amino or carboxy
terminus or both to facilitate expression, solubilization, stabilization, isolation, and/or
detection. For example, when immunogenic peptides, dimeric peptides, or fusion
polypeptides are produced according to recombinant methods, an initiating methionine
residue at the amino terminus is typically included (see, for example, SEQ ID NOS: 5,
6, 7, and 8 corresponding to the first, second, third, and fourth fusion polypeptides,
respectively). Examples of amino acid sequences that may be added at either the amino
or carboxy terminal ends (also called polypeptide tags) to facilitate isolation,
solubilization, stabilization, and/or detection of the peptide or fusion polypeptide
include polyhistidine tags (e.g., 6-His tag), glutathione-S-transferase (GST), FLAG
epitope tag (DYKDDDDK, SEQ ID NO:60), beta-galactosidase, alkaline phosphatase,
chitin binding protein (CBP), XPRESS epitope tag (DLYDDDDK, SEQ ID NO:61;
Invitrogen Life Technologies, Carlsbad, CA) maltose binding protein (MBP),
thioredoxin (a solubilization tag), and poly(NANP) (a solubilization tag) (see, e.g., U.S.
Patent No. 5,011,912; Hopp et al., (Bio/Technology 6:1204 (1988)). If the peptide,
dimeric peptide, or fusion polypeptide is recombinantly produced, the affinity sequence
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may be supplied by a vector, such as, for example, a hexa-histidine tag that is provided
in pBAD/His (Invitrogen). Alternatively, the affinity sequence may be added either
synthetically or engineered into the primers used to recombinantly generate the nucleic
acid coding sequence (e.g., using the polymerase chain reaction). Additional methods
and techniques for using polypeptide tags are routinely practiced in the art and available
from commercial vendors who manufacture systems and kits for adding tags to a
peptide or polypeptide of interest. By way of illustration, the first, second, third, and
fourth fusion polypeptides may incorporate a polyhistidine tag to facilitate purification
(see, e.g., SEQ ID NOS:9-12, respectively).
A fusion polypeptide variant includes a fusion polypeptide, that has at
least 85%, 90%, 92%, 95%, 97%, 98%, or 99% amino acid sequence identity to any of
the exemplary fusion polypeptide amino acid sequences provided herein, including in
the sequence listing (e.g., SEQ ID NOS:1-12). Fusion polypeptides useful for inducing
an immune response against GAS may include substitutions, insertions, and deletions of
one or more amino acids of any of the aforementioned amino acid sequences and that
do not adversely affect or alter (decrease or reduce) the immunogenicity of the fusion
polypeptide in a statistically, biologically, or clinically significant manner. Stated
another way, a variant of a fusion polypeptide described herein retains immunogenicity
exhibited by the parent or non-variant fusion polypeptide. As described herein,
retention of immunogenicity includes the capability to evoke an immune response
against GAS, such as production of antibodies that specifically bind to the cognate
immunogenic peptide and/or fusion polypeptide, cognate mature or full-length M
protein or Spa protein (either isolated or present on the cell surface of GAS bacteria),
and which antibodies include those with bactericidal and phagocytic activity.
As described in the Examples, immunization of animals with an
immunogenic composition comprising each of the above described exemplary first,
second, third, and fourth fusion polypeptides evoked production of bactericidal
antibodies that specifically bound to each GAS serotype represented by an M protein
immunogenic peptide and Spa immunogenic peptide (i.e., GAS serotypes 1; 2; 3; 4; 5;
6; 11; 12; 14; 18; 19; 22; 24; 28; 29; 44; 49; 58; 73; 75; 77; 78; 81; 82; 83; 87; 89; 92;
114; 118). In addition, antisera from immunized animals was bactericidal against more
than half of GAS serotypes for which an immunogenic peptide from an M protein or
Spa protein was not represented in any one of the fusion polypeptides. This improved
efficacy is greater than a person skilled in the art may have predicted on the basis of
previously described results with other multivalent compositions. When compared with
a previously described 26-valent vaccine (see, e.g., U.S. Patent No. 7,270,827; Hu et al.,
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supra), the particular combination of immunogenic peptides described herein
recognized significantly more non-vaccine GAS serotypes (see Example 4). Serotypes
represented in the 30-valent immunogenic composition described herein that were not
included in the 26-valent composition include GAS M serotypes 4, 29, 58, 44, 49, 73,
78, 81, 82, 83, 87, 118. Serotypes in the 26-valent composition not represented by
immunogenic peptides in the composition described herein include GAS M serotypes
1.2, 13, 33, 43, 59, 76, and 101.
Two different immunogenic peptides may be directly linked in tandem to
form dimeric peptides (also called dipeptides), which in certain embodiments, are
useful in methods for detecting antibodies are specific for an immunogenic peptide.
Dimeric peptides may be recombinantly produced as described herein and in the art
according to methods and techniques routinely used and practiced in the art (see, e.g.,
Hu et al., supra). Alternatively, dimeric peptides may be chemically synthesized
according to methods described herein and in the art. Dimeric peptides may be
comprised of any two different immunogenic peptides described herein. By way of
example, a dimeric peptide (which includes an immunogenic dimeric peptide), is
composed of two immunogenic peptides and each peptide of the dimeric peptide is
different and comprises at least 25 contiguous amino acids of the amino terminal
portion of a different M protein or a Spa protein, wherein each different M protein is
independently selected from the M protein of GAS serotype 1, 2, 3, 4, 5, 6, 11, 12, 14,
18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118,
and the Spa protein is from GAS serotype 18.
Production of Immunogenic Peptides and Fusion Polypeptides
Provided herein are immunogenic peptides comprising at least 25
contiguous amino acids from the amino terminal portion of an M protein or Spa protein
from different serotypes of group A streptococcus and fusion polypeptides comprising
these immunogenic peptides. Each of the immunogenic peptides, immunogenic dimeric
peptides (i.e., polypeptides comprising two different immunogenic peptides), and fusion
polypeptides may be produced recombinantly or may be chemically synthesized.
Alternatively, polynucleotides encoding individual immunogenic peptides,
immunogenic dimeric peptides, and fusion polypeptides may be constructed by
recombinant methods or chemically synthesized, and the constructed or synthesized
polynucleotides may be incorporated into expression vectors for production of the
peptide, dimeric peptide, or fusion polypeptide in a host cell into which the expression
vector has been introduced.
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Peptides and polypeptides may be chemically synthesized by manual
techniques or by automated procedures. Equipment for automated synthesis of
polypeptides is commercially available from suppliers such as Perkin-Elmer, Inc. and
Applied BioSystems Division (Foster City, CA), and may be operated according to the
manufacturer's instructions. By way of example, solid phase polypeptide synthesis has
been performed since the early 1960’s (see, e.g., Merrifield, J. Am. Chem. Soc.
85:2149-2154 (1963)). Numerous improvements to synthesis methods have been
developed, and many methods have been automated and chemistries have been
developed to protect the terminal ends and other reactive groups (see, e.g., Geysen et
al., J. Immun. Meth. 102:259-274 (1987); Miranda et al., Proc. Natl. Acad. Sci. USA
96:1181-86 (1999); Frank et al., Tetrahedron 44:6031-6040 (1988); Hyrup et al.,
Bioorg. Med. Chem. 4:5-23 (1996); Perry-O'Keefe et al., Proc. Natl. Acad. Sci. USA
93:14670-675 (1996); Schnölzer, et al. Int. J. Pept. Protein Res. 40, 180-193 (1992);
Hackeng et al., Proc. Natl. Acad. Sci. USA 94:7845-50 (1997); Creighton, T. E. Protein:
Structures and Molecular Properties, pp. 55-60, W. H. Freeman and Co., New York,
NY (1984)). Equipment for performing automated synthesis is available from a variety
of manufacturers. Synthesized peptides, dimeric peptides, and fusion polypeptides may
be obtained from any number of different custom peptide synthesizing manufacturers.
If required, synthesized peptides or polypeptides may be purified using preparative
reverse phase chromatography, partition chromatography, gel filtration, gel
electrophoresis, or ion-exchange chromatography or other methods used in the art.
Polynucleotides may also be chemically synthesized or may be
constructed by recombinant methods familiar to a person skilled in the art.
Polynucleotides can also be synthesized using an automatic synthesizer. The nucleotide
sequence can be designed with the appropriate codons for the particular amino acid
sequence desired. In general, preferred codons may be selected for the intended host in
which the nucleotide sequence will be expressed. One recombinant method of preparing
a polynucleotide includes assembly from overlapping oligonucleotides prepared by
standard methods to provide a complete coding sequence (see, e.g., Au et al., Biochem.
Biophys. Res. Commun. 248:200-203 (1998); Stemmer et al., Gene 164:49-53 (1995);
Ausubel et al. (eds.), Current Protocols in Molecular Biology, (Greene Publ. Assoc.
Inc. & John Wiley & Sons, Inc., 1993)); Sambrook et al., et al. Molecular Cloning: A
Laboratory Manual, 3rd Ed., (Cold Spring Harbor Laboratory 2001; and elsewhere).
Methods for purifying polynucleotides after either chemical synthesis or recombinant
synthesis are known to persons skilled in the art (see, e.g., Ausubel et al., supra;
Sambrook et al., supra).
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Chemical synthesis of oligonucleotides for primers and probes has long
been practiced in the art (see, e.g., Letsinger et al., J. Am. Chem. Soc. 98:3655-61
(1976); Matteucci et al., J. Am. Chem. Soc. 103:3185-9 (1981)). Improved methods for
synthesizing oligonucleotides and polynucleotides, which provide more rapid results,
greater yields, and longer polynucleotides, have since been developed and automated
(see, e.g., Gao et al., Biopolymers 73:579-96 (2004); Mueller et al., Chem. Biol. 16:337-
47 (2009); Lee et al., Nucleic Acids Res. 38:2514-21 (2010)). Polynucleotides that
encode the immunogenic peptides, dimeric peptides, and fusion polypeptides described
herein may be synthesized commercially (see, e.g., GENSCRIPT, Piscataway, NJ).
Polynucleotides that encode an immunogenic peptide, dimeric peptide,
or fusion polypeptide described herein may be recombinantly expressed in a variety of
different host cells. Host cells containing recombinant expression constructs may be
genetically engineered (transduced, transformed, or transfected) with the vectors and/or
expression constructs (for example, a cloning vector, a shuttle vector, or an expression
construct). The vector or construct may be in the form of a plasmid, a viral particle, a
phage, etc. The engineered host cells can be cultured in conventional nutrient media
modified as appropriate for activating promoters, selecting transformants, or amplifying
particular genes or encoding-nucleotide sequences. Selection and maintenance of
culture conditions for particular host cells, such as temperature, pH and the like, will be
readily apparent to the ordinarily skilled artisan. In general, the desired host cell is one
that can be adapted to sustained propagation in culture to yield a stable cell line that can
express sufficient amount of the peptide, dimeric peptide, or fusion polypeptide. In
certain embodiments, the cell line is an immortal cell line, which refers to a cell line
that can be repeatedly passaged (at least ten times while remaining viable) in culture
following log-phase growth. In other embodiments the host cell used to generate a cell
line is a cell that is capable of unregulated growth, such as a cancer cell, or a
transformed cell, or a malignant cell.
Useful bacterial expression constructs are prepared by inserting into an
expression vector a structural DNA sequence encoding the desired peptide or
polypeptide together with suitable translation initiation and termination signals in an
operative reading phase with a functional promoter. The construct may comprise one or
more phenotypic selectable markers and an origin of replication to ensure maintenance
of the vector construct and, if desirable, to provide amplification within the host.
Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis,
Salmonella typhimurium and various species within the genera Pseudomonas,
Streptomyces, and Staphylococcus, although others may also be employed as a matter
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of choice. Any other plasmid or vector may be used as long as the plasmid or vector is
replicable and viable in the host. Thus, for example, the polynucleotides as provided
herein may be included in any one of a variety of expression vector constructs as a
recombinant expression construct for expressing the peptide, dimeric peptide, or fusion
polypeptide. Such vectors and constructs include chromosomal, nonchromosomal, and
synthetic DNA sequences, e.g., bacterial plasmids; phage DNA; baculovirus; yeast
plasmids; vectors derived from combinations of plasmids and phage DNA; viral DNA,
such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
The appropriate DNA sequence(s) may be inserted into the vector by a
variety of procedures with which the skilled person is familiar. In certain instances, the
DNA sequence is inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Notably, the fusion polypeptides described herein lack
any restriction enzyme sites between any one of the immunogenic peptides that
comprise the fusion polypeptide. The omission of restriction sites and the amino acid
sequence encoded by the restriction site is intended to remove the possibility that a
desired immunogenic epitope will be adversely altered or that an epitope will be
inadvertently added that may have an undesirable immunogenicity (for example,
inducing production of an antibody that recognizes a normal, human protein). Standard
techniques for cloning, DNA isolation, amplification and purification, for enzymatic
reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the
like, and various separation techniques are those known and commonly employed by
those skilled in the art. Numerous standard techniques are described, for example, in
Ausubel et al. (Current Protocols in Molecular Biology (Greene Publ. Assoc. Inc. &
John Wiley & Sons, Inc., 1993)) and in Sambrook et al. (Molecular Cloning: A
Laboratory Manual, 3rd Ed., (Cold Spring Harbor Laboratory 2001)).
The DNA sequence encoding a peptide or polypeptide in the expression
vector is operatively linked to at least one appropriate expression control sequences
(e.g., a promoter or a regulated promoter) to direct mRNA synthesis. Representative
examples of such expression control sequences include LTR or SV40 promoter, E. coli
lac or trp, the phage lambda P promoter, and other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their viruses. Promoter regions
can be selected from any desired gene using CAT (chloramphenicol transferase) vectors
or other vectors with selectable markers. Particular bacterial promoters include lacI,
lacZ, T3, T5, T7, gpt, lambda P , P , and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine kinase, early and late SV40, LTRs from retroviruses,
and mouse metallothionein-I. Selection of the appropriate vector and promoter and
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preparation of certain recombinant expression constructs comprising at least one
promoter or regulated promoter operatively linked to a polynucleotide described herein
is well within the level of ordinary skill in the art.
Design and selection of inducible, regulated promoters and/or tightly
regulated promoters are known in the art and will depend on the particular host cell and
expression system. The pBAD Expression System (Invitrogen Life Technologies,
Carlsbad, CA) is an example of a tightly regulated expression system that uses the E.
coli arabinose operon (P or P ) (see Guzman et al., J. Bacteriology 177:4121-30
BAD ARA
(1995); Smith et al., J. Biol. Chem. 253:6931-33 (1978); Hirsh et al., Cell 11:545-50
(1977)), which controls the arabinose metabolic pathway. A variety of vectors
employing this system are commercially available. Other examples of tightly regulated
promoter-driven expression systems include PET Expression Systems (see U.S. Patent
No. 4.952,496) available from Stratagene (La Jolla, CA) or tet-regulated expression
systems (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-51 (1992); Gossen et al.,
Science 268:1766-69 (1995)). The pLP-TRE2 Acceptor Vector (BD Biosciences
Clontech, Palo Alto, CA) is designed for use with CLONTECH’s Creator Cloning
Kits to rapidly generate a tetracycline-regulated expression construct for tightly
controlled, inducible expression of a gene of interest using the site-specific Cre-lox
recombination system (see, e.g., Sauer, Methods 14:381-92 (1998); Furth, J. Mamm.
Gland Biol. Neoplas. 2:373 (1997)), which may also be employed for host cell
immortalization (see, e.g., Cascio, Artif. Organs 25:529 (2001)).
Polynucleotide sequences that encode four exemplary fusion
polypeptides (SEQ ID NOS:1-4) are provided in SEQ ID NOS:13, 17, 21, and 25
(encoding the fusion polypeptide comprising SEQ ID NO:1); SEQ ID NOS:14, 18, 22,
and 26 (encoding the fusion polypeptide comprising SEQ ID NO:2); SEQ ID NOS: 15,
19, 23, and 27 (encoding the fusion polypeptide comprising SEQ ID NO:3); and SEQ
ID NOS:16, 20, 24, and 28 (encoding the fusion polypeptide comprising SEQ ID
NO:4). Each of SEQ ID NOS:25, 26, 27, and 28 lack the expression control sequences,
the codon encoding the initiating methionine residue, and the codons encoding the poly-
histidine tag. Each of SEQ ID NOS:21, 22, 23, and 24 include the nucleotide sequence
encoding the initiating methionine residue of the respective fusion polypeptide but lack
expression control sequences and the poly-histidine tag encoding sequence. Each of
SEQ ID NOS:17, 18, 19, and 20 include the expression control sequences, the codon
encoding the initiating methionine residue, and the codons encoding the poly-histidine
tag (see also Figures 2A, 2C, 2E, and 2F) Each of SEQ ID NOS:13, 14, 15, and 16
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include the expression control sequences and the codon encoding the initiating
methionine residue, but lack the codons encoding the poly-histidine tag.
Exemplary nucleotide sequences that encode M proteins and Spa
proteins can be readily obtained as described herein from public databases (see, e.g.,
GenBank; GenEBML; CDC emm typing center website accessed via Internet at
cdc.gov/ncidod/biotech/strep/emmtypes). The nucleotide sequence an M protein
variant or Spa protein variant (or the amino terminal portion of the M protein variant or
Spa protein variant) can be determined and/or identified by comparing the nucleotide
sequence of a polynucleotide encoding the variant with a polynucleotide described
herein or known in the art that encodes the particular M protein or Spa protein using
any one of the alignment algorithms described herein and used in the art. The percent
identity between two polynucleotides may thus be readily determined. Polynucleotides
have 100% nucleotide sequence identity if the nucleotide residues of the two sequences
are the same when aligned for maximal correspondence. In particular embodiments, the
nucleotide sequence of an M protein immunogenic peptide variant-encoding
polynucleotide or a Spa protein immunogenic peptide variant-encoding polynucleotide
or the nucleotide sequence of the region of the polynucleotide of an M protein or Spa
protein that encodes the amino terminal portion of the M protein or Spa protein from
which the immunogenic peptide is derived, respectively), is at least 70%, 75%, 80%,
85%, 90%, 95%, 97%, or 98% identical to one or more of the polynucleotide sequences
that encode the respective immunogenic peptide of each M protein and Spa protein,
which are described herein. Polynucleotide variants also include polynucleotides that
differ in nucleotide sequence identity due to the degeneracy of the genetic code and
encode the amino terminal portion of an M protein or Spa protein having an amino acid
sequence disclosed herein or known in the art. A polynucleotide sequence that is
complementary to the encoding polynucleotide can be readily determined by a person
skilled in the art. Certain polynucleotides that encode any immunogenic peptide or
variant thereof may also be used as probes, primers, short interfering RNA (siRNA), or
antisense oligonucleotides. Polynucleotides may be single-stranded DNA or RNA
(coding or antisense) or double-stranded RNA (e.g., genomic or synthetic) or DNA
(e.g., cDNA or synthetic).
Polynucleotide variants may be identified by alignment procedures
described herein and in the art and also may be identified by hybridization methods.
Hybridization of two polynucleotides may be performed using methods that incorporate
the use of suitable moderately stringent conditions, for example, pre-washing in a
solution of 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50 °C-70 °C,
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5X SSC for 1-16 hours; followed by washing once or twice at 22-65 °C for 20-40
minutes with one or more each of 2X, 0.5X, and 0.2X SSC containing 0.05-0.1% SDS.
For additional stringency, conditions may include a wash in 0.1X SSC and 0.1% SDS at
50-60 C for 15 minutes. As understood by persons having ordinary skill in the art,
variations in stringency of hybridization conditions may be achieved by altering the
time, temperature, and/or concentration of the solutions used for pre-hybridization,
hybridization, and wash steps. Suitable conditions may also depend in part on the
particular nucleotide sequences of the probe used (i.e., for example, the guanine plus
cytosine (G/C) versus adenine plus thymidine (A/T) content). Accordingly, a person
skilled in the art will appreciate that suitably stringent conditions can be readily selected
without undue experimentation when a desired selectivity of the probe is identified.
If desired, immunogenic peptide variants and fusion polypeptide variants
may be readily prepared by genetic engineering and recombinant molecular biology
methods and techniques. Analysis of the primary and secondary amino acid sequence
of the immunogenic peptide or fusion polypeptide and computer modeling of same to
analyze the tertiary structure of the polypeptide may aid in identifying specific amino
acid residues that can be substituted, added, or deleted without altering the structure and
as a consequence, potentially the immunogenicity of the peptide or polypeptide.
Modification of a polynucleotide, such as DNA, encoding an immunogenic peptide or
fusion polypeptide variant may be performed by a variety of methods, including site-
specific or site-directed mutagenesis of the DNA, which methods include DNA
amplification using primers to introduce and amplify alterations in the DNA template,
such as PCR splicing by overlap extension (SOE). Mutations may be introduced at a
particular location by synthesizing oligonucleotides containing a mutant sequence,
flanked by restriction sites enabling ligation to fragments of the non-variant sequence.
Following ligation, the resulting reconstructed sequence encodes a variant having the
desired amino acid insertion, substitution, or deletion. While restriction sites may be
introduced during mutagenesis procedures, as described herein, inclusion of amino
acids that are encoded by the nucleotide sequences of a restriction site in the encoded
peptide or fusion polypeptide is avoided to reduce the likelihood of introducing an
undesired epitope.
Site directed mutagenesis of a polynucleotide such that it encodes a variant
of an immunogenic peptide or of a fusion polypeptide may be performed according to any
one of numerous methods described herein and practiced in the art (Kramer et al., Nucleic
Acids Res. 12:9441 (1984); Kunkel Proc. Natl. Acad. Sci. USA 82:488-92 (1985); Kunkel
et al., Methods Enzymol. 154:367-82 (1987)). Random mutagenesis methods to identify
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residues that, when mutated (e.g., substituted or deleted), alter binding of the immunogenic
peptide or fusion polypeptide to a ligand (e.g., a monoclonal or polyclonal antibody) can
also be performed according to procedures that are routinely practiced by a person skilled
in the art (e.g., alanine scanning mutagenesis; error prone polymerase chain reaction
mutagenesis; and oligonucleotide-directed mutagenesis (see, e.g., Sambrook et al.
Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory
Press, NY (2001)). When a variant or an immunogenic peptide or fusion polypeptide is
intended for use in an immunogenic composition to induce an immune response against
GAS, the variant desirably retains the capability in a substantially similar manner (i.e.,
in a statistically, biologically, or clinically significant manner) as the non-variant
immunogenic peptide or fusion polypeptide to bind to specific antibodies and to evoke
production of antibodies that bind to GAS bacteria and that are bactericidal.
Characterization of Immunogenic Peptides and Fusion Polypeptides
As described in detail herein, the immunogenic peptides and fusion
polypeptides comprising immunogenic peptides are intended for use as immunogens to
induce an immune response, particularly a protective immune response, against a
majority of GAS serotypes that cause GAS infections in humans. To determine and
characterize the immunogenicity of the immunogenic peptides and fusion polypeptides,
any number of immunoassays, phagocytosis and bactericidal assays, and animal studies
may be performed according to methods routinely practiced by a person skilled in the
art. Additional pre-clinical studies that evaluate the safety of the immunogenic
composition to be administered to a subject are typically performed. Ultimately, the
safety and efficacy of immunogenic compositions comprising immunogenic peptides or
fusion polypeptides as described herein will be determined by clinical studies, which
are monitored by regulatory agencies.
The immunogenicity of an immunogenic peptide or fusion polypeptide
(or immunogenic composition comprising same) may be determined by administering
an immunogenic composition described herein to a host (or subject, patient) according
to immunization protocols described herein and in the art. Typically, after
administering an initial dose of the immunogenic composition (also called the primary
immunization) to a host, one, two or more doses of the immunogenic composition (also
called boosting or booster doses) are administered.
In general, to monitor the immune response of an immunized host during
pre-clinical studies in animals, sera is obtained from the animals prior to the first dose
(i.e., pre-immune sera) and obtained after the final boosting dose. Sera may also be
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obtained after any one or more of the boosting doses between the primary dose and
final boosting dose. To monitor the immune response of an immunized host during
clinical studies or during post-marketing studies, sera may also be obtained from
humans before the first immunization and after one or more administrations of the
immunogenic compositions.
An antibody that specifically binds to an immunogenic peptide (and to a
fusion polypeptide, dimeric peptide, full length or mature protein, or GAS bacteria
expressing the protein) may belong to any immunoglobulin class, for example IgG, IgE,
IgM, IgD, or IgA. For characterizing the immunogenic peptides and fusion polypeptides
described herein, use of polyclonal and/or monoclonal antibodies may be desired. The
antibody may be obtained from or derived from an animal, for example, fowl (e.g.,
chicken) and mammals, which include but are not limited to a mouse, rat, hamster, rabbit,
or other rodent, a cow, horse, sheep, goat, camel, human, or other primate. As described
herein, polyclonal antisera are obtained from an animal by immunizing the animal with
an immunogenic composition comprising an immunogenic peptide, plurality of
immunogenic peptides or a fusion polypeptide or plurality of fusion polypeptides.
Production of immunogenic peptide-specific antibodies in an immunized
host (including a human host) may include production of any class of immunoglobulin,
including IgG, IgA, IgM, and/or IgE, and isotypes within the classes. The presence of
specific IgG, IgM, IgE, and IgA (particularly in mucosal secretions) may be detected in
a biological sample (e.g., serum, nasal wash, lung lavage, or other tissues) obtained
from an immunized host. For detection of immunogenic peptide- and GAS-specific
antibodies in an immunoassay, the biological sample may be permitted to interact with
or contact an antigen that is purified, isolated, partially isolated, or a fragment thereof,
or to interact with or contact a microorganism, which may be fixed (such as with
ethanol or formaldehyde) or unfixed or non-denatured. Mucosal secretions include
those collected from the respiratory tract, including the nasopharynx and lungs.
Functional assays may also be performed, such as the ability of an immunogen-specific
antibody to facilitate phagocytosis or opsonization of GAS, inhibit growth of GAS or
kill the bacteria, or to prevent entry of GAS into a host cell. Such methods are
described herein and are routinely practiced by persons skilled in the art.
Immune sera (i.e., sera obtained from a host after immunization with one
or more doses of the immunogenic composition) or other biological sample may be
evaluated for the presence of antibodies (immunoglobulins) that specifically bind to any
one or more of the immunogenic peptides included in the immune composition
(including those that are incorporated into fusion polypeptides). An assay to detect
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presence of a specific antibody in a biological sample, such as immunoassays described
herein, may also use a mature or full-length M protein or Spa protein and/or whole GAS
bacteria. The level to which antibodies in the immune sera bind to GAS serotypes
represented by an immunogenic peptide in the immunogenic composition and the level
to which antibodies bind to GAS serotypes not represented by an immunogenic peptide
in the composition (also called non-vaccine GAS serotypes) may be determined. The
level to which antibodies bind to an immunogenic peptide, fusion polypeptide, M
protein, Spa protein, or GAS bacteria (typically referred in the art as titer) can be
readily determined using any one or more immunoassays that are routinely practiced by
the person having ordinary skill in the art. By way of non-limiting example,
immunoassays include ELISA, immunoblot, radioimmunoassay,
immunohistochemistry, fluorescence activated cell sorting (FACS), Ouchterlony, and
the like. Immunoassays may be performed using one or more of the immunogenic
peptides included in the immunizing composition, and which may be used in the assays
as individual immunogenic peptides, dimeric peptides, or fusion polypeptides.
In one embodiment, a method is provided for detecting an antibody,
which may be a monoclonal antibody or polyclonal antibody, and which may be of any
class or isotype, and may be present in or suspected of being present in a biological
sample described herein, which antibody binds to any one of the immunogenic peptides
(including, for example, an immunogenic peptide comprising the amino acid sequence
of any one of SEQ ID NOS: 29-59), dimeric peptides, or fusion polypeptides (including
any one of the exemplary fusion polypeptides described herein, such as the fusion
polypeptide comprising the amino acid sequence of any one of SEQ ID NOS:1, 2, 3, 4
and SEQ ID NOS:5-12). The method comprises contacting the biological sample with
at least one of the immunogenic peptides, dimeric peptides, or fusion polypeptides
described above and herein under conditions and for a time sufficient for an antibody in
the sample to interact with the peptide, dimeric peptide, or fusion polypeptide (i.e.,
mixing, combining, or in some manner permitting the biological sample and the
immunogenic peptide, fusion polypeptide, or dimeric peptide to interact). An antibody
present in the biological sample that specifically binds to the peptide, dimeric peptide,
or fusion polypeptide can be detected using any one of the exemplary detection
methods described herein and in the art for detecting antibody-antigen binding. By way
of non-limiting example, antibody bound to the peptide, dimeric peptide, or fusion
polypeptide may be detected using a reagent specific for a conserved region of the
antibody, such as the Fc portion of the antibody, which reagent is typically selected
depending on the source of the antibody (i.e., whether the antibody is from an animal,
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such as a mouse, rat, goat, or sheep, etc or whether the antibody is from a human).
Such reagents typically comprise a detectable label, such as for example an enzyme,
fluorescent label, luminescent label, or radioactive label. Additional exemplary
reagents include those that detect a specific isotype or class of antibody. Many such
reagents may be obtained from commercial sources.
Conditions for a particular assay including temperature, buffers
(including salts, cations, media), and other components, which maintain the integrity of
the antibodies within the pre-immune and immune sera and the integrity of the antigen
(which may be an immunogenic peptide, dimeric peptide, or fusion polypeptide, M
protein, Spa protein, or bacteria) used in the assay, are familiar to a person skilled in the
art and/or which can be readily determined. A biological sample, such as serum, is
contacted (mixed, combined with, or in some manner permitted to interaction) with the
antigen, under conditions and for a time sufficient to permit interaction between the
antigen and antibodies present in the sample. The interaction, or level of binding, of the
antigen to an antibody present in an immune serum sample (or other biological sample)
may be determined and compared to a level of binding of the respective antigen to
antibodies present in a pre-immune sample (or an otherwise suitable negative control).
An increase in the level of binding of the antigen to the immune serum sample
compared with the pre-immune serum sample indicates that the immunogenic
composition evoked production of specific antibodies. As noted herein, the level of
binding of an immunogen to antibodies present in a sample from an immunized host is
typically referred to in the art as the titer.
Interaction or binding of an antibody to a specific antigen generally
involves electrostatic interactions, hydrogen bonding, Van der Waals interactions, and
hydrophobic interactions. Any one of these or any combination thereof can play a role
in the binding between an antibody and its antigen. As used herein, an antibody is said
to be “specific for” or to “specifically bind” an immunogenic peptide, fusion
polypeptide comprising the immunogenic peptide, M protein, Spa protein, or GAS
bacteria when the antibody reacts at a detectable level with the respective immunogen,
4 -1
preferably with an affinity constant, K of greater than or equal to about 10 M , or
-1 6 -1
greater than or equal to about 10 M , greater than or equal to about 10 M , greater
7 -1 8
than or equal to about 10 M , or greater than or equal to 10 M . The ability of the
antibody to bind to its cognate ligand (in this instance, the immunogenic peptide, fusion
polypeptide, M protein, Spa protein, or bacteria) may also be expressed as a
dissociation constant K , and an antibody is said to specifically bind its cognate ligand
if it binds with a K of less than or equal to 10 M, less than or equal to about 10 M,
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less than or equal to about 10 M, less than or equal to 10 M, or less than or equal to
M.
Affinities of an antibody for an immunogenic peptide or a polypeptide
comprising the immunogenic peptide, such as fusion polypeptide described herein, can
be readily determined using conventional techniques, for example, those described by
Scatchard et al. (Ann. N.Y. Acad. Sci. USA 51:660 (1949)) and by surface plasmon
resonance (SPR; BIAcore , Biosensor, Piscataway, NJ). For surface plasmon
resonance, target molecules are immobilized on a solid phase and exposed to ligands in
a mobile phase running along a flow cell. If ligand binding to the immobilized target
occurs, the local refractive index changes, leading to a change in SPR angle, which can
be monitored in real time by detecting changes in the intensity of the reflected light.
The rates of change of the surface plasmon resonance signal can be analyzed to yield
apparent rate constants for the association and dissociation phases of the binding
reaction. The ratio of these values gives the apparent equilibrium constant (affinity)
(see, e.g., Wolff et al., Cancer Res. 53:2560-65 (1993)).
Several in vitro assays described herein and routinely practiced in the art
may be used to determine activity of specific antibodies evoked by the immunogenic
and fusion polypeptides described herein. An exemplary assay is an
opsonophagocytosis assay, which detects phagocytosis facilitated by the presence of
opsonic antibodies present in test antisera. Briefly, the assay measures the level of
phagocytosis of bacterial by neutrophils after preincubating the bacteria in the presence
of immune sera. Pre-immune or other suitable negative control is also included in such
an assay. Preincubated bacteria are then mixed with whole blood from a suitable host,
such as a host for which opsonic protection is sought (e.g., a human), to determine the
percent of neutrophils that associate with the bacterial cells, which is a measure of
phagocytic activity facilitated by opsonic antibodies. The percent of neutrophils
associated with the bacteria preincubated with immune sera can be compared to the
percent of neutrophils associated with the bacteria that is preincubated with preimmune
sera (other other suitable sera believed or known not to contain anti-GAS antibodies).
A greater percent (i.e., a statistically, biologically, or clinically significant increased
percent) of neutrophils associated with the bacteria pre-incubated with immune sera
compared with the percent of neutrophils associated with bacteria pre-incubated with
pre-immune sera indicates that the test immune sera contains opsonic antibodies.
Another exemplary in vitro assay that determines bactericidal activity of
antibodies present in a sample is a bactericidal assay (see, e.g., Hu et al., supra;
Examples herein). GAS bacteria are incubated with immune serum or an appropriate
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control serum (e.g., pre-immune serum) and then plated on medium that permits growth
of GAS (e.g., sheep blood agar). Typically after an overnight incubation, the number of
viable bacteria is quantified and the results are expressed as percent killing. A
commonly used formula used in an indirect bactericidal assay for expressing percent
killing is [(CFU (colony forming units) of bacteria sample pre-incubated with
preimmune serum) – (CFU of bacteria sample pre-incubated with immune serum)
‚CFU of bacteria sample pre-incubated with preimmune serum] x 100.
Opsonization, phagocytosis, and bactericidal assays are art-accepted in
vitro animal models for characterization of potential anti-GAS prophylactic and
therapeutic treatments. Results obtained in one or more of these assays that suggest to a
person skilled in the art the usefulness of a vaccine for prophylactic or therapeutic use
have been supported by clinical study findings (see, e.g., U.S. Patent No. 7,270,827;
Kotloff et al., supra; McNeil et al., supra). Animal models that may be used for
characterizing the immunogenicity of the immunogenic peptides, fusion polypeptides,
and immunogenic compositions comprising same include those that are considered
direct immunotherapy models (i.e., animals are immunized with a potential candidate
immunogenic composition and then challenged with GAS) and those that are indirect or
passive immunotherapy models (i.e., antiserum, or antibodies purified or isolated from
antisera, obtained from animals immunized with a candidate immunogenic composition
or a monoclonal antibody that is specific for a GAS antigen is administered prior to or
concurrently with GAS challenge bacteria).
Animal models that mimic non-invasive GAS disease, such as
pharyngitis, have been difficult to establish in rodent models. A mouse model for
studying GAS impetigo has been described by Scaramuzzino et al. (Infect. Immun.
68:2880-87 (2000)). An non-human primate model that has been successfully
developed for studying pharyngitis includes a cynomolgus macaque model of acute
pharyngitis that mimics human disease (see, e.g., Ashbaugh et al., Cellular Microbiol.
2:283-92 (2000); Sumby et al., in Meth. Molec. Biol. 431:255-67 (DeLeo et al. (ed.)
Humana Press, Totowa NJ (2008))). Other animal models available in the art have been
developed to evaluate therapeutics and prophylactics for invasive diseases, such as an
invasive soft tissue infection (see, e.g., Ashbaugh et al., J. Clin. Investig. 102:550-60
(1998); Boyle et al., J. Infect. Dis. 177:991-97 (1998)); sepsis (see, e.g., Goldmann et
al., J. Inf. Dis. 187:854-61 (2003); Kapur et al., Microbiol. Pathogenesis 16:443-50
(1994); Medina et al., J. Infect. Dis. 184:846-52 (2001)); and necrotizing fasciitis (Patel
et al., J. Inf. Dis. 181:230-34 (2000)).
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Polyclonal antibodies that bind specifically to an immunogenic peptide
(and immune sera that comprise such polyclonal antibodies), can be prepared using
methods described herein and practiced by persons skilled in the art (see, for example,
Green et al., “Production of Polyclonal Antisera,” in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Harlow et al., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory (1988); Williams et al.,
“Expression of foreign proteins in E. coli using plasmid vectors and purification of
specific polyclonal antibodies,” in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995)). See also, for example,
U.S. Patent Nos. 7,270,827; 6,716,433; 7,402,316; 7,063,850. Although polyclonal
antibodies are typically raised in animals such as rats, mice, rabbits, goats, cattle, or
sheep, an antibody may also be obtained from a subhuman primate. General techniques
for immunizing baboons may be found, for example, in International Patent Application
Publication No. WO 91/11465 (1991) and in Losman et al., Int. J. Cancer 46:310, 1990.
Non-human animals that may be immunized with any one of the
immunogenic peptides, dimeric peptides, fusion polypeptides, or immunogenic
compositions comprising same include by way of non-limiting example, mice, rats,
rabbits, hamsters, ferrets, dogs, cats, camels, sheep, cattle, pigs, horses, goats, chickens,
and non-human primates (e.g., cynomolgus macaque, chimpanzee, rhesus monkeys,
orangutan, and baboon). Any one of the immunogenic compositions described herein
may be administered to immunize an animal by a parenteral (e.g., intravenous),
intraperitoneal, intramuscular, intradermal, intraocular, or subcutaneous route. The
immunogenic composition may further comprise a suitable adjuvant to enhance the
immune response to the immunogen. See, e.g., Harlow et al., Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory (1988). Adjuvants typically used for
immunization of non-human animals include but are not limited to Freund’s complete
adjuvant, Freund’s incomplete adjuvant, montanide ISA, Ribi Adjuvant System (RAS)
(GlaxoSmithKline, Hamilton, MT), and nitrocellulose-adsorbed antigen. In general,
after the first injection, animals receive one or more booster immunizations according
to a preferred schedule that may vary according to, inter alia, the immunogen, the
adjuvant (if any) and/or the particular animal species. The immune response may be
monitored by periodically bleeding the animal, separating the sera from the collected
blood, and analyzing the sera in an immunoassay, such as an ELISA or Ouchterlony
diffusion assay, or the like, to determine the specific antibody titer. When an adequate
antibody titer is established, the animals may be bled periodically to accumulate the
polyclonal antisera.
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Polyclonal antibodies that bind specifically to the immunogen may then
be purified from immune antisera, for example, by affinity chromatography using
protein A or protein G immobilized on a suitable solid support (see, e.g., Coligan,
supra, p. 2.7.1-2.7.12; 2.9.1-2.9.3; Baines et al., Purification of Immunoglobulin G
(IgG), in Methods in Molecular Biology, 10:9-104 (The Humana Press, Inc. (1992)).
Alternatively, affinity chromatography may be performed wherein an antibody specific
for an Ig constant region of the particular immunized animal species is immobilized on
a suitable solid support. Affinity chromatography may also incorporate use of one or
more immunogenic peptides, dimeric peptides, or fusion proteins, which may be useful
for separating polyclonal antibodies by their binding activity to a particular
immunogenic peptide.
Monoclonal antibodies that specifically bind to an immunogenic peptide
and immortal eukaryotic cell lines (e.g., hybridomas) that produce monoclonal
antibodies having the desired binding specificity, may also be prepared, for example,
using the technique of Kohler and Milstein (Nature, 256:495-97 (1976), Eur. J.
Immunol. 6:511-19 (1975)) and improvements thereto (see, e.g., Coligan et al. (eds.),
Current Protocols in Immunology, 1:2.5.1-2.6.7 (John Wiley & Sons 1991); U.S. Patent
Nos. 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies, Hybridomas: A
New Dimension in Biological Analyses, Plenum Press, Kennett et al. (eds.) (1980); and
Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor
Laboratory Press (1988); see also, e.g., Brand et al., Planta Med. 70:986-92 (2004);
Pasqualini et al., Proc. Natl. Acad. Sci. USA 101:257-59 (2004)). Monoclonal
antibodies may be used as reagents to determine and monitor immunogenicity of
immunogenic peptides, fusion polypeptides, and immunogenic compositions
comprising a plurality of immunogenic peptides or fusion polypeptides.
Monoclonal antibodies may be isolated from the supernatants of
eukaryotic cell cultures such as hybridoma cultures. An alternative method for
production of a murine monoclonal antibody is to inject the hybridoma cells into the
peritoneal cavity of a syngeneic mouse, for example, a mouse that has been treated
(e.g., pristane-primed) to promote formation of ascites fluid containing the monoclonal
antibody. Contaminants may be removed from the subsequently harvested ascites fluid
(usually within 1-3 weeks) by conventional techniques, such as chromatography (e.g.,
size-exclusion, ion-exchange), gel filtration, precipitation, extraction, or the like (see,
e.g., Coligan, supra, p. 2.7.1-2.7.12; 2.9.1-2.9.3; Baines et al., Purification of
Immunoglobulin G (IgG), in Methods in Molecular Biology, 10:9-104 (The Humana
Press, Inc. (1992)). Monoclonal antibodies may be purified by affinity chromatography
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using an appropriate ligand selected based on particular properties of the monoclonal
antibody (e.g., heavy or light chain isotype, binding specificity, etc.). Examples of a
suitable ligand, immobilized on a solid support, include Protein A, Protein G, an anti-
constant region (light chain or heavy chain) antibody, an anti-idiotype antibody, or the
peptide or polypeptide used for immunizing the animal that is the source of the B cells.
If desired, human monoclonal antibodies may be generated by any
number of techniques with which those having ordinary skill in the art will be familiar.
Such methods include, but are not limited to, Epstein Barr Virus (EBV) transformation
of human peripheral blood cells (e.g., containing B lymphocytes) (see, e.g., U.S. Patent
No. 4,464,456; Glasky et al., Hybridoma 8:377-89 (1989)), in vitro immunization of
human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted
human immunoglobulin genes, isolation from human immunoglobulin V region phage
libraries, or other procedures as known in the art and based on the disclosure herein.
For example, methods for obtaining human antibodies from transgenic mice are described,
for example, by Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature 368:856
(1994); Taylor et al., Int. Immun. 6:579 (1994); U.S. Patent No. 5,877,397; Bruggemann
et al., Curr. Opin. Biotechnol. 8:455-58 (1997); Jakobovits et al., Ann. N. Y. Acad. Sci.
764:525-35 (1995). In certain embodiments, a B cell that is producing the desired
antibody is selected, and the light chain and heavy chain variable regions are cloned
from the B cell according to molecular biology techniques known in the art (WO
92/02551; U.S. Patent No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-48 (1996)) and described herein. Chimeric antibodies, including humanized
antibodies, may also be generated. A chimeric antibody has at least one constant region
domain derived from a first mammalian species and at least one variable region domain
derived from a second, distinct mammalian species (see, e.g., Morrison et al., Proc.
Natl. Acad. Sci. USA, 81:6851-55 (1984); Shin et al., Methods Enzymol. 178:459-76
(1989); Walls et al., Nucleic Acids Res. 21:2921-29 (1993); U.S. Patent No. 5,482,856).
A non-human/human chimeric antibody may be further genetically engineered to create
a “humanized” antibody (see, e.g., Jones et al., Nature 321:522-25 (1986); Riechmann
et al., Nature 332:323-27 (1988)). Designing a humanized antibody may include
determining CDR loop conformations and structural determinants of the non-human
variable regions, for example, by computer modeling, and then comparing the CDR
loops and determinants to known human CDR loop structures and determinants (see,
e.g., Padlan et al., FASEB 9:133-39 (1995); Chothia et al., Nature, 342:377-83 (1989);
Bajorath et al., Ther. Immunol. 2:95-103 (1995); Davies et al., Ann. Rev. Biochem.
59:439-73, (1990); EPA3).
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For particular uses, antigen-binding fragments of antibodies may be
desired. Antibody fragments, F(ab') Fab, Fab', Fv, and Fd, can be obtained, for
example, by proteolytic hydrolysis of the antibody, for example, pepsin or papain
digestion of whole antibodies according to conventional methods. An antibody
fragment may also be any synthetic or genetically engineered protein that acts like an
antibody in that it binds to a specific antigen to form a complex according to numerous
methods described in the art (see, for example, Larrick et al., Methods: A Companion to
Methods in Enzymology 2:106, (1991); Courtenay-Luck, “Genetic Manipulation of
Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and
Clinical Application, Ritter et al. (eds.), page 166 (Cambridge University Press 1995);
and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page 137 (Wiley-Liss, Inc.
1995))
Antibodies may also be identified and isolated from human
immunoglobulin phage libraries, from rabbit immunoglobulin phage libraries, from
mouse immunoglobulin phage libraries, and/or from chicken immunoglobulin phage
libraries (see, e.g., Winter et al., Annu. Rev. Immunol. 12:433-55 (1994); Burton et al.,
Adv. Immunol. 57:191-280 (1994); U.S. Patent No. 5,223,409; Huse et al., Science
246:1275-81 (1989); Schlebusch et al., Hybridoma 16:47-52 (1997) and references
cited therein; Rader et al., J. Biol. Chem. 275:13668-76 (2000); Popkov et al., J. Mol.
Biol. 325:325-35 (2003); Andris-Widhopf et al., J. Immunol. Methods 242:159-31
(2000)). Antibodies isolated from non-human species or non-human immunoglobulin
libraries may be genetically engineered according to methods described herein and
known in the art to “humanize” the antibody or fragment thereof. Immunoglobulin
variable region gene combinatorial libraries may be created in phage vectors that can be
screened to select Ig fragments (Fab, Fv, scFv, or multimers thereof) that bind
specifically to an immunogenic peptide as described herein (see, e.g., U.S. Patent No.
,223,409; Huse et al., Science 246:1275-81 (1989); Sastry et al., Proc. Natl. Acad. Sci.
USA 86:5728-32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1-9
(1990); Kang et al., Proc. Natl. Acad. Sci. USA 88:4363-66 (1991); Hoogenboom et al.,
J. Molec. Biol. 227:381-388 (1992); Schlebusch et al., Hybridoma 16:47-52 (1997) and
references cited therein; U.S. Patent No. 6,703,015).
Preparation of Immunogenic Compositions
The immunogenic compositions described herein may be used for
immunizing a subject (a human or non-human animal) to induce an immune response
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against GAS. The immunogenic compositions may be formulated such that the
compositions are pharmaceutically or physiologically acceptable or suitable
compositions, preparations, or formulations for administration to a human or non-
human animal. Immunogenic compositions may be combined with a pharmaceutically
acceptable (i.e., physiologically suitable or acceptable) excipient(s), which are
described in greater detail herein. Any physiological or pharmaceutically suitable
excipient or carrier (i.e., a non-toxic material that does not interfere with the activity of
the active ingredient) known to those of ordinary skill in the art for use in
pharmaceutical compositions may be employed in the compositions described herein.
Exemplary excipients include diluents and carriers that maintain stability and integrity
of the component(s) of the composition. Exemplary excipients include diluents and
carriers that maintain stability and integrity of proteins. Excipients for therapeutic use
are well known, and are described, for example, in Remington: The Science and
Practice of Pharmacy (Gennaro, 21 Ed. Mack Pub. Co., Easton, PA (2005)), which are
described in greater detail herein. The choice of an excipient depends on several factors,
including the stability of the immunogenic peptides or fusion polypeptides; the route of
administration; and the dosing schedule. For example, saline and phosphate buffered
saline at physiological pH may be used. Preservatives, stabilizers, dyes and even
flavoring agents (if administered orally) may be provided in the composition.
The immunogenic compositions described herein may also comprise a
suitable adjuvant. An adjuvant is intended to enhance (or improve, augment) the
immune response to the immunogenic peptides and fusion polypeptides comprising the
peptides (i.e., increase the level of the specific immune response to the immunogenic
peptide and in a statistically, biologically, or clinically significant manner compared
with the level of the specific immune response in the absence of administering the
adjuvant).
For administration in humans, a pharmaceutically acceptable adjuvant is
one that has been approved or is approvable for human administration by pertinent
regulatory bodies. For example, as discussed herein and known in the art, Complete
Freund's adjuvant is not suitable for human administration. Desired adjuvants augment
the response to the immunogenic peptide or fusion polypeptide without causing
conformational changes in the immunogen that might adversely affect the qualitative
immune response. Suitable adjuvants include aluminum salts, such as alum (potassium
aluminum sulfate), or other aluminum containing adjuvants such as aluminum
hydroxide, aluminum phosphate, or aluminum sulfate. Other pharmaceutically suitable
adjuvants include nontoxic lipid A-related adjuvants such as, by way of non-limiting
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example, nontoxic monophosphoryl lipid A (see, e.g., Persing et al., Trends Microbiol.
:s32-s37 (2002)), for example, 3 De-O-acylated monophosphoryl lipid A (MPL) (see,
e.g., United Kingdom Patent Application No. GB 2220211). Other useful adjuvants
include QS21 and QuilA that comprise a triterpene glycoside or saponin isolated from
the bark of the Quillaja saponaria Molina tree found in South America (see, e.g.,
Kensil et al., in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell and
Newman, Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). Other suitable
adjuvants include oil in water emulsions, optionally in combination with immune
stimulants, such as monophosphoryl lipid A (see, e.g., Stoute et al., N. Engl. J. Med.
336, 86-91 (1997)). Other suitable adjuvants include polymeric or monomeric amino
acids such as polyglutamic acid or polylysine, liposomes, and CpG (see, e.g., Klinman,
Int. Rev. Immunol. 25(3-4):135-54 (2006); U.S. Patent No. 7,402,572; European Patent
No. 772 619).
The immunogenic compositions described herein may be formulated by
combining the plurality of immunogenic peptides or the plurality of fusion polypeptides
with at least one pharmaceutically acceptable excipient. As described herein the
immunogenic compositions may further comprise a pharmaceutically suitable adjuvant.
Typically, all immunogenic peptides or all fusion polypeptides intended to be
administered to a host are combined in a single immunogenic composition, which may
include at least one pharmaceutically acceptable excipient and which may further
include a pharmaceutically suitable adjuvant. Alternatively, for example, multiple
immunogenic compositions may be formulated separately for separate administration,
which could be by any route described herein or in the art and which could be
sequential or concurrent.
The immunogenic compositions described herein may be formulated as
sterile aqueous or non-aqueous solutions, suspensions or emulsions, which as described
herein may additionally comprise a physiologically acceptable excipient (which may
also be called carrier) and/or a diluent. The immunogenic compositions may be in the
form of a solid, liquid, or gas (aerosol). Alternatively, immunogenic compositions
described herein may be formulated as a lyophilate (i.e., a lyophilized composition), or
may be encapsulated within liposomes using technology known in the art.
Immunogenic compositions may also contain other components, which may be
biologically active or inactive. Such components include, but are not limited to, buffers
(e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose,
mannose, sucrose or dextrans), mannitol, proteins (such as albumin), polypeptides or
amino acids such as glycine, antioxidants, chelating agents such as EDTA or
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glutathione, stabilizers, dyes, flavoring agents, and suspending agents and/or
preservatives.
In general, as discussed herein, the type of excipient is selected on the
basis of the mode of administration. The compositions and preparations described
herein may be formulated for any appropriate manner of administration, including, for
example, topical, buccal, lingual, oral, intranasal, intrathecal, rectal, vaginal,
intraocular, subconjunctival, transdermal, sublingual or parenteral administration,
including subcutaneous, intravenous, intramuscular, intrasternal, intracavernous,
intrameatal or intraurethral injection or infusion.
For parenteral administration, such as subcutaneous injection or
intramuscular injection, the carrier or excipient preferably comprises water, saline,
alcohol, a fat, a wax or a buffer, and the immunogenic composition is sterile. For oral
administration, any of the above excipients or a solid carrier, such as mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and
magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactic
galactide) as well as nanoparticles may also be used as carriers for the compositions
described herein. Suitable biodegradable microspheres described, for example, in U.S.
Patent Nos. 4,897,268 and 5,075,109. In particular embodiments in which the
composition or preparation is combined with a microsphere, the microsphere is larger
than approximately 25 microns. An immunogenic composition described herein may
be lyophilized or otherwise formulated as a lyophilized product using one or more
appropriate excipient solutions (e.g., sucrose, physiological saline) as diluents upon
administration. Nanoparticles may be used to deliver the lyphophilized product and
appropriate excipient(s).
The immunogenic compositions disclosed herein may be intended for
topical administration, such as directly to mucosal tissue, in which case the carrier may
suitably comprise a solution, emulsion, ointment or gel base. The base, for example,
may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols,
beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and
stabilizers. Thickening agents may be present in a pharmaceutical composition for
topical administration (e.g., oral or vaginal). The immunogenic compositions described
herein may be administered topically by using any one of several delivery vehicles
described herein and used in the art, including but not limited to a sponge, gel cap,
suppository, gauze (or other suitable fabric for application to the tissue to be treated),
nanoparticles, and a lozenge. With respect to certain delivery vehicles, such as a
sponge, fabric, or gauze, the composition or preparation is attached to, absorbed by,
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adsorbed to, or in some manner applied to the vehicle that permits release of the
composition or preparation upon contact with the tissue to be treated.
An immunogenic composition disclosed herein may be intended for
rectal, oral, or vaginal administration, in the form, e.g., of a suppository or lozenge,
which will melt in the rectum, oral, or vaginal space, respectively, and release the drug
or components of the composition. A composition or preparation described herein that
is administered orally may also be in the form of a liquid. The composition or
preparation for rectal administration may contain an oleaginous base as a suitable
nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter
and polyethylene glycol.
The immunogenic compositions described herein are preferably
endotoxin free, particularly when delivered parenterally. An endotoxin free
composition is substantially free of endotoxins and/or related pyrogenic substances (i.e.,
an endotoxin is not detectable by methods accepted by regulatory agencies to
demonstrate with sufficient sensitivity whether an endotoxin is present). Endotoxins
include toxins that are present in viable microorganisms and include toxins that are
released only when the microorganisms lack cell integrity or die. Pyrogenic substances
include fever-inducing, thermostable substances (lipopolysaccharides and
glycoproteins) located in the outer membrane of bacteria and other microorganisms.
These substances can cause fever, hypotension, and shock when administered to
humans. Manufacturing compositions that are endotoxin-free can require special
equipment, expert artisans, and can be significantly more expensive than making
formulations that are not endotoxin-free.
In another embodiment, a method of manufacture of the immunogenic
compositions described herein is provided. Methods of manufacture comprise
combining or mixing together the desired plurality of immunogenic peptides or desired
fusion polypeptides to provide the immunogenic compositions described herein. The
methods of manufacture may further comprise combining or mixing one or more
physiologically suitable (or pharmaceutically suitable) excipients as described herein.
The methods may further comprise combining or mixing the immunogenic composition
comprising the desired immunogenic peptides or desired fusion polypeptides with a
pharmaceutically suitable adjuvant; at least one pharmaceutically suitable excipient
may also be combined or mixed with the immunogenic composition comprising an
adjuvant. In still further embodiments, a method of manufacture comprises chemical
synthesis or recombinant production of the desired immunogenic peptides or desired
fusion polypeptides. Chemical synthesis and recombinant production of the
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immunogenic peptides and fusion polypeptides are described in detail herein. During
manufacture of each immunogenic peptide and fusion protein, appropriate manufactures
processes (such as Good Manufacturing Practices (GMP)) as required by a regulatory
agency are employed. In addition, persons skilled in the art is familiar with techniques
and steps to be taken to maintain stability and integrity of the peptides or fusion
polypeptides during manufacture of an immunogenic composition.
Methods of Using Immunogenic Peptides, Fusion Polypeptides, and Immunogenic
Compositions
The immunogenic compositions described herein that comprise a
plurality of GAS immunogenic peptide, for example, least 31 different immunogenic
peptides derived from GAS M proteins or GAS Spa proteins, and immunogenic
compositions described herein that comprise at least four fusion polypeptides that
comprise the plurality of immunogenic peptides (e.g., the at least 31 different
immunogenic peptides each independently comprising at least 25 contiguous amino
acids from the amino terminal portions of one of the M proteins from GAS M serotypes
1; 2; 3; 4; 5; 6; 11; 12; 14; 18; 19; 22; 24; 28; 29; 44; 49; 58; 73; 75; 77; 78; 81; 82; 83;
87; 89; 92; 114; 118, or Spa protein from GAS serotype 18) may be used for inducing
an immune response against GAS. Accordingly, in certain embodiments, methods are
provided herein that comprise immunizing a host (or subject), which may be a human
host, in a manner appropriate to evoke an immune response (including a humoral
response) against GAS bacteria. These compositions described herein are therefore
useful for prophylactic and/or therapeutic treatment of a host in need thereof who has
inadequate immunity to GAS and is susceptible to infection.
Methods described herein for inducing an immune response against GAS
and for preventing (i.e., reducing the likelihood of occurrence) and/or treating a GAS
infection comprise administering an immunogenic composition described herein to the
host once, twice, three times, four times, or more times at appropriate time intervals to
evoke and maintain the desired anti-GAS immune response. As long known in the art,
protection of a host against GAS infection generally correlates with the production of
opsonizing antibodies against GAS serotype-specific M protein (see, e.g., Lancefield, J.
Immunol. 89:307, 1962). In addition, the presence of secretory or mucosal anti-GAS
antibodies in the host may prevent (reduce or decrease likelihood of occurrence) initial
colonization by streptococci. Because the immunogenic compositions described herein
that comprise a plurality of immunogenic peptides or comprise fusion polypeptides
comprising the plurality of immunogenic peptides may be used for prevention,
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amelioration, or treatment of GAS infection, the immunogenic compositions may also
be referred to as vaccines by a person skilled in the art.
The dose of each immunogenic composition, the number of doses
administered to the host, and the time intervals between two doses of the composition
may be determined by a person skilled in the medical art. The appropriate amount of
each immunogenic peptide or the amount of each fusion polypeptide in the
immunogenic composition administered to the host may depend upon the host’s or
patient's (e.g., human’s) condition, that is, stage of the disease, general health status, as
well as age and weight, and other factors familiar to a person skilled in the medical art.
Hosts or subjects who may be immunized with the immunogenic compositions
described herein include human and non-human hosts and subjects. Human
hosts/subjects include infants, children, or an adult. Immunogenic compositions
suitable for administration to an adult may further be prepared dependent on whether
the adult is a young adult, middle-aged, or a senior adult.
Immunogenic compositions may be administered in a manner
appropriate to the disease to be treated (or prevented) as determined by persons skilled
in the medical art. An appropriate dose and a suitable duration and frequency of
administration will be determined by factors such as the condition of the patient, age of
the patient, the type and severity of the patient's disease to be treated or prevented, the
particular form of the active ingredient, and the method of administration. In general,
an appropriate dose and treatment regimen provides the immunogenic composition in
an amount sufficient to provide therapeutic and/or prophylactic benefit (e.g., an
improved clinical outcome, overall survival, or a lessening of symptom severity). For
prophylactic use, a dose should be sufficient to prevent or delay the onset of, and/or to
diminish the severity of an infection in a statistically, biologically, or clinically
significant manner.
Optimal doses may generally be determined using experimental in vitro,
in vivo animal models, and/or human clinical trials. The optimal dose may depend
upon the body mass, weight, or blood volume of the host. In general, the amount of an
immunogenic peptide or fusion polypeptide as described herein present in a dose,
ranges from about 10 mg to about 10 mg, from about 100 mg to 1 mg, from about 150
mg to 500 mg, or from about 200 mg to about 400 mg. The use of the minimum dosage
that is sufficient to provide effective therapy and/or prophylaxis is usually preferred.
Patients may generally be monitored for therapeutic or prophylactic effectiveness using
assays suitable for the condition being treated or prevented, which assays will be
familiar to those having ordinary skill in the art. When administered in a liquid form,
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suitable dose sizes will vary with the size of the patient, but will typically range from
about 1 ml to about 500 ml (comprising an appropriate dose) for a 10-60 kg subject.
Booster immunizations may be administered multiple times (e.g., two
times or three times or four times or more), at desired time intervals ranging from about
2 weeks to about 26 weeks, such as 2, 4, 8, 12, 16, or 26 week intervals. The time
intervals between different doses (e.g., between the primary dose and second dose, or
between the second dose and a third dose) may not be the same, and the time interval
between each two doses may be determined independently.
The immunogenic compositions described herein may be administered a
route including oral, enteral, parenteral, transdermal/transmucosal, and inhalation. The
term enteral, as used herein, is a route of administration in which the agent is absorbed
through the gastrointestinal tract or oral mucosa, including oral, rectal, and sublingual.
The term parenteral, as used herein, describes administration routes that bypass the
gastrointestinal tract, and are typically administered by injection or infusion, including
intraarterial, intradermal, subdermal, intramuscular, intranasal, intraocular,
intraperitoneal, intravenous, subcutaneous, submucosal, intravaginal, intrasternal,
intracavernous, intrathecal, intrameatal, and intraurethral injection. The term
transdermal/transmucosal, as used herein, is a route of administration in which the agent
is administered through or by way of the skin, including topical. The term inhalation
encompasses techniques of administration in which an agent is introduced into the
pulmonary tree, including intrapulmonary or transpulmonary and includes intranasal
administration. In more particular embodiments, the immunogenic compositions
described herein may be administered orally, intramuscularly, or intranasally. All doses
of the immunogenic compositions may not be administered by the same route. In
certain embodiments, different doses of the immunogenic compositions may be
delivered by different routes, such as by two or more of oral, intramuscular, and
intransal routes.
The immunogenic compositions described herein may be used to reduce
the likelihood of occurrence of a GAS infection or to treat a GAS infection that causes
any one of the following: pharyngitis, scarlet fever, necrotizing fasciitis, cellulitis,
meningitis, pneumonia, streptococcal toxic shock syndrome, bacteremia, septicemia,
septic arthritis, pyoderma, skin infections (invasive and non-invasive), impetigo,
erysipelas, soft-tissue infection, nephritis, and GAS pyrogenic reaction. Methods
described herein for inducing an immune response against GAS and reducing the
likelihood of occurrence or treating a GAS infection may also effectively reduce the
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likelihood of occurrence or severity of nonsuppurative sequelae such as acute rheumatic
fever, rheumatic heart disease, reactive arthritis, and glomerulonephritis.
Immunized subjects may generally be monitored for therapeutic or
prophylactic effectiveness using assays suitable for the infection or condition being
treated or prevented, which assays will be familiar to those having ordinary skill in the
art and which are described herein. The immune response evoked by administering the
immunogenic compositions described herein according to the methods described above
comprises an adaptive immune response that includes a humoral response and may also
include a cellular response (which comprises a CD4 immune response and a CD8
immune response) specific for each immunogen peptide represented in the
immunogenic composition. The humoral immune response (i.e., antibody response)
can be monitored throughout an immunization protocol using any one of the
immunoassays (e.g., ELISA, immunoblotting), in vitro functional assays (e.g., opsonic,
phagocytic and killing assays, indirect bactericidal assays) and the like. Such methods
are useful for monitoring and determining the level of binding (i.e., titer) of specific
antibodies present in a biological sample (e.g., sera) from an immunized host. Based on
the results from one or more of these assays, the dose or timing of the next dose or the
necessity for an additional dose can be determined.
A cell-mediated immune response involves various types of T cells (i.e.,
T lymphocytes). In a cell mediated response, T cells act to eliminate an antigen by a
number of mechanisms. For example, helper T cells that are capable of recognizing
specific antigens may respond by releasing soluble mediators such as cytokines to
recruit additional cells of the immune system to participate in an immune response.
Also, cytotoxic T cells are capable of specifically recognizing an antigen and may
respond by binding to and destroying or damaging an antigen-bearing cell such as a
GAS bacterial cell.
Assays routinely practiced in the art to examine cellular immune
response include determining the presence and level of soluble mediators such as
cytokines, lymphokines, chemokines, hormones, growth factors, as well as other
mediators. Immunoassays also include determining cellular activation state changes of
immune cells by analyzing altered functional or structural properties of the immune
cells, for example, cell proliferation, altered motility, induction of specialized activities
such as specific gene expression or cytolytic behavior; cell maturation, and alteration in
relationship between a Th1 response and a Th2 response. Procedures for performing
these and similar assays are may be found, for example, in Lefkovits (Immunology
Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also
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Current Protocols in Immunology; Weir, Handbook of Experimental Immunology,
Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in
Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed,
Science 281:1309 (1998) and references cited therein).
As described briefly herein, a biological sample may be obtained from
the subject for determining the presence and level of an immune response to
immunogenic peptide(s), to a fusion polypeptide comprising the immunogenic peptides,
and/or to a full-length or mature, or GAS bacteria in the subject who has received the
immunogenic compositions described herein. A “biological sample” as used herein
may be a blood sample (from which serum or plasma may be prepared), biopsy
specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid),
bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell
preparation from the subject or a biological source. Biological samples may also be
obtained from the subject prior to receiving any immunogenic composition, which
biological sample is useful as a control for establishing baseline (i.e., pre-immunization)
data.
Determining the effectiveness of immunization with the immunogenic
compositions described herein may also include clinical evaluation. By way of
example, the presence of a GAS infection may be determined by performing routine
assays (bacteria cell culture; immunofluorescence assays) available to the clinician to
determine quickly if GAS are present in a body fluid or at a site on the body, such as the
throat, mucosal tissue, or skin. Symptomatology, such as fever, inflammation, pain,
and various other and numerous symptoms of GAS infections can be monitored by
persons skilled in the clinical art.
Immunogenic peptides, dimeric peptides, and fusion polypeptides
described herein may be used as reagents for detecting the presence and level of
specific antibody in a sample. A biological sample, such as by way of non-limiting
example, a biological sample described herein from an immunized host, or a cell
supernatant or cell lysate from cell lines that are known to or suspected of producing a
specific monoclonal antibody is obtained. The biological sample is contacted with (i.e.,
mixed with, combined with, or in some manner permitted to interact with) an
immunogenic peptide (or dimeric peptide, fusion polypeptide, mature or full-length
protein, or GAS bacteria which each may comprise the immunogenic peptide) for a
time sufficient and under conditions suitable to permit an antibody in the biological
sample and the immunogenic peptide (alone or as part of a larger molecule) to interact.
The level of interaction between the biological sample and the immunogenic peptide is
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detected and compared with the level of interaction of the immunogenic peptide with a
control biological sample that serves as a baseline or negative control. The level of
interaction (i.e., binding of an antibody and immunogenic peptide) can be determined
by any one of the numerous immunoassay methods described herein and the art and
with which a person skilled in the art is familiar.
Immunogenic peptides, dimeric peptides, and fusion polypeptides
described herein may also be used for methods for producing polyclonal antibodies or
monoclonal antibodies that bind specifically to an immunogenic peptide. Exemplary
methods are described in detail herein.
EXAMPLES
EXAMPLE 1
MPROTEIN FROM GAS SEROTYPE 4
This example describes characterization of a GAS antigen expressed by
and present on the cell surface of GAS serotype 4.
The potential protective efficacy of the 30-valent vaccine described
below and herein was enhanced by the discovery of opsonic epitopes within the N-
terminus of the protein Arp4, which is a component of protein 2. Arp4 was previously
shown to bind IgA but was not required for virulence or resistance to phagocytosis (see,
e.g., Husmann et al., Infect. Immun. 63:345-348 (1995)). The most recent epidemiologic
studies found that Type 4 streptococcal infections account for up to 9% of all GAS
infections. To detect opsonic epitopes within the N-terminus of Arp4 (M4), rabbit
antisera were raised against (1) a synthetic peptide representing the N-terminal 30
amino acids of the protein, (2) a recombinant fusion protein containing 50 amino-
terminal residues of Arp4 as well as 5 other M peptides, or (3) the intact recombinant
Arp4 protein. All three antisera provided significant bactericidal activity when tested
using in vitro bactericidal assays (see Example 3 for experimental protocol): anti-
sM4(1-30) antisera resulted in 52% killing; antisera obtained from rabbits immunized
with recombinant 50 aa Arp4 peptide as a component of a hexavalent fusion protein had
82% killing; and antisera from animals immunized with intact recombinant Arp4
provided 85% killing. The results of the functional killing assays indicated that the N-
terminal peptide of Arp4 (M4) was a suitable candidate for inclusion in the 30-valent
vaccine.
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EXAMPLE 2
DESIGN AND CONSTRUCTION OF MULTIVALENT GAS PEPTIDE COMPOSITIONS
This example describes selection of GAS serotypes from which M
proteins immunogenic fragments would be selected and describes construction of fusion
polypeptides comprising these immunogenic fragments.
M peptides were selected from serotypes of GAS based on the
epidemiology of 1) pharyngitis in pediatric subjects in North America (see, e.g.,
Shulman et al., Clin. Infect. Dis. 2009, supra); (2) invasive GAS infections (see, e.g.,
Active Bacterial Core Surveillance of the Emerging Infections Program Network,
supported by the Centers for Disease Control and Prevention) (see, e.g., O'Loughlin et
al., supra); and (3) invasive infections in Europe (for example, as reported by the
StrepEuro consortium, see Luca-Harari et al., supra). Also included in the 30-valent
vaccine composition were M peptides from serotypes of GAS that are currently or have
been historically considered “rheumatogenic” (see, e.g., Shulman et al., Clin. Infect.
Dis. 42:441-47 (2006)) and the amino-terminal peptide fragment of Spa18 (also called
Spa protein from GAS serotype 18, a protective antigen that is expressed by at least
several serotypes of group A streptococci (see, e.g., Dale et al., J. Clin. Invest.
103:1261-68 (1999); U.S. Patent No. 7,063,850; International Patent Application
Publication No. WO 00/37648).
The amino acid sequences of the M proteins selected for inclusion in the
immunogenic composition were obtained from the (Center for Disease Control (CDC)
emm typing center website that may be found by accessing the Internet at
cdc.gov/ncidod/biotech/strep/emmtypes. When sub-types were present, in most
instances, the sequence of the M protein from the predominant subtype was selected.
The amino-terminal regions of the mature M proteins and Spa were searched by BlastP
in the GenBank database to identify regions of homology with human proteins. Amino-
terminal regions that had five or more contiguous amino acid that were identical with
human proteins were not incorporated into the vaccine design.
Construction and expression recombinant multivalent fusion proteins.
The specific 5’ sequences of each emm gene were used to design four hybrid DNA
molecules. Each gene was chemically synthesized (GENSCRIPT®, Piscataway, NJ) to
contain 7 or 8 emm gene fragments linked in tandem, an upstream T7 promoter, and a 3'
poly-histidine motif followed by a stop codon. Unlike the 26-valent vaccine (see Hu et
al., supra; U.S. Patent No. 7,270,827), no linking codons were inserted between the
emm sequences. Optimization of codons for E. coli was accomplished during synthesis
rather than by subsequent site mutation. The DNA strands were annealed and ligated
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into pUC57; the integrity of the synthetic gene sequences was verified by sequencing
using the ABI dye termination method. Expression of each multivalent fusion protein
was detected by SDS-PAGE analysis using whole cell lysates before and after IPTG
induction. A polypeptide tag of six histidine residues (i.e., His-tag) has been added at
the carboxy (also called herein and in the art, COOH) terminus for ease of purification
by nickel affinity chromatography according to methods routinely practiced in the art.
Figure 1 provides a schematic of the encoded M protein immunogenic
peptides for each of four fusion proteins. The serotypes from which M proteins were
included in the vaccine composition included GAS serotypes that accounted for 98% of
all cases of pharyngitis in the U.S. and Canada, 90% of invasive disease in the U.S., and
78% of invasive disease in Europe. In Figure 1, the numbers below the depiction of
each immunogenic peptide refer to the location of the amino acids in the amino terminal
portion of the M protein or Spa protein. By way of illustration and explanation, in
Protein 1, the M1 immunogenic peptide contains amino acids at positions 1-50 from the
amino terminus of the M protein from GAS serotype 1; the M3.1 immunogenic peptide
contains amino acids at positions 22-71 from the amino terminal portion of the M
protein from GAS serotype 3.1; M6.4 contains a tandem repeat of amino acids at
positions 1-25 from the amino terminal portion of GAS serotype 6.4, etc. At the
carboxy terminal end of Protein 1, the M1 immunogenic peptide (amino acids 1-50) is
repeated. The total number of amino acids in each fusion protein is provided at the
right hand end of the schematic for each protein; the initiating methionine residue at the
amino terminus is included in the total amino acid number.
The amino acid sequences of the immunogenic peptides are presented in
Table 1. The initiating amino acid (typically a methionine) of the full-length M protein
is not included in the immunogenic fragment peptide sequence for each peptide of the
fusion proteins (see Table 1). The nucleotide sequence of polynucleotides encoding the
four fusion proteins and the amino acid sequences of the encoded polypeptides are
presented in Figures 2A-2H. The nucleotide sequences provided in Figure 2 include
expression control sequences upstream of the coding region and nucleotides at the
carboxy terminus that encode the His-tag (see also SEQ ID NOS: 17-20,
polynucleotides encoding Fusion Proteins 1-4, respectively). Nucleotide sequences
encoding fusion proteins 1-4 that include the expression control sequences but exclude
the His-tag encoding nucleotides are provided in SEQ ID NOS: 13-16, respectively.
Nucleotide sequences encoding fusion proteins 1-4 that exclude the expression control
sequences, exclude the His-tag encoding nucleotides, and include nucleotides encoding
the amino terminus methionine (ATG) are provided in SEQ ID NOS: 17-20,
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respectively. Nucleotide sequences encoding fusion proteins 1-4 that exclude the
expression control sequences, exclude the His-tag encoding nucleotides, and exclude
nucleotides encoding the amino terminus methionine are provided in SEQ ID NOS: 21-
24, respectively.
Purification of recombinant multivalent vaccine component fusion
proteins. Each fusion protein was purified separately according to procedures
previously reported (see, e.g., Hu et al., supra). Purity was monitored using SDS-
PAGE, and fractions containing pure recombinant protein were pooled and stored at –
C until use.
Construction and expression of individual recombinant dimeric M
peptides. Individual recombinant dimeric peptides comprising vaccine component
peptides were expressed and purified to use as serologic reagents, as previously
described (see, e.g., Hu et al., supra). In addition, seven new M peptides were
chemically synthesized for these studies (GENSCRIPT®, Piscataway, NJ).
TABLE 1: AMINO ACID SEQUENCES OF M PROTEIN IMMUNOGENIC PEPTIDES
M protein
(NH Fusion Protein Sequence
Amino Acid Sequence
terminal Location Identifier
residues)
NGDGNPREVI EDLAANNPAI
M1 (1-50) 1-50 and 401- SEQ ID
QNIRLRHENK DLKARLENAM 450 of Protein 1 NO:29
EVAGRDFKRA
(SEQ ID NO:1)
LLDQVTQLYT KHNSNYQQYN
M3.1 (22-71) 51-100 of SEQ ID
AQAGRLDLRQ KAEYLKGLND Protein 1 (SEQ NO:30
WAERLLQELN
ID NO:1)
RVFPRGTVEN PDKARELLNK
M6.4 (1-25) 101-150 of SEQ ID
YDVENRVFPR GTVENPDKAR Protein 1 (SEQ NO:31
ELLNKYDVEN
ID NO:1)
M2 (2-26) SKNPVPVKKE AKLSEAELHD 151-200 of SEQ ID
KIKNLSKNPV PVKKEAKLSE Protein 1 (SEQ NO:32
AELHDKIKNL
ID NO:1)
M18 (1-50) APLTRATADN KDELIKRAND 201-250 of SEQ ID
YEIQNHQLTV ENKKLKTDKE
Protein 1 (SEQ NO:33
QLTKENDDLK
ID NO:1)
M28 (1-50) AESPKSTETS ANGADKLADA 251-300 of SEQ ID
YNTLLTEHEK LRDEYYTLID
Protein 1 (SEQ NO:34
AKEEEPRYKA
ID NO:1)
M12 (1-50) DHSDLVAEKQ RLEDLGQKFE 301-350 of SEQ ID
RLKQRSELYL QQYYDNKSNG
Protein 1 (SEQ NO:35
YKGDWYVQQL
ID NO:1)
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M protein
(NH Fusion Protein Sequence
Amino Acid Sequence
terminal Location Identifier
residues)
SPA (1-50) DSVSGLEVAD PSDSKKLIEL 351-400 of SEQ ID
GLAKYLNDKL PFKTKEDSEI Protein 1 (SEQ NO:36
LSELRDVLKN
ID NO:1)
M4 (1-50) AEIKKPQADS AWNWPKEYNA 1-50 and 401- SEQ ID
LLKENEELKV EREKYLSYAD 450 of Protein 2 NO:37
DKEKDPQYRA
(SEQ ID NO:2)
M5.0 (1-25) AVTRGTINDP QRAKEALDKY 51-100 of SEQ ID
ELENHAVTRG TINDPQRAKE
Protein 2 (SEQ NO:38
ALDKYELENH
ID NO:2)
M11 (1-50) TEVKAAGQSA PKGTNVSADL 101-150 of SEQ ID
YNSLWDENKT LREKQEEYIT
Protein 2 (SEQ NO:39
KIQNEETKNK
ID NO:2)
M75 (1-50) EEERTFTELP YEARYKAWKS 151-200 of SEQ ID
ENDELRENYR RTLDKFNTEQ
Protein 2 (SEQ NO:40
GKTTRLEEQN
ID NO:2)
RVRYTRHTPE DKLKKIIDDL
M19 (1-25) 201-250 of SEQ ID
DAKEHRVRYT RHTPEDKLKK
Protein 2 (SEQ NO:41
IIDDLDAKEH
ID NO:2)
RVYITRRMTK EDVEKIANDL
M29.2 (1-50) 251-300 of SEQ ID
DTENHGLKQQ NEQLSTEKQG
Protein 2 (SEQ NO:42
LEEQNKQLST ID NO:2)
DRVSRSMSRD DLLNRAQDLE
M14.3 (1-50) 301-350 of SEQ ID
AKNHGLEHQN TKLSTENKTL Protein 2 (SEQ NO:43
QEQAEARQKE
ID NO:2)
VATRSQTDTL EKVQERADKF
M24 (1-50) 351-400 of SEQ ID
EIENNTLKLK NSDLSFNNKA Protein 2 (SEQ NO:44
LKDHNDELTE
ID NO:2)
EGVSVGSDAS LHNRITDLEE
M77 (1-50) 1-50 and 401- SEQ ID
EREKLLNKLD KVEEEHKKDH 450 of Protein 3 NO:45
EQLEKKSEDV
(SEQ ID NO:3)
M22 (1-50) ESSNNAESSN ISQESKLINT 51-100 of SEQ ID
LTDENEKLRE ELQQYYALSD Protein 3 (SEQ NO:46
AKEEEPRYKA
ID NO:3)
M73 (1-50) DNQSPAPVKK EAKKLNEAEL 101-150 of SEQ ID
YNKIQELEEG KAELFDKLEK
Protein 3 (SEQ NO:47
VEEENKKVKE
ID NO:3)
M89 (1-50) DSDNINRSVS VKDNEKELHN 151-200 of SEQ ID
KIADLEEERG EHLDKIDELK
Protein 3 (SEQ NO:48
EELKAKEKSS
ID NO:3)
M58 (1-50) DSSREVTNEL TASMWKAQAD 201-250 of SEQ ID
SAKAKAKELE KQVEEYKKNY
Protein 3 (SEQ NO:49
ETLEKGYDDL
ID NO:3)
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M protein
(NH Fusion Protein Sequence
Amino Acid Sequence
terminal Location Identifier
residues)
M44 (1-50) AESRSVSQGS VSLELYDKLS 251-300 of SEQ ID
DENDILREKQ DEYLTKIDGL Protein 3 (SEQ NO:50
DKENKEYASQ
ID NO:3)
M78 (1-50) ESQNSRSITN EQLIDKLVEE 301-350 of SEQ ID
NNDLKEERAK YLDLLDNREK Protein 3 (SEQ NO:51
DPQYRALMGE
ID NO:3)
M118 (1-50) AEKKVEVADS NASSVAKLYN 351-400 of SEQ ID
QIADLTDKNG EYLERIEELE
Protein 3 (SEQ NO:52
ERQKNLEKLE
ID NO:3)
M83.1 (1-50) DNPRYTDAHN AVTQGRTVPL 1-50 and 351- SEQ ID
QNLLHEMDKN GKLRSENEEL
400 of Protein 4 NO:53
KADLQKKEQE
(SEQ ID NO:4)
M82 (1-50) DSSSRDITEA GVSKFWKSKF 51-100 of SEQ ID
DAEQNRANEL EKKLSGYEKD
Protein 4 (SEQ NO:54
YKTLEQEYEN
ID NO:4)
AGSEENVPKQ QYNALWEENE
M81 (1-50) 101-150 of SEQ ID
DLRGRERKYI AKLEKEEIQN
Protein 4 (SEQ NO:55
GELNEKNRKL
ID NO:4)
ESPREVTNEL AASVWKKKVE
M87 (1-50) 151-200 of SEQ ID
EAKEKASKLE KQLEEAQKDY
Protein 4 (SEQ NO:56
SEIEGKLEQF ID NO:4)
VEKKVEAAEN NVSSVARREK
M49.1 (1-50) 201-250 of SEQ ID
ELYDQIADLT DKNGEYLERI Protein 4 (SEQ NO:57
GELEERQKNL
ID NO:4)
DDRSVSTNSG SVSTPYNNLL
M92 (1-50) 251-300 of SEQ ID
NEYDDLLAKH GELLSEYDAL Protein 4 (SEQ NO:58
KEKQDKNQEE
ID NO:4)
NSKNPAPAPA SAVPVKKEAT
M114 (1-50) 301-350 of SEQ ID
KLSEAELYNK IQELEEGKAE Protein 4 (SEQ NO:59
LFDKLEKVEE
ID NO:4)
EXAMPLE 3
IMMUNOGENICITY OF MULTIVALENT GAS PEPTIDE COMPOSITIONS
This example describes studies performed to characterize the
immunological properties of the multivalent immunogenic compositions.
Vaccine formulation and immunization of rabbits. The four vaccine
fusion polypeptides were mixed in equimolar amounts and adsorbed to alum
(REHYDRAGEL®, low viscosity, Reheis, Inc., Berkeley Heights, NJ). Vaccines were
formulated so that each 0.5 ml dose contained 200 μg, 400 μg, 800 μg, or 1000 μg of
protein and approximately equal amounts of alum by weight. Groups of three New
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Zealand white rabbits were immunized with the four vaccine doses via the
intramuscular route at 0, 4, and 8 weeks according to described protocols (see, e.g.,
Dale, Vaccine 17:193-200 (1999)). Serum was obtained prior to the first injection and
two weeks after the final injection.
Detection of type-specific antibodies. ELISAs were performed using
preimmune and immune rabbit sera by methods previously described (see, e.g., Dale,
Vaccine 17:193-200 (1999)). The purified recombinant dimeric M peptides or synthetic
peptides comprising the vaccine peptides were used as solid-phase antigens.
Assays for tissue cross-reactive antibodies. Immune sera were tested for
the presence of tissue cross-reactive antibodies by indirect immunofluorescence assays
using frozen sections of human heart, brain, and kidney by methods previously
described (see, e.g., McNeil et al., supra).
The four recombinant vaccine proteins that comprised the 30-valent
vaccine were purified and formulated on alum in four different concentrations. Dose
response studies were conducted using sets of three rabbits that received three injections
of 200 μg, 400 μg, 800 μg or 1,000 μg at times 0, 4 weeks and 8 weeks. Sera obtained 2
weeks after the final injection were assayed by ELISA using the vaccine subunit
peptides as test antigens. Recombinant dimeric peptides or synthetic peptides were used
as the source for the antigens bound to the plate. Synthetic peptides used in the assay
were M44, M78, M118, M83, M81, M87, and M49 peptides. A dose-response was
determined by calculating the average geometric mean antibody titer for the three
antisera against the vaccine subunit peptides: 200 μg dose group = 5651 average
antibody titer, 400 μg = 8868, 800 μg = 9619, and 1,000 μg = 8071. A graph depicting
these data is provided in Figure 3. None of the immune sera contained antibodies that
cross-reacted with human brain, kidney or heart, as determined by indirect
immunofluorescence assays. Based on the dose-response curve, all subsequent
experiments were performed using antisera from rabbits that received the 800 μg dose
of vaccine, which was highly immunogenic and evoked significant levels of antibodies
against each subunit peptide contained in the vaccine as shown in Figure 4A.
Indirect bactericidal tests. Bactericidal assays were performed as
previously described (see, e.g., Hu et al., supra). Briefly, 0.05 ml of Todd-Hewitt broth
containing bacteria were added to 0.1 ml of test serum and 0.35 ml of blood and the
mixture was rotated for three hours at 37 C. Then 0.1 ml aliquots of this mixture were
added to melted sheep’s blood agar, pour plates were prepared, and viable organisms
(CFU) were counted after overnight incubation at 37 C. For each serotype tested, three
different inocula were used to assure that the growth in blood containing preimmune
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serum was optimal and quantifiable. The results were expressed as percent killing,
which was calculated using the following formula: [(CFU after three hours growth with
preimmune serum) – (CFU after three hours growth with immune serum) ‚ CFU after
three hours growth with preimmune serum] x 100. Only those assays that resulted in
growth of the test strain to at least seven generations in the presence of preimmune
serum were used to express percent killing in the presence of immune serum.
The 800 g dose of vaccine elicited significant levels of bactericidal
antibodies against the vaccine serotypes of GAS, as determined by in vitro
opsonophagocytic killing assays in whole human blood. Results are presented in Figure
4B. Bactericidal killing of >50% was observed with 28/30 vaccine serotypes of GAS
using serum from one of the immunized rabbits. With respect to the two vaccine
serotypes with less than 50% killing, bactericidal killing was between 35-40% (see
Figure 4B, GAS M serotypes 77 and 44). The average killing observed against all
vaccine serotypes was 83%.
EXAMPLE 4
BACTERICIDAL ACTIVITY OF IMMUNE SERA AGAINST NON-VACCINE SEROTYPES
This example describes that the immune sera from animals immunized
with the 30-valent vaccine composition exhibited bactericidal activity against GAS
serotypes not represented in the vaccine. These data are contrasted with data obtained
using immune sera obtained from animals immunized with a previously described 26-
valent vaccine.
The GAS M serotypes represented in the 30-valent vaccine that were not
contained in the 26-valent vaccine are M4, M29, M73, M58, M44, M78, M118, M82,
M83, M81, M87, and M49. Serotypes represented in the 26-valent vaccine that are not
components of the 30-valent vaccine are M1.2, M43, M13, M59, M33, M101, and
M76. See Hu et al., supra; U.S. Patent No. 7,270,827. Certain serotypes, such as M1
serotype, were subtyped prior to the currently used emm-typing system (see Facklam et
al., Emerg. Infect. Dis. 5:247-53 (1999)). The serotype designated M1.2 has significant
amino acid variation from serotype M1.0 and under the newer typing criteria would
likely not be considered an M1 serotype (see, e.g., Dale et al., Clin. Diagn. Lab.
Immunol. 12:833-36 (2005)).
Bactericidal activity of antisera against non-vaccine serotypes.
Bactericidal assays were performed as described in Example 3. The immune sera
against the 30-valent vaccine (see Example 3) were also used in indirect bactericidal
assays against a panel of GAS serotypes that were not represented in the vaccine. In a
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first experiment as shown in Figure 5A, bactericidal activity of >50% was observed
against 11/21 serotypes randomly selected from an internal laboratory collection. The
average observed for the 11 serotypes that displayed >50% killing was 80% killing and
for all 21 serotypes tested was 50%. The immune sera lacked bactericidal activity
against only 4 of the 21 non-vaccine serotypes tested.
In a second experiment, a greater number of GAS serotypes not
represented in the vaccine were included in a bactericidal activity assay. As shown in
Figure 5B, bactericidal activity of >50% was observed against 33/47 serotypes selected
from the laboratory collection. The average observed for the 33 serotypes that displayed
>50% killing was 80% killing and for all 47 serotypes tested was 63%. The immune
sera lacked bactericidal activity against only 4 of the 47 non-vaccine serotypes tested.
Comparison of bactericidal antibody activity evoked by the 30-valent vs.
the 26-valent vaccine against non-vaccine serotypes. To determine if the cross-opsonic
antibodies evoked by the 30-valent vaccine could potentially afford broader protection
against infections then the previously reported 26-valent vaccine (see Hu et al., supra;
U.S. Patent No. 7,270,827), bactericidal assays were performed with both antisera in the
same experiment. Twenty-one GAS serotypes not included in either the 30-valent or
26-valent vaccines were randomly chosen for inclusion in this experiment. The results
are presented in Figure 6. The level of bactericidal killing promoted by the 30-valent
vaccine against 10 serotypes that were not represented in either vaccine construct was
significantly greater than that observed with the 26-valent antiserum (average 64.9% vs.
23.6%, p=0.0008, Student's t-test, paired, two-tailed).
The various embodiments described above can be combined to provide
further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent
applications, foreign patents, foreign patent applications, and non-patent publications
referred to in this specification and/or listed in the Application Data Sheet are
incorporated herein by reference, in their entirety. Aspects of the embodiments can be
modified, if necessary to employ concepts of the various patents, applications, and
publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the
above-detailed description. In general, in the following claims, the terms used should
not be construed to limit the claims to the specific embodiments disclosed in the
specification and the claims, but should be construed to include all possible
embodiments along with the full scope of equivalents to which such claims are entitled.
Accordingly, the claims are not limited by the disclosure.
SHR510264NZPR
303884893
The reference to any prior art in the specification is not, and should not
be taken as, an acknowledgement or any form of suggestion that the prior art
forms part of the common general knowledge in New Zealand.
Claims (40)
1. An immunogenic composition comprising at least 31 immunogenic peptides, wherein each immunogenic peptide is different and comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein or a Spa protein, wherein each different M protein is independently selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18, and wherein the immunogenic composition induces an immune response against at least each of GAS 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118 serotypes.
2. The immunogenic composition of claim 1, wherein at least four of the different immunogenic peptides are linked in tandem to form a fusion polypeptide.
3. The immunogenic composition of claim 1 or claim 2 comprising a first fusion polypeptide, a second fusion polypeptide, a third fusion polypeptide, and a fourth fusion polypeptide that each comprises at least six of the different immunogenic peptides linked in tandem.
4. The immunogenic composition of claim 3, wherein the first fusion polypeptide comprises eight of the different immunogenic peptides linked in tandem, wherein each of the eight immunogenic peptides comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein or the Spa protein, wherein each different M protein is independently selected from the M protein of GAS serotype 1, 2, 3, 6, 12, 18, and 28, and the Spa protein is from GAS serotype 18.
5. The immunogenic composition of claim 3 or 4, wherein the immunogenic peptide located at the carboxy terminal end of the first fusion polypeptide is a duplicate of the immunogenic peptide located at the amino terminal end of the first fusion polypeptide.
6. The immunogenic composition of claim 5, wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous amino acids from the amino terminal portion of the M protein of GAS serotype 1.
7. The immunogenic composition of any one of claims 3 to 6, wherein the second fusion polypeptide comprises eight of the different immunogenic peptides linked in tandem, and wherein each of the eight immunogenic peptides comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein independently selected from the M protein of GAS serotype 4, 5, 11, 14, 19, 24, 29, and 75.
8. The immunogenic composition of any one of claims 3 to 7, wherein the immunogenic peptide located at the carboxy terminal end of the second fusion polypeptide is a duplicate of the immunogenic peptide located at the amino terminal end of the second fusion polypeptide.
9. The immunogenic composition of claim 8, wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous amino acids from the amino terminal portion of the M protein of GAS serotype 4.
10. The immunogenic composition of any one of claims 3 to 9, wherein the third fusion polypeptide comprises eight of the different immunogenic peptides linked in tandem, and wherein each of the eight immunogenic peptides comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein independently selected from the M protein of GAS serotype 22, 44, 58, 73, 77, 78, 89, and 118.
11. The immunogenic composition of any one of claims 3 to 10, wherein the immunogenic peptide located at the carboxy terminal end of the third fusion polypeptide is a duplicate of the immunogenic peptide located at the amino terminal end of the third fusion polypeptide.
12. The immunogenic composition of claim 11, wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous amino acids from the amino terminal portion of the M protein of GAS serotype 77.
13. The immunogenic composition of any one of claims 3 to 12, wherein the fourth fusion polypeptide comprises seven of the different immunogenic peptides linked in tandem, and wherein each of the seven immunogenic peptides comprises at least 25 contiguous amino acids from the amino terminal portion of a different M protein independently selected from the M protein of GAS serotype 49, 81, 82, 83, 87, 92, and 114.
14. The immunogenic composition of any one of claims 3 to 13, wherein the immunogenic peptide located at the carboxy terminal end of the fourth fusion polypeptide is a duplicate of the immunogenic peptide located at the amino terminal end of the fourth fusion polypeptide.
15. The immunogenic composition of claim 14, wherein the immunogenic peptide that is duplicated comprises at least 25 contiguous amino acids from the amino terminal portion of the M protein of GAS serotype 83.
16. The immunogenic composition of any one of claim 1 to 15, wherein each of the immunogenic peptides comprises (a) the at least 25 contiguous amino acids from the amino terminal portion of the different M protein or the Spa protein in duplicate; (b) at least 40 contiguous amino acids from the amino terminal portion of the different M protein or the Spa protein; (c) at least 45 contiguous amino acids from the amino terminal portion of the different M protein or the Spa protein; or (d) at least 50 contiguous amino acids from the amino terminal portion of the different M protein or the Spa protein.
17. An immunogenic composition comprising: (a) a first fusion polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) a second fusion polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) a third fusion polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:3; and (d) a fourth fusion polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:4, wherein the immunogenic composition induces an immune response against at least each of GAS 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118 serotypes.
18. The immunogenic composition of claim 17 wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4.
19. The immunogenic composition of claim 17 or 18, wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4.
20. The immunogenic composition of any one of claims 17 to 19, wherein (a) the first fusion polypeptide comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion polypeptide comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:3; and (d) the fourth fusion polypeptide comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:4.
21. The immunogenic composition of any one of claims 17 to 20, wherein (a) the first fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1; (b) the second fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2; (c) the third fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3; and (d) the fourth fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:4.
22. The immunogenic composition of any one of claims 1 to 21, further comprising a pharmaceutically acceptable excipient.
23. The immunogenic composition of any one of claims 1 to 22, further comprising a pharmaceutically acceptable adjuvant.
24. The immunogenic composition of any one of claims 1 to 23, wherein the immune response against group A streptococcus comprises an immune response against at least each of GAS 8, 9, 15, 25, 30, 33, 36, 40, 48, 51, 52, 53, 59, 60, 65, 66, 68, 76, 79, 85, 94, 95, 97, 102, 105, 109, 111, and 122 serotypes.
25. The immunogenic composition of any one of claims 1 to 23, wherein the immune response against group A streptococcus comprises an immune response against GAS serotype 8, 30, 36, 51, 63, 71, and 102.
26. Use of the immunogenic composition of any one of claims 1 to 25 in the manufacture of a vaccine for inducing an immune response against group A streptococcus in a subject.
27. Use of the immunogenic composition of any one of claims 1 to 25 in the manufacture of a vaccine for reducing the likelihood of occurrence of a group A streptococcus infection in a subject.
28. The immunogenic composition of any one of claims 1 to 25 for use in preventing or treating a group A streptococcus infection.
29. Use of the immunogenic composition of any one of claims 1 to 25 in the manufacture of a vaccine to prevent or treat a group A streptococcus infection.
30. A fusion polypeptide comprising: (a) an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:3; or (d) an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:4, wherein the fusion polypeptide induces an immune response against group A streptococcus.
31. The fusion polypeptide of claim 30 comprising: (a) an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:3; or (d) an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:4.
32. The fusion polypeptide of claim 30 or 31 comprising: (a) an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:3; or (d) an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:4.
33. The fusion polypeptide of any one of claims 30 to 32 comprising: (a) an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:1; (b) an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:3; or (d) an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO:4.
34. The fusion polypeptide of any one of claims 30 to 33 comprising: (a) the amino acid sequence set forth in SEQ ID NO:1; (b) the amino acid sequence set forth in SEQ ID NO:2; (c) the amino acid sequence set forth in SEQ ID NO:3; or (d) the amino acid sequence set forth in SEQ ID NO:4.
35. An isolated polynucleotide encoding the fusion polypeptide of any one of claims 30 to 34.
36. A recombinant expression vector comprising the isolated polynucleotide of claim 35 operatively linked to at least one expression control region.
37. An isolated host cell transfected, transduced, or transformed with the recombinant expression vector of claim 36.
38. A process for producing a fusion polypeptide, said method comprising: (a) culturing the isolated host cell of claim 37; and (b) isolating the fusion polypeptide from the host cell culture.
39. An in vitro method for detecting an antibody that specifically binds to the fusion polypeptide of any one of claims 30 to 34 in a biological sample suspected of containing the antibody, said method comprising: (a) contacting the biological sample with (i) an immunogenic peptide comprising at least 25 contiguous amino acids of the amino terminal portion of an M protein or a Spa protein, wherein the M protein is selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18; (ii) a dimeric peptide, wherein each peptide of the dimeric peptide is different and comprises at least 25 contiguous amino acids of the amino terminal portion of a different M protein or a Spa protein, wherein each different M protein is independently selected from the M protein of group A streptococcus (GAS) serotype 1, 2, 3, 4, 5, 6, 11, 12, 14, 18, 19, 22, 24, 28, 29, 44, 49, 58, 73, 75, 77, 78, 81, 82, 83, 87, 89, 92, 114, and 118, and the Spa protein is from GAS serotype 18; or (iii) the fusion polypeptide of any one of claims 31-35; and (b) detecting specific binding of the immunogenic peptide, dimeric peptide, or fusion polypeptide with the biological sample, thereby indicating that the biological sample contains the antibody.
40. An immunogenic composition as claimed in claim 1, 17 or 28 substantially as herein before described with particular reference to the examples,
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161498397P | 2011-06-17 | 2011-06-17 | |
US61/498,397 | 2011-06-17 | ||
US201261641448P | 2012-05-02 | 2012-05-02 | |
US61/641,448 | 2012-05-02 | ||
PCT/US2012/042782 WO2012174455A2 (en) | 2011-06-17 | 2012-06-15 | Group a streptococcus multivalent vaccine |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ618243A NZ618243A (en) | 2015-08-28 |
NZ618243B2 true NZ618243B2 (en) | 2016-04-06 |
Family
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