U.S. patent application number 13/931028 was filed with the patent office on 2014-09-11 for fusion proteins and methods of use.
This patent application is currently assigned to VaxInnate Corporation. The applicant listed for this patent is VaxInnate Corporation. Invention is credited to Uma Kavita, Hong Li, Ge Liu, Xiangyu Liu, Langzhou Song, Lynda Tussey, Scott Umlauf, Bruce Weaver.
Application Number | 20140255438 13/931028 |
Document ID | / |
Family ID | 49162234 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140255438 |
Kind Code |
A9 |
Song; Langzhou ; et
al. |
September 11, 2014 |
FUSION PROTEINS AND METHODS OF USE
Abstract
Compositions that include a fusion protein comprising flagellin
and at least one antigen that has an isoelectric point greater than
about 7.0 and that is fused to at least one domain 3 of the
flagellin activate Toll-like Receptor 5. Methods of stimulating an
immune response, in particular, a protective immune response
include administering a composition that includes an antigen fused
to a loop of domain 3 of flagellin.
Inventors: |
Song; Langzhou; (Cranbury,
NJ) ; Liu; Ge; (Cranbury, NJ) ; Umlauf;
Scott; (Cranbury, NJ) ; Kavita; Uma;
(Cranbury, NJ) ; Li; Hong; (Cranbury, NJ) ;
Liu; Xiangyu; (Cranbury, NJ) ; Weaver; Bruce;
(Cranbury, NJ) ; Tussey; Lynda; (Cranbury,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
VaxInnate Corporation |
Cranbury |
NJ |
US |
|
|
Assignee: |
VaxInnate Corporation
Cranbury
NJ
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20140065177 A1 |
March 6, 2014 |
|
|
Family ID: |
49162234 |
Appl. No.: |
13/931028 |
Filed: |
June 28, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61743165 |
Aug 28, 2012 |
|
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|
Current U.S.
Class: |
424/186.1 ;
530/350 |
Current CPC
Class: |
A61K 39/145 20130101;
A61K 2039/70 20130101; A61K 39/12 20130101; C07K 2319/00 20130101;
C07K 14/005 20130101; C07K 2319/40 20130101; C07K 14/255 20130101;
C12N 2760/16134 20130101; C12N 2760/16234 20130101; C12N 2760/16222
20130101; C07K 14/195 20130101; A61K 39/39 20130101; A61K 38/164
20130101; A61K 2039/6068 20130101; C12N 7/00 20130101; C07K 2319/74
20130101 |
Class at
Publication: |
424/186.1 ;
530/350 |
International
Class: |
C07K 14/255 20060101
C07K014/255; C07K 14/005 20060101 C07K014/005 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under
HHS01002011000011C from the Biomedical Advanced Research and
Development Authority. The government has certain rights in the
invention.
Claims
1. A composition that includes a fusion protein, comprising: (a) a
flagellin; and (b) at least one antigen that has an isolectric
point greater than about 7.0 and is fused to at least one loop of
domain 3 of the flagellin, wherein the fusion protein activates a
Toll-like Receptor 5.
2. The composition of claim 1, wherein the isoelectric point of the
antigen is at least one member selected from the group consisting
of about 7.5, about 8.0, about 8.5, about 9.0, about 9.5 and about
10.0.
3. The composition of claim 1, wherein the flagellin lacks at least
one of a portion of a carboxy-domain 0 and portion of an
amino-domain 0.
4. The composition of claim 1, further including an additional
antigen fused to the flagellin at a site that is distinct from
fusion of the antigen to the loop of domain 3 of the flagellin.
5. The composition of claim 1, wherein the flagellin includes at
least one member selected from the group consisting of Salmonella
typhimurium flagellin, an E coli flagellin, a S. muenchen
flagellin, a Yersinia flagellin, a P. aeruginosa flagellin and a L.
monocytogenes flagellin.
6. The composition of claim 5, wherein the flagellin is the S.
typhimurium flagellin.
7. The composition of claim 6, wherein the S. typhimurium flagellin
is at least one member selected from the group consisting of SEQ ID
NOs. 1 and 2.
8. The composition of claim 7, wherein the S. typhimurium flagellin
is SEQ ID NO: 2.
9. The composition of claim 8, wherein the antigen to fused between
amino acid residue 277 and amino acid residue 278 of SEQ ID NO:
2.
10. The composition of claim 8, wherein the antigen to fused
between amino acid residue 259 and amino acid residue 260 of SEQ ID
NO: 2.
11. The composition of claim 1, wherein the antigen is an influenza
viral antigen.
12. The composition of claim 11, wherein the influenza viral
antigen is an influenza B viral antigen.
13. The composition of claim 11, wherein the influenza viral
antigen is an influenza A antigen subtype, and the influenza A
antigen subtype is at least one member selected from the group
consisting of H3, H7 and H9.
14. The composition of claim 13, wherein the influenza A antigen
subtype is H3.
15. The composition of claim 11, wherein the influenza viral
antigen is a hemagglutinin antigen.
16. The composition of claim 15, wherein the hemagglutinin antigen
is at least one member selected from the group consisting of an
influenza A viral hemagglutinin antigen and an influenza B viral
hemagglutinin antigen.
17. The composition of claim 15, wherein the hemagglutinin antigen
is a portion of the hemagglutinin antigen that includes at least a
portion of a globular head.
18. The composition of claim 17, wherein the portion of the
hemagglutinin lacks a transmembrane domain and a cytoplasmic
domain.
19. The composition of claim 18, wherein the portion of the
hemagglutinin further lacks an HA2 subunit.
20. The composition of claim 19, wherein the portion of the
globular head has at least one .beta.-sheet at a bottom of the
portion of the globular head.
21. The composition of claim 20, wherein the portion of the
globular head further includes at least one .beta.-sandwich at the
bottom of the portion of the globular head.
22. The composition of claim 21, wherein the portion of the
globular head further includes at least two .beta.-strands at the
bottom of the portion of the globular head.
23. The composition of claim 1, further including an amino acid
linker between at least one of an amino-terminus or a
carboxy-terminus of the antigen and the loop of domain 3 of the
flagellin.
24. A composition comprising at least three fusion proteins that
each activate a Toll-like Receptor 5, wherein: (a) a first fusion
protein includes a first flagellin and a first influenza A viral
hemagglutinin antigen that has an isoelectric point greater than
about 6.0 fused to a portion of the first flagellin; (b) a second
fusion protein includes a second flagellin and a second influenza A
viral hemagglutinin antigen that is distinct from the first
influenza A viral hemagglutinin antigen and that has an isoelectric
point greater than about 7.0 fused to a portion of the second
flagellin; and (c) a third fusion protein includes a third
flagellin and a first influenza B viral hemagglutinin antigen that
has an isolectric point greater than about 8.0 and is fused to at
least one loop of domain 3 of the third flagellin.
25. The composition of claim 24, further including at least one
adjuvant.
26. The composition of claim 24, wherein: (a) the first influenza A
viral hemagglutinin antigen includes at least a portion or the
entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head; (b) the second influenza A viral
hemagglutinin antigen includes at least a portion or the entirety
of an HA1 subunit having at least a portion of a globular head that
includes at least one .beta.-sheet, at least one .beta.-sandwich
and at least two .beta.-strands at the bottom of the portion of the
globular head; and (c) the first influenza B viral hemagglutinin
antigen includes at least a portion or the entirety of an HA1
subunit that has at least a portion of a globular head that
includes at least one .beta.-sheet at a bottom of the globular
head.
27. The composition of claim 24, further including a fourth fusion
protein that activates a Toll-like Receptor 5, the fourth fusion
protein comprising a second influenza B viral hemagglutinin antigen
that is distinct from the first influenza B viral hemagglutinin
antigen and that has an isoelectric point greater than about 8.0
fused to at least one loop of domain 3 in the fourth flagellin.
28. The composition of claim 27, wherein the second influenza B
viral hemagglutinin antigen includes at least a portion or the
entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head.
29. A method of stimulating an immune response to an antigen in a
subject, comprising the step of administering to the subject a
composition that includes at least one fusion protein that
activates Toll-like Receptor 5, the fusion protein comprising a
flagellin and at least one antigen that has an isolectric point
greater than about 7.0 and that is fused to at least one loop of
domain 3 of the flagellin.
30. The method of claim 29, wherein the fusion protein of the
composition administered to the subject is in a dose of at least
one member selected from the group consisting of about 1 .mu.g,
about 2 .mu.g, about 3 .mu.g, about 4 .mu.g, about 5 .mu.g, about 6
.mu.g, about 7 .mu.g, about 8 .mu.g, about 9 .mu.g, about 10 .mu.g
dose, about 15 .mu.g dose, about 20 .mu.g dose, about 25 .mu.g
dose, about 30 .mu.g dose, about 35 .mu.g dose, about 40 .mu.g
dose, about 45 .mu.g dose and about 50 .mu.g dose.
31. The method of claim 29, wherein the composition is administered
to the subject in a single dose.
32. The method of claim 29, wherein the composition is administered
to the subject in multiple doses.
33. The method of claim 29, wherein the antigen is an influenza
viral antigen.
34. The method of claim 29, wherein the influenza viral antigen is
at least a portion of hemagglutinin.
35. The method of claim 29, wherein the composition includes an
adjuvant.
36. A method of stimulating an immune response in a subject,
comprising the step of administering to the subject a composition
comprising at least three fusion proteins each that activate a
Toll-like Receptor 5, wherein: a) a first fusion protein includes a
first flagellin and a first influenza A viral hemagglutinin antigen
that has an isoelectric point greater than about 6.0 fused to a
portion of the first flagellin; (b) a second fusion protein
includes a second flagellin and a second influenza A viral
hemagglutinin antigen that is distinct from the first influenza A
viral hemagglutinin antigen and that has an isoelectric point
greater than about 7.0 fused to a portion of the second flagellin;
and (c) a third fusion protein includes a third flagellin and a
first influenza B viral hemagglutinin antigen that has an
isolectric point greater than about 8.0 and is fused to at least
one loop of domain 3 of the third flagellin.
37. The method of claim 36, wherein: (a) the first influenza A
viral hemagglutinin antigen administered to the subject includes at
least a portion or the entirety of an HA1 subunit that has at least
a portion of a globular head that includes at least one
.beta.-sheet at a bottom of the globular head; (b) the second
influenza A viral hemagglutinin antigen administered to the subject
includes a at least a portion or the entirety of an HA1 subunit
having at least a portion of a globular head that includes at least
one .beta.-sheet, at least one .beta.-sandwich and at least two
.beta.-strands at the bottom of the portion of the globular head;
and (c) the first influenza B viral hemagglutinin antigen
administered to the subject includes at least a portion or the
entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head.
38. The method of claim 36, wherein the composition administered to
the subject further includes a fourth fusion protein that activates
a Toll-like Receptor 5, the fourth fusion protein comprising a
second influenza B viral hemagglutinin antigen that is distinct
from the first influenza B viral hemagglutinin antigen and that has
an isoelectric point greater than about 8.0 fused to at least one
loop of domain 3 in the fourth flagellin.
39. The method of claim 38, wherein the second influenza B viral
hemagglutinin antigen administered to the subject includes at least
a portion or the entirety of an HA1 subunit that has at least a
portion of a globular head that includes at least one .beta.-sheet
at a bottom of the globular head.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/743,165, filed on Aug. 28, 2012. The entire
teachings of the above application are incorporated herein by
reference.
INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE
[0003] This application incorporates by reference the Sequence
Listing contained in the following ASCII text file being submitted
concurrently herewith: [0004] a) File name:
37101054001SEQUENCELISTING.txt; created Jun. 28, 2013, 1,173 KB in
size.
BACKGROUND OF THE INVENTION
[0005] Compositions that include antigens can be employed to
stimulate immunity to disease consequent to or infection from
exposure to an organism that includes the antigen. Antigens
employed in compositions to stimulate immunity, specifically
protective immunity to a particular disease, often resemble the
disease-causing organism from which the antigen is a source.
Compositions that include antigens may also include adjuvants that
augment the immune response to the antigen to maximize the
production of antibodies that neutralize the related antigen in a
disease-causing organism. Recombinant DNA technology has been
employed to generate fusion proteins that include antigens and
Toll-like Receptor agonists, specifically, flagellin, which is a
Toll-like Receptor .delta. agonist, to augment an immune response
to the antigen. However, not all antigens are suitable for existing
formats of fusion proteins to flagellin. Therefore, a need exists
for new and improved designs of fusion protein that include
flagellin and an antigen for use in methods of stimulating an
immune response, specifically a protective immune response to the
antigen.
SUMMARY OF THE INVENTION
[0006] The present invention generally relates to compositions that
include fusion proteins that stimulate an immune response,
specifically a protective immune response in a subject, and methods
of using the fusion proteins.
[0007] In an embodiment, the invention is a composition that
includes a fusion protein comprising a flagellin and at least one
antigen that has an isoelectric point greater than about 7.0 and is
fused to at least one loop of domain 3 of the flagellin. The fusion
protein of the invention activates a Toll-like Receptor 5.
[0008] In another embodiment, the invention is a composition that
includes a fusion protein comprising a flagellin and at least one
antigen that has an isoelectric point greater than about 7.5 and is
fused to at least one loop of domain 3 of the flagellin. The fusion
protein of the invention activates a Toll-like Receptor 5.
[0009] In another embodiment, the invention is a composition
comprising at least three fusion proteins that each activate a
Toll-like Receptor 5, wherein: (a) a first fusion protein includes
a first flagellin and a first influenza A viral hemagglutinin
antigen that has an isoelectric point greater than about 6.0 fused
to a portion of the first flagellin; (b) a second fusion protein
includes a second flagellin and a second influenza A viral
hemagglutinin antigen that is distinct from the first influenza A
viral hemagglutinin antigen and that has an isoelectric point
greater than about 7.0 fused to a portion of the second flagellin;
and (c) a third fusion protein includes a third flagellin and a
first influenza B viral hemagglutinin antigen that has an
isolectric point greater than about 8.0 and is fused to at least
one loop of domain 3 of the third flagellin.
[0010] In yet another embodiment, the invention is a composition
comprising at least four fusion proteins that each activate a
Toll-like Receptor 5, wherein: (a) a first fusion protein includes
a first flagellin and a first influenza A viral hemagglutinin
antigen that has an isoelectric point greater than about 6.0 fused
to a portion of the first flagellin; (b) a second fusion protein
includes a second flagellin and a second influenza A viral
hemagglutinin antigen that is distinct from the first influenza A
viral hemagglutinin antigen and that has an isoelectric point
greater than about 7.0 fused to a portion of the second flagellin;
(c) a third fusion protein includes a third flagellin and a first
influenza B viral hemagglutinin antigen that has an isolectric
point greater than about 8.0 and is fused to at least one loop of
domain 3 of the third flagellin; and (d) a fourth fusion protein
that activates a Toll-like Receptor 5 includes a second influenza B
viral hemagglutinin antigen that is distinct from the first
influenza B viral hemagglutinin antigen and that has an isoelectric
point greater than about 8.0 fused to at least one loop of domain 3
in the fourth flagellin.
[0011] In another embodiment, the invention is a method of
stimulating an immune response to an antigen in a subject,
comprising the step of administering to the subject a composition
that includes a fusion protein that activates a Toll-like Receptor
5, the fusion protein comprising a flagellin and at least one
antigen that has an isoelectric point greater than about 7.0 and
that is fused to at least one loop of domain 3 of the
flagellin.
[0012] In still another embodiment, the invention is a method of
stimulating an immune response to an antigen in a subject,
comprising the step of administering to the subject a composition
that includes a fusion protein that activates a Toll-like Receptor
5, the fusion protein comprising a flagellin and at least one
antigen that has an isoelectric point greater than about 7.5 and
that is fused to at least one loop of domain 3 of the
flagellin.
[0013] In yet another embodiment, the invention is a method of
stimulating an immune response in a subject, comprising the step of
administering to the subject a composition that includes at least
three fusion proteins each of which activates a Toll-like Receptor
5, wherein: (a) a first fusion protein includes a first flagellin
and a first influenza A viral hemagglutinin antigen that has an
isoelectric point greater than about 6.0 fused to a portion of the
first flagellin; (b) a second fusion protein includes a second
flagellin and a second influenza A viral hemagglutinin antigen that
is distinct from the first influenza A viral hemagglutinin antigen
and that has an isoelectric point greater than about 7.0 fused to a
portion of the second flagellin; and (c) a third fusion protein
includes a third flagellin and a first influenza B viral
hemagglutinin antigen that has an isolectric point greater than
about 8.0 and is fused to at least one loop of domain 3 of the
third flagellin.
[0014] In still another embodiment, the invention is a method of
stimulating an immune response in a subject, comprising the step of
administering to the subject a composition that includes at least
four fusion proteins each of which activates a Toll-like Receptor
5, wherein: (a) a first fusion protein includes a first flagellin
and a first influenza A viral hemagglutinin antigen that has an
isoelectric point greater than about 6.0 fused to a portion of the
first flagellin; (b) a second fusion protein includes a second
flagellin and a second influenza A viral hemagglutinin antigen that
is distinct from the first influenza A viral hemagglutinin antigen
and that has an isoelectric point greater than about 7.0 fused to a
portion of the second flagellin; (c) a third fusion protein
includes a third flagellin and a first influenza B viral
hemagglutinin antigen that has an isolectric point greater than
about 8.0 and is fused to at least one loop of domain 3 of the
third flagellin; and (d) a fourth fusion protein that activates a
Toll-like Receptor 5 includes a second influenza B viral
hemagglutinin antigen that is distinct from the first influenza B
viral hemagglutinin antigen and that has an isoelectric point
greater than about 8.0 fused to at least one loop of domain 3 in
the fourth flagellin.
[0015] The compositions and methods of the invention can be
employed to stimulate an immune response, in particular, a
protective immune response in the subject. Advantages of the
claimed invention include, for example, the production of fusion
proteins that have antigens with isoelectric points greater than
about 7.0 that stimulate an adaptive immune response to the antigen
sufficient to generate antibodies that provide protective immunity
to a disease-causing organism that includes the antigen. The
compositions of the invention provide an appropriate balance of
innate and adaptive immune stimulating properties for use in
vaccines that include antigens having an isoelectric point greater
than about 7.0.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 depicts the hemagglutinin (HA) trimer, the full
length HA1 subunit monomer with an HA1 globular head subunit
(membrane distal), and an HA2 membrane subunit (proximal stalk
subunit). Portions of the HA1 subunit, referred to as "HA1-1" and
"HA1-2" are depicted. The portion of HA referred to as "HA1-3" has
been employed as a negative control and lacks sufficient secondary
structure to essentially maintain the tertiary structure of the
globular head of the portion of HA.
[0017] FIG. 2A shows that a fusion protein (SEQ ID NO: 122) that
includes an R3 format of flagellin and a portion of HA (SEQ ID NO:
45), without a negatively charged linker (SEQ ID NO: 123) between
the HA antigen and flagellin, did not induce an immunogenic
response to the HA component of the fusion protein. Serum HAI
antibody titers were measured by HAI test against B/Florida/4/06,
and plotted as individual values with geometric mean titers (GMTs,
horizontal lines) with 10 .mu.g ( ) or 3 .mu.g (.box-solid.) of
HL199 (SEQ ID NO: 45) or with 10 .mu.g (.tangle-solidup.) or 3
.mu.g () of HL098 (SEQ ID NO: 122). F147 formulation buffer was
used a negative control (.diamond-solid.). BV (baculovirus)
expressed HA0 (.largecircle., full-length HA) was used as a
positive control.
[0018] FIG. 2B shows that the inclusion of an amino acid linker
with a net negative charge (SEQ ID NO: 123) in the fusion protein
depicted in FIG. 2A (SEQ ID NO: 122) improved immunogenicity to the
HA component of the fusion protein. Serum antibody titers were
measured by MN test against B/Florida/4/06 virus, and plotted
individually with GMTs (horizontal lines) with HL199 ( , SEQ ID NO:
145) or HL352 (, SEQ ID NO: 125). Control groups included the
formulation buffer (+, F147, negative) and BV (baculovirus)
expressed HA0 (*, full-length HA, positive).
[0019] FIGS. 3A and 3B show in vivo TLR5 stimulated cytokine
production: Comparison of fusion proteins for BNFL (Yamagata
lineage) compositions: R3.HA1-2 B (.box-solid., HL098, SEQ ID NO:
122) and R3.HA1-2 B with a 9 amino acid linker B (.largecircle.,
HL352, SEQ ID NO: 125). R3.HA1 ( , HL185, SEQ ID NO: 143) was
included as a positive control. Dotted lines indicate (1) mean+3
standard deviations (SD) of naive mice (.sub.------); (2) mean+1 SD
of HL077 ( . . . , SEQ ID NO: 146); or (3) mean+3 SD of HL185 (_._,
, SEQ ID NO: 143). IL-6 (FIG. 3A) and TNF (FIG. 3B) levels of
individual mice are shown, along with bars representing the mean
and standard error.
[0020] FIGS. 4A and 4B show in vivo TLR5 stimulated cytokine
production: Comparison of fusion proteins for B/BR (Victoria
lineage) compositions: R3.HA1-2 B (, HL169, SEQ ID NO: 147) and
R3.HA1-2 B with a 9 amino acid linker B (.box-solid., HL483, SEQ ID
NO: 148). R3.HA1 ( , HL185, SEQ ID NO: 143) was included as a
positive control. Dotted lines indicate (1) mean+3 standard
deviations (SD) of naive mice (.sub.------); (2) mean+1 SD of HL077
( . . . , SEQ ID NO: 146); and (3) mean+3 SD of HL185 (_._, , SEQ
ID NO: 143). IL-6 (FIG. 4A) and TNF (FIG. 4B) levels of individual
mice are shown, along with bars representing the mean and standard
error.
[0021] FIGS. 5A and 5B show in vivo TLR5 stimulated cytokine
production: Comparison of fusion protein compositions with
additional negative charges. Dotted lines indicate thresholds of
cytokines levels correlated with immunogenicity. IL-6 (FIG. 5A) and
TNF (FIG. 5B) levels of individual mice are shown, along with bars
representing the mean and standard error. Formats for B/FL
(Yamagata lineage) were compared: R3.HA1-2 B with a 9aa (amino
acid) linker (SEQ ID NO: 123) B (.box-solid., HL352, SEQ ID NO:
125) and the same format with additional negative charges
introduced in the linker ( , HL610, SEQ ID NO: 149). Formats for
B/BR (Yamagata lineage) were compared: R3.HA1-2 B with a 9aa linker
(SEQ ID NO: 123) B (.tangle-solidup., HL169, SEQ ID NO: 147) and
the same format with additional negative charges introduced in the
linker (.diamond-solid., HL611, SEQ ID NO: 150). R3.HA1 ( , HL185,
SEQ ID NO: 143) was included as a positive control and naive
(.box-solid.) as a negative control.
[0022] FIG. 6 shows similar NIA activities of the two B/FL
compositions that contain negatively charged amino acids in the
linkers: B/Florida/4/06 R3 fusion protein compositions HL352
(.largecircle., SEQ ID NO: 125) and HL610 (.quadrature., SEQ ID NO:
149).
[0023] FIG. 7 shows a comparison of NIA activities of B/BR
compositions with or without the use of additional negative charged
amino acid residues (SEQ ID NO: 153) in the portion of HA that
includes a portion of the globular head of HA: B/Brisbane/60/08 R3
fusion protein compositions HL169 (.largecircle., SEQ ID NO: 147),
HL483 (SEQ ID NO: 148) (.quadrature., with 9 amino acid linker of
SEQ ID NO: 123), and HL611 (SEQ ID NO: 150) (.DELTA., with a 9
amino acid extension and other negatively charged amino acids of
SEQ ID NO: 153).
[0024] FIGS. 8A and 8B show in vivo TLR5 stimulated cytokine
production: Comparison of fusion protein compositions: B/FL
(Yamagata lineage) R3.HA B (.box-solid., HL352, SEQ ID NO: 125) and
D3Ins.HA B (.tangle-solidup., HL656, SEQ ID NO: 151) and B/BR
(Victoria lineage) R3.HA B (.tangle-solidup., HL169, SEQ ID NO:
147) and D3Ins.HA B (, HL657, SEQ ID NO: 128). R3.HA1 ( , HL185,
SEQ ID NO: 143) was included as a positive control. Dotted lines
indicate thresholds of cytokines levels correlated with
immunogenicity. IL-6 (A) and TNF (B) levels of individual mice are
shown, along with bars representing the mean and standard
error.
[0025] FIGS. 9A through 9C show a comparison of NIA activities of
fusion protein compositions of B/Florida/4/06 (.largecircle.,
HL352, SEQ ID NO: 125 and .quadrature., HL656, SEQ ID NO: 151) (A),
B/Brisbane/60/08 (.quadrature., HL611, SEQ ID NO: 150 and
.largecircle., HL657, SEQ ID NO: 128) (B), or B/Wisconsin/1/10
(.tangle-solidup., HL724, SEQ ID NO: 152 and .largecircle., HL772,
SEQ ID NO: 126) (C).
[0026] FIG. 10 shows D3Ins B/Fl composition elicits better MN
titers than fusion proteins that include HA fused to R3 formats of
flagellin: HL352 (SEQ ID NO: 125) ( , R3 with 9 an extension of SEQ
ID NO: 123), HL610 (SEQ ID NO: 149) (.largecircle., R3 with 9 amino
acid linker and additional negatively charged amino acids of SEQ ID
NO: 153), or HL656 (SEQ ID NO: 151) (.largecircle., D3Ins with 9
amino acid linker of SEQ ID NO: 123). Data are shown as titers of
individual mice with GMTs and seroconversion rates (percent (%)
mice with a MN titer greater than about or equal to 80) above each
group.
[0027] FIG. 11 shows relative immunogenicity of B/Wisconsin/1/2010
formats: R3 B/WI ( , HL719, SEQ ID NO: 154, R3 with negative
linker), R3 B/WI (.box-solid., HL724, SEQ ID NO: 152, R3 construct
format with additional negative charges in the amino acid
extension), D3Ins B WI (.tangle-solidup., HL772, SEQ ID NO: 126 or
F147 buffer ( ). Data are shown as titers of individual mice with
GMTs. Statistical differences were determined in 1-way ANOVA/Tukey
test. *, p<0.05, ***, p<0.001.
[0028] FIG. 12 shows HAI titers of monovalent flu B (influenza B)
compositions: R3 formats (HL611, SEQ ID NO: 150; HL753, SEQ ID NO:
155; HL742, SEQ ID NO: 156) or D3Ins format compositions of
B/Brisbane/60/08-like viruses (B/Brisbane/60/08 or B/BR, HL657, SEQ
ID NO: 128; B/Hong Hong/259/10 or B/HK, HL774, SEQ ID NO: 157; and
B/Bangladesh/5945/09 or B/BD, HL787, SEQ ID NO: 158). Controls
included FLUVIRIN.RTM. or F147 buffer. Data are shown as titers of
individual mice with GMTs. Statistical differences were determined
in 2-way ANOVA/Tukey tests, *, p<0.05, **, p<0.01.
[0029] FIGS. 13A and 13B show in vivo TLR5 stimulated cytokine
production:
[0030] Comparison of variations of D3Ins format fusion proteins: B
Wisconsin (Yamagata lineage) D3I-o1 (HL772, SEQ ID NO: 126), D3I-s1
(HL848, SEQ ID NO: 160), and D3I-i1 (HL849, SEQ ID NO: 159). R3.HA1
(HL185, SEQ ID NO: 143) was included as a positive control.
[0031] FIG. 14 depicts the structure of flagellin, domains 0, 1, 2
and 3 and insertion sites in flagellin (SEQ ID NO: 2) for
generation of fusion proteins that include antigens. Reference to
amino acid numbers are made to SEQ ID NO: 2.
[0032] FIGS. 15A and 15B show the in vivo TLR5 stimulated cytokine
production: Comparison of variations of D3I and D2I fusion protein
constructs: B Wisconsin (Yamagata lineage) D3I-o1 (HL772, SEQ ID
NO: 126), D2I-o1 (HL825, SEQ ID NO: 161), D2I-o2 (HL826, SEQ ID NO:
162), D2I-o3 (HL827, SEQ ID NO: 163), D1I-o1 (HL828, SEQ ID NO:
164) and D2I-i1 (HL850, SEQ ID NO: 165) and B Brisbane (Victoria
lineage) D3I-o1 (HL657, SEQ ID NO: 128), D2I-o1 (HL733, SEQ ID NO:
166), D2I-o1 (HL856, SEQ ID NO: 167) and D2I-o2 (HL857, SEQ ID NO:
168). R3.HA1 (HL185, SEQ ID NO: 143) was included as a positive
control. IL-6 (FIG. 15 A) and TNF-alpha (FIG. 15B) levels of
individual mice are shown, along with bars representing the mean
and standard error.
[0033] FIGS. 16A and 16B show in vivo TLR5 stimulated cytokine
production: Comparison of variations of D3I and D2I fusion protein
constructs: B Wisconsin (Yamagata lineage) were compared in FIG.
16A: D3I-o1 (HL772, SEQ ID NO: 126), D3I-o2 (HL888, SEQ ID NO:
169), D2I-c1 (HL890, SEQ ID NO: 170), D2I-i2 (HL892, SEQ ID NO:
171) and B Brisbane (Victoria lineage) were compared in FIG. 16B:
D3I-o1 (HL657, SEQ ID NO: 128), D2I-o3 (HL858, SEQ ID NO: 172),
D3I-s1 (HL860, SEQ ID NO: 173), D3I-o2 (HL889, SEQ ID NO: 174),
D2I-c1 (HL891, SEQ ID NO: 175) and D2I-i2 (HL893, SEQ ID NO: 176).
R3.HA1 (HL185, SEQ ID NO: 143) was included as a positive control.
The concentrations of IL6 from individual mice are shown, along
with bars representing the mean and standard error.
[0034] FIG. 17 shows NIA assay for insertion variants
(B/Wisconsin/1/2010): D3Ins (.largecircle., HL772, D3I-o1, SEQ ID
NO: 126), various D2Ins (.DELTA., HL825, D2I-o1, SEQ ID NO: 161;
.diamond., HL826, D2I-o2, SEQ ID NO: 162; , HL827, D2I-o3, SEQ ID
NO: 163), and D1Ins (.box-solid., HL828, D1I-o1, SEQ ID NO: 164),
in comparison to R3 format (.tangle-solidup., HL724, SEQ ID NO:
152).
[0035] FIGS. 18A and 18B show NIA assay for B/Wisconsin/1/2010 D2I
variants in comparison to D3I format. A) B/Wisconsin/1/2010 D3I
insertion fusion proteins: D3I-o1 (.largecircle., HL772, SEQ ID NO:
126), D3I-s1 (.DELTA., HL848, SEQ ID NO: 160), D3I-i1 (.diamond.,
HL849, SEQ ID NO: 159), and D3I (.tangle-solidup., HL864, SEQ ID
NO: 177) in comparison to the D2I-i1 ( , HL850, SEQ ID NO: 165) and
D2I (.box-solid., HL854, SEQ ID NO: 179) fusion proteins. B)
B/Wisconsin/1/2010 D3I insertion fusion proteins: D3I-o1
(.largecircle., HL772, SEQ ID NO: 126), D3I-o2 (.quadrature.,
HL888, SEQ ID NO: 169), and D3I-c1 (.DELTA., HL890, SEQ ID NO:
170), in comparison to D2I-i2 (.diamond., HL892, SEQ ID NO:
171).
[0036] FIG. 19A shows NIA assay data for (B/Brisbane/60/2008) D3I
fusion proteins: D3I-o1 (.largecircle., HL657, SEQ ID NO: 128) and
D3I-i1 (.box-solid., HL861, SEQ ID NO: 180), compared to the R3
(.quadrature., HL611, SEQ ID NO: 150) fusion protein.
[0037] FIG. 19B shows NIA assay for B/Brisbane/60/2008 R3
(.quadrature., HL611, SEQ ID NO: 150) fusion protein in comparison
to fusion protein insertion variants: D3I-o1 (.largecircle., HL657,
SEQ ID NO: 128), D2I-o1 (.DELTA., HL733, SEQ ID NO: 166), D2I-o1
(.box-solid., HL856, SEQ ID NO: 167), D2I-o2 (.tangle-solidup.,
HL857, SEQ ID NO: 168), and D2I-i1 (.diamond-solid., HL862, SEQ ID
NO: 181).
[0038] FIG. 20 shows NIA assay for B/Brisbane/60/2008 D3I fusion
proteins: D3I-o1 (.largecircle., HL657, SEQ ID NO: 128) and D2I-o3
(.quadrature., HL858, SEQ ID NO: 172), in comparison to D2I fusion
proteins: D3I-s1 (.DELTA., HL860, SEQ ID NO: 173), D3I-o2 (HL889,
SEQ ID NO: 174), D2I-c1 (HL891, SEQ ID NO: 175), D2I-i2 (HL893, SEQ
ID NO: 176), and D2I-i1 (HL862, SEQ ID NO: 181).
[0039] FIGS. 21A and 21B show HAI studies expressed as GMT in mice
to treatment with A) B/Wisconsin/1/2010 (B/WI) fusion protein
compositions comprising D3I-o1, HL772, SEQ ID NO: 126; D3I-i1,
HL849, SEQ ID NO: 159 and D2I-i1, HL850, SEQ ID NO: 165 or B)
B/Sichuan/379/1999 D3I-o1 (HL863, SEQ ID NO: 182) or D3I-i1 (HL903,
SEQ ID NO: 183). Positive (HA HU and HA0 WI) and negative F147
(buffer) controls were also included. Seroconversion rates
(percentage of mice show a HAI greater than about or equal to 40)
are also given. ***, p<0.001 vs F147 group in ANOVA test.
[0040] FIG. 22 shows HAI titers of mouse sera following treatment
with B/Wisconsin/1/2010 (D3Ins B/WI, HL772, SEQ ID NO: 126);
B/Hubei-Wujiagang/158/2009 (D3Ins B/HU HL869, SEQ ID NO: 184) and
B/Texas/6/2011 (D3Ins B/TX HL871, SEQ ID NO: 185). Horizontal lines
represent GMTs (numbers above). Seroconversion rates are shown.
[0041] FIG. 23 shows HAI titers elicited by D3Ins fusion protein
compositions of B Brisbane or Bangladesh in rabbits. FLUVIRIN.RTM.
was included as a positive control, and F147 buffer was a negative
control. Data are shown as titers of individual rabbits with bars.
Horizontal lines represented GMTs (numbers above). Seroconversion
percentage is shown.
[0042] FIG. 24 shows HAI titers of mouse sera following treatment
with fusion protein composition STF2D3InsB/SI (HL863, SEQ ID NO:
182) or formulation buffer (F147). Data were plotted individually
with GMTs and seroconversion indicated above.
[0043] FIGS. 25A and 25B show efficacy of treatment with fusion
protein D3Ins B/SI 99 (HL863, SEQ ID NO: 182) compositions in mice
challenged with B/Sichuan/379/99 virus. Survival rates (FIG. 25A)
and weights (mean percentage of initial weight) (FIG. 25B) are
shown.
[0044] FIG. 26 shows HAI Titers elicited by fusion proteins R3 B
Wisconsin (HL724, SEQ ID NO: 152) and D3Ins B Wisconsin (HL772, SEQ
ID NO: 126). CBER HA was included as a positive control and F147
buffer was included as a negative control. Data are shown as titers
of individual rabbits with bars, and numbers representing geometric
means and seroconversion percentages.
[0045] FIG. 27 shows HAI titers of rabbit sera following treatment
with fusion protein STF2D3Ins B/WI (HL772, SEQ ID NO: 126),
positive control Hubei virus, or formulation buffer (F147). Values
are plotted individually with GMTs and seroconversion.
[0046] FIGS. 28A-28C show reactogenicity of the HL772 fusion
protein (SEQ ID NO: 126) or buffer control (F147) in rabbits.
Groups of 10 rabbits were immunized once with the dose indicated on
the x-axis. FIG. 28A depicts food consumption measured about 24
hours after immunization. FIG. 28B depicts temperature measured
rectally about 6 hours post-immunization. FIG. 28C depicts CRP was
measured from serum taken about 24 hours after prime after the
initial immunization (also referred to as "prime"). Data for all
measures are shown as results of individual rabbits with lines
representing means and standard error of the mean. Dotted lines
represent the safety threshold calculated using the data from
formula control rabbits.
[0047] FIG. 29 depicts loops in domain 3 for S. typhimurium FliC
(SEQ ID NO: 1) based on a known crystal structure and predicted
loops in S. typhimurium FljB (SEQ ID NO: 2).
[0048] FIG. 30 depicts predicted insertion sites in domains 0, 1, 2
and 3 of S. typhimurium FljB (SEQ ID NO: 2) for fusion with
antigens, including insertion sites in a loop of domain 3. Domain 0
is predicted at amino acid residues 1-46 and amino acid residues
465-506; Domain 1 is predicted at amino acid residues 47-176 and
amino acid residues 415-464; Domain 2 is predicted at amino acid
residues 177-190 and amino acid residues 292-414; and Domain 3 is
predicted at amino acid residues 191-291. The amino acid number of
boundaries between domains 0, 1, 2 and 3 and insertion sites are
also indicated.
[0049] FIG. 31 depicts the domains (D0, D1, D2, D3) of a flagellin
construct (SEQ ID NO: 283) and a fusion protein that includes, in
sequence, the amino-domain 0, the amino-domain 1, the amino-domain
2, the carboxy-domain 2, the carboxy-domain 1 and the
carboxy-domain 0 of flagellin. The antigen (Ag) is inserted between
the amino-terminus and carboxy-terminus of domain 2 of the
flagellin construct. The flagellin construct is referred to herein
as "the R3 construct" (SEQ ID NO: 283) and lacks a domain 3, which
is present in the naturally occurring flagellin (SEQ ID NO: 2).
[0050] FIG. 32 depicts the domains (D0, D1, D2, D3) of a flagellin
construct (SEQ ID NO: 284) and a fusion protein that includes, in
sequence, the amino-domain 0, the amino-domain 1, the amino-domain
2, the carboxy-domain 2, the carboxy-domain 1 and the
carboxy-domain 0 of the flagellin. Two antigens (Ag) are in the
fusion protein. An antigen is fused between the amino-domain 2 and
the carboxy-domain 2 of the flagellin construct. Another antigen is
fused to the carboxy-terminal amino acid of the Domain 0 of the
flagellin construct. The flagellin construct is referred to herein
as "the R3-2xAg" construct, which lacks a domain 3 of the naturally
occurring flagellin (SEQ ID NO: 2).
DETAILED DESCRIPTION OF THE INVENTION
[0051] The features and other details of the invention, either as
steps of the invention or as combinations of parts of the
invention, will now be more particularly described and pointed out
in the claims. It will be understood that the particular
embodiments of the invention are shown by way of illustration and
not as limitations of the invention. The principle features of this
invention can be employed in various embodiments without departing
from the scope of the invention.
[0052] The invention is generally directed to compositions that
include fusion proteins having an antigen with an isoelectric point
greater than about 7.0 that activate Toll-like Receptor 5 and use
of the fusion proteins in compositions to stimulate immune
responses in a subject, such as protective immune responses.
[0053] A threshold of TLR5 signaling must be reached to promote the
immunogenicity of fusion proteins that include flagellin and
antigens. However, excess TLR5 signaling heightens proinflammatory
cytokine response that can elicit systemic reactogenicity (e.g.,
unwanted side-effects, such as fever, weight loss) in vivo. Fusion
proteins that include flagellin must activate TLR5 signaling to
promote an adaptive immune response to the antigen with an
isoelectric point greater than about 7.0, while simultaneously
remaining below a threshold of TLR5 signaling that results in
unacceptable reactogenicity.
[0054] Mouse and rabbit animal models to assess the minimal TLR5
activity required for a fusion protein that includes flagellin to
be immunogenic (mouse model) and the maximal TLR5 activity
associated with a well tolerated composition that includes a fusion
protein of flagellin for use in a vaccine (rabbit model) have been
developed, as described herein. In the mouse TLR5 activity model,
when a mouse is immunized with about 1 .mu.g of a fusion protein,
the fusion protein must elicit at least about 32 pg/ml of IL6 and
about 80 pg/ml of TNF in the serum about 3 hours post immunization
to be considered immunogenic. In the rabbit model, a dose that
elicits levels of food consumption that are greater than about 1
standard deviation below the mean of a buffer control, rises in
body temperature that are greater than about 3 standard deviations
away from the mean of the control and rises in C-reactive protein
(CRP) that are greater than about 10 standard deviations away from
the mean of the buffer control are likely to be reactogenic (e.g.,
have negative, unwanted side effects) in a human. The dose range
for which a composition of the invention would be considered useful
as a vaccine remains non-reactogenic in the rabbit model and is
described as the "safety window." The dose range for which a
composition of the invention would be considered useful as a
vaccine is immunogenic, yet remains non-reactogenic in preclinical
and clinical models is described as the "therapeutic window." The
fusion proteins properly balance activation of TLR5 signaling and
adaptive immune responses to the antigen to achieve a therapeutic
window that is acceptable for use in methods to stimulate
protective immunity.
[0055] Full length flagellin, without fusion to any antigen, or
flagellin fused to an antigen at the carboxy-terminus of the
flagellin have the narrowest safety window for use in compositions
to stimulate immune responses in subjects because such compositions
trigger a maximal TLR5 signaling response. Antigens with an
isoelectric point less than about 7.0 fused to R3 constructs of
flagellin, in which domain 3 of flagellin is entirely replaced with
an antigen (U.S. application Ser. No. 12/905,584), have a wider
safety window, believed to be due, in part, to the ability of the
antigen to sterically hinder the fusion protein binding to TLR5 to
thereby rendering it a "partial agonist." Partial agonist activity
of R3 flagellin fusion proteins have been described for A/Puerto
Rico/08/34 and A/California/07/09 (Taylor, et al., Vaccine
30:5761-5769 (2012)).
[0056] There must be sufficient partial TLR5 agonist activity for a
fusion protein that includes flagellin to generate an immune
response to the antigen component. As described herein, antigens
with isoelectric points greater than about 7.0 when fused to R3 and
R32x constructs of flagellin, result in decreased TLR5 signaling in
the mouse model of TLR5 activity (decreased stimulation of adaptive
immune responses), and, thus, are poorly immunogenic. Antigens with
isolectric points greater than about 7.0 may result in
intramolecular interactions between the negatively charged
flagellin and the antigen. Fusion of an antigen with an isoelectric
point greater than about 7.0 (e.g., about 7.0, about 7.5, about
8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about
11.0, about 11.5) to at least one loop in domain 3 of flagellin, is
believed to form a configuration that maximizes the distance of the
antigen from the TLR5 binding domain of flagellin to thereby
preserve the ability of the fusion protein to activate TLR5 by
flagellin binding to TLR5.
[0057] In an embodiment, compositions of the invention include a
fusion protein comprising a flagellin and at least one antigen that
has an isoelectric point greater than about 7.5 and that is fused
to at least one loop of domain 3 of the flagellin. The fusion
protein of the invention activates the Toll-like Receptor 5.
[0058] Fusion of the antigen to a loop of domain 3 of flagellin
essentially retains domain 3 of flagellin in its tertiary
structure. The phrase "essentially retains domain 3 of flagellin in
its tertiary structure," as used herein, refers to maintenance of
the tertiary structure of domain 3 of flagellin, which can be
assessed by well-established in vivo and in vitro assays described
herein that are known to one of ordinary skill in the art,
including the ability of flagellin to activate TLR5 and to assess
protective immunity.
[0059] "Fusion protein," as used herein, refers to a protein
generated from at least two distinct components, a flagellin or a
portion of a flagellin and an antigen or a portion of an antigen
having an isoelectric point greater than or equal to 7.0, including
antigens with an isoelectric point of at least one member selected
from the group consisting of about 7.0, about 7.5, about 8.0, about
8.5, about 9.0, about 9.5, about 10, about 10.5 and about 11.0.
[0060] Fusion proteins of the invention can be generated
recombinantly or by chemical conjugation using well-established
techniques. A recombinant fusion protein can be generated by
operably linking a nucleic acid sequence encoding a flagellin, or a
portion of a flagellin that includes a domain 3, to a nucleic acid
sequence encoding an antigen, such as an antigen that is at least a
portion of an influenza viral antigen. Fusion proteins of the
invention can include, for example, one, two, three, four or five,
antigens fused to, for example, one, two, three, or four loops of
domain 3 of one or more flagellin.
[0061] The isoelectric point (pI) of a protein is the pH at which a
particular antigen carries no net charge. The isoelectric point of
the antigen fused to the flagellin can be at least one member
selected from the group consisting of about 7.0, about 7.5, about
8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5 and
about 11.0.
[0062] The flagellin in the fusion proteins of the invention can be
an S. typhimurium flagellin (UniProt accession number P06179 or
P52616), such as an S. typhimurium flagellin selected from the
group consisting of SEQ ID NOS: 1 and 2; E. coli flagellin (UniProt
accession number A0PCV8), such as, for example, SEQ ID NO: 3; P.
aeruginosa flagellin (UniProt accession number P72151), such as SEQ
ID NO: 4; Aquifex aeolicus VF5 flagellin (UniProt accession number
067803), such as SEQ ID NO: 5; Helicobacter pylori J99 Flagellin A
(UniProt accession number P0A052), such as SEQ ID NO: 6; and
Legionella pneumophila flagellin (UniProt accession number Q48824),
such as SEQ ID NO: 7.
[0063] The flagellin employed in the fusion proteins of the
invention can lack at least a portion of a carboxy-domain 0 or a
portion of an amino-domain 0, for example, at least one member
selected from the group consisting of about 5, about 10, about 14,
about 15 and about 20 amino acids of the carboxy-domain 0 or a
portion of an amino-domain 0. As shown in FIG. 14, flagellin
includes a carboxy-domain 0, a carboxy-domain 1, a carboxy-domain
2, a domain 3, an amino-domain 2, an amino-domain 1, and an
amino-domain 0. When flagellin assumes its tertiary structure,
domains 2 and 3 form a juncture in which the flagellin folds on
itself so that the most carboxy-amino acid of the carboxy-domain 0
of flagellin is adjacent to the most amino-amino acid of the
amino-domain 0 of flagellin. Crystallographic studies of flagellin
have identified the secondary and tertiary structure of S.
typhimurium FliC flagellin (SEQ ID NO: 1) (PDB code: 1UCU).
[0064] The phrase "a loop of domain 3 of flagellin," as used
herein, refers to a stretch of amino acids within domain 3 of
flagellin that is, itself, devoid of secondary structures (e.g.,
.beta.-sheets, .alpha.-helices), yet flanks adjacent stretches of
amino acids in domain 3 that include secondary structures, such as
.beta.-sheets, .alpha.-helices. Loops of domain 3 in flagellin can
be about 2, about 3, about 4, about 5, about 6, about 7 and between
about 5 to about 30 amino acids in length.
[0065] Flagellin (FljB) from Salmonella typhimurium is depicted in
SEQ ID NO: 2. Domain 3 of Salmonella typhimurium flagellin is
between amino acid residue 191 and amino acid residue 291 of SEQ ID
NO: 2. Flagellin from E. coli (UniProt accession number A0PCV8) is
depicted in SEQ ID NO: 3. Domain 3 of E. coli flagellin of SEQ ID
NO: 3 is predicted between amino acid residue 191 and amino acid
residue 283 of SEQ ID NO. 3. P. aeruginosa flagellin (UniProt
accession number P72151) is depicted in SEQ ID NO: 4 with domain 3
predicted between amino acid residue. Flagellin from Aquifex
aeolicus VF5 (UniProt accession number 067803) is depicted in SEQ
ID NO: 5. Domain 3 of Aquifex aeolicus flagellin is predicted
between amino acid residue 197 and amino acid residue 302 of SEQ ID
NO. 5. The flagellin A from Helicobacter pylori J99 (UniProt
accession number P0A052) is depicted in SEQ ID NO: 6. Domain 3 of
Helicobacter pylori J99 of SEQ ID NO: 6 is predicted between amino
acid residue 189 and amino acid residue 283 of SEQ ID NO: 6. The
flagellin from Legionella pneumophila (UniProt accession number
Q48824) is depicted in SEQ ID NO: 7. Domain 3 of Legionella
pneumophila flagellin of SEQ ID NO: 7 is predicted between amino
acid residue 189 and amino acid residue 283 of SEQ ID NO: 7.
[0066] X-ray crystallography of Salmonella typhimurium FliC
flagellin (SEQ ID NO: 1) shows that domain 3 of flagellin includes
6 loops (FIG. 29). The loops in domain 3 of Salmonella typhimurium
flagellin SEQ ID NO: 1 are from amino acid residues 211 to 212
(loop 1); amino acid residues 217 to 219 (loop 2); amino acid
residues 223 to 229 (loop 3); amino acid residues 237 to 242 (loop
4); amino acid residues 250 to 255 (loop 5) and amino acid residues
259 to 275 (loop 6).
[0067] FIG. 29 identifies the predicted loops (gray) in domain 3 of
Salmonella typhimurium FljB flagellin (SEQ ID NO: 2) and compares
the location in the sequence to the loops in domain 3 of Salmonella
typhimurium FliC flagellin (SEQ ID NO: 1). Darkly shaded arrows
depict secondary structures in domain 3 of S. typhimurium FliC,
such as .beta.-sheets and .alpha.-helices.
[0068] FIG. 30 depicts the amino acid sequence of Salmonella
typhimurium FljB flagellin (SEQ ID NO: 2), boundaries of domain 0,
1, 2 and 3, and potential sites of fusion of antigens having
isoelectric points greater than about 7.0 in loops of domain 3 of
the flagellin. In an embodiment, the site of fusion of an antigen
having an isoelectric point greater than about 7.0 is D3I-i1 (also
referred to as "D3Ins-i1") between amino acid residues 259 and 260
of SEQ ID NO: 2 in loop 5 of domain 3 (see FIG. 29). In another
embodiment, the site of fusion of an antigen having an isoelectric
point greater than about 7.0 is D3I-o1 (also referred to as
"D3Ins-o1") between amino acid residues 277 and 278 of SEQ ID NO: 2
in loop 6 of domain 3 (see FIG. 29).
[0069] In another embodiment, the D3I-o1 site of fusion of an
antigen having an isoelectric point greater than about 7.0 is
between amino acid residues 274 and 275 of SEQ ID NO: 4 in
predicted loop 6 of domain 3. In still another embodiment, the
D3I-o1 site of fusion of an antigen having an isoelectric point
greater than about 7.0 is between amino acid residues 274 and 275
of SEQ ID NO: 5 in predicted loop 6 of domain 3.
[0070] In another embodiment, the D3I-i1 site of fusion of an
antigen having an isoelectric point greater than about 7.0 is
between amino acid residues 258 and 259 of SEQ ID NO: 4 in
predicted loop 5 of domain 3. In still another embodiment, the
D3I-o1 site of fusion of an antigen having an isoelectric point
greater than about 7.0 is between amino acid residues 260 and 261
of SEQ ID NO: 5 in predicted loop 5 of domain 3.
[0071] The antigen can be fused to domain 3 of flagellin to
generate constructs referred to as D3I-o1 and D3I-i1. "D3I-o1," as
used herein, refers to insertion into a loop of domain 3 (Domain 3
Insertion) on the outer (o) or concave surface of flagellin, such
as loop 6 OF SEQ ID NO: 2 or SEQ ID NO: 1. "D3I-i1," as used
herein, refers to a loop of domain 3 on the inner (i) or convex
surface of flagellin, such as loop 3 of SEQ ID NO: 2 and SEQ ID NO:
1. The tertiary structure of flagellin and portions of flagellin
that would be considered concave and convex surfaces of flagellin
are described, for example, by Samatey, et al., Nature 410:331-337
(2001).
[0072] The designation "c," with respect to an insertion of an
antigen into a loop of domain 3 of flagellin (D3I-c1), refers to a
"side-way" portion of domain 3 (FIG. 14).
[0073] The designation "s," with respect to an insertion of an
antigen into a loop of domain 3 of flagellin (D3I-s1), means the
"tip" of domain 3 of flagellin (FIG. 14).
[0074] Exemplary fusion proteins of the invention are shown in SEQ
ID NOS: 126, 128-130, 151, 157-193.
[0075] The junction of the flagellin component and antigen
component of fusion proteins of the invention results in a sequence
of unique amino acids. For fusion proteins employed in methods of
the invention to treat humans, if this unique sequence of amino
acids at the juncture of the fusion of the flagellin component and
the antigen component shares homology with a known human protein,
the fusion protein has the potential to elicit an unwanted immune
response to the human protein or a portion of the human protein.
Upon selection of an insertion site in flagellin (e.g., a loop of
domain 3, an adjacent portion of the carboxy- or amino-terminus of
domain 2 of an R3 construct), the sequence of unique amino acids
that would be created by fusion of the antigen to flagellin is
assessed for its potential ability to elicit an unwanted immune
response. In the evaluation, a probe of about 10 to about 12 amino
acids in length, which includes the flagellin antigen junction is
used to probe a database of known human genome sequences. If
homology is identified for a stretch of amino acids greater than
about 5 amino acids, then the junction sequence is modified with an
amino acid substitution of, for example, 1, 2, 3, 4, 5, or 6 amino
acids to decrease the homology. The order of preferred amino acids
for use in the modification is serine, threonine, alanine and
glycine. Generally, a single amino acid substitution is sufficient
to modify the homology. For example see SEQ ID NO: 283 for the
native FljB sequence without domain 3. For example, in an R3
construct of flagellin, Arg405 of SEQ ID NO: 283 can be modified to
an alanine residue to generate SEQ ID NO: 284. Alternatively,
Ala191 of SEQ ID NO: 283 can be modified to a serine residue,
Arg405 of SEQ ID NO: 283 can be modified to an alanine and a serine
residue can be added to the modified alanine residue (i.e., the
most carboxy-terminus amino acid) to generate SEQ ID NO: 285. An
exemplary fusion protein with modified amino acid residues at the
junction of fusion of the antigen and the flagellin is SEQ ID NO:
268, where an HA1-2 portion of SEQ ID NO: 286 is fused to a
flagellin of SEQ ID NO: 285. In SEQ ID NO: 268, the junctions of
the fusion of the antigen and the flagellin are at Ala190 and
Gly191; Ser413 and Ser414; and Ala628 and Ser629 with Gly630.
[0076] In an embodiment, the flagellin employed in the fusion
protein of the invention lacks at least one member selected from
the group consisting of at least a portion of the carboxy-domain 0
and at least a portion of an amino-domain 0.
[0077] In another embodiment at least one additional antigen is
fused to the flagellin at a site that is distinct from fusion of
the antigen to a loop of domain 3 of the flagellin. The additional
antigen can be an antigen similar to the antigen fused to at least
one loop of domain 3 of the flagellin. Alternatively, the
additional antigen can be an antigen that is different (also
referred to herein as "distinct") from the antigen that is fused to
at least one loop of domain 3 of the flagellin. For example, at
least a portion of an influenza hemagglutinin antigen, such as an
HA1-1 or an HA1-2 portion, can be fused to a loop of domain 3
(e.g., between amino acid 277 and amino acid 278 of loop 3 of SEQ
ID NO: 2) of flagellin and a different portion of an influenza
hemagglutinin antigen, such as HA1-1L, can be fused to a different
loop of domain 3, such as between amino acids 259 and 260 of loop 5
of SEQ ID NO: 2.
[0078] In another embodiment, the site of insertion of an antigen
in a loop of domain 3 of flagellin can be 1, about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10,
amino acids towards the carboxy-domain 2 or an amino-domain 2 of
flagellin from the insertion sites identified in FIGS. 14, 29 and
30. For example, with reference to SEQ ID NO: 2, a loop of domain 3
to which the antigen is fused is between amino acid residues 277
and 278 (e.g., D3I-o1) (FIG. 30). Alternatively, fusion of the
antigen can occur between amino acid 266 and 282 of SEQ ID NO:
2.
[0079] Likewise, with reference to SEQ ID NO: 2, a loop of domain 3
to which the antigen is fused is between amino acid residues 259 to
260 (D3I-i1) or amino acid residues 260 to 261. The insertion site
in a loop of domain 3 in the D3I-i1 fusion protein of SEQ ID NO: 2
is between amino acid residues 190 to 191 or between amino acid
residues 291 to 292.
[0080] In another embodiment, about 2 to about 4 amino acid
residues in the loop of domain 3 can be deleted prior to fusion
with the antigen with an isoelectric point greater than about 7.0.
The deletions would be designed so that adjacent secondary
structures in the flagellin would not be disrupted. Such deletions
in at least one loop of domain 3 of flagellin may be employed when
fusing flagellin to relatively large antigens.
[0081] In a preferred embodiment, the flagellin for use in the
fusion proteins is a flagellin that includes at least one member
selected from the group consisting of SEQ ID NOS: 1-7. The antigen
is fused between amino acid residue 191 and amino acid residue 285
of SEQ ID NO: 1, which is within a loop of domain 3 of
flagellin.
[0082] In a particular embodiment, the antigen that is fused to a
loop of domain 3 of flagellin is an influenza viral antigen, in
particular, an influenza B viral antigen.
[0083] In another embodiment the influenza viral antigen is at
least a portion of an influenza A antigen subtype (H3, H7, H5 or
H9). The influenza A antigen subtype can be at least one member
selected from the group consisting of the H3, H5, H7 and H9. An
additional influenza A antigen subtype can be at least one member
selected from the group consisting of an H1 and an H2 subtype, (H1
subtype or H2 subtype).
[0084] In a particular embodiment, the influenza viral antigen is
hemagglutinin, in particular, a portion of a hemagglutinin antigen
that includes at least a portion of a globular head of the
hemagglutinin antigen. The portion of an influenza viral
hemagglutinin employed in the fusion proteins of the invention
preferably includes at least a portion of a globular head of
hemagglutinin that includes a sialic acid binding site. The portion
of the globular head of the hemagglutinin includes at least one
.beta.-sheet at the bottom of the globular head that causes the
globular head to essentially retain its tertiary structure.
[0085] "A sialic acid binding site," as that phrase is used herein
in reference to the portion of the protein from the naturally
occurring viral hemagglutinin, means a part of the influenza viral
hemagglutinin that has the capacity to interact with sialic acid
residues. "A sialic acid binding site" is also referred to herein
as "a sialic acid binding domain."
[0086] "At least a portion," as used herein, refers to any part of
the antigen or flagellin that is less than the entirety of the
antigen or flagellin.
[0087] "A globular head," as that phrase is used herein, refers to
a portion of a protein of an influenza viral hemagglutinin that
includes the receptor or sialic acid binding regions. "Globular
head," is also referred to herein as a "globular domain." The
globular head of viral hemagglutinin proteins has been determined
based on x-ray crystallography as described, for example, by Wilson
I. A., et al. Nature 289:366-373 (1981); Chen, J., et al., Cell
95:409-417 (1998); Ha Y., et al., The EMBO Journal 21:865-875
(2002); Russell, R. J., et al., Virology 325:287-296 (2004); and
Cox, N. J., et al., In: Toply and Wilson's Microbiology and
Microbial Infections, eds. B W J Mathy, et al., Vol. 1 (9.sup.th
ed.) New York, N.Y., Oxford Univ. Press, Ch. 32, p. 634 (1998). The
globular head of an influenza viral hemagglutinin is a component of
the HA1 subunit of influenza viral hemagglutinin. In addition to
the receptor binding domain, the globular head can include the
E.sup.-subdomain and F.sup.-subdomain as described, for example, by
Ha, Y., et al. The EMBO Journal 21:865-875 (2002).
[0088] The phrase, "causes the globular head to essentially retain
its tertiary structure," as used herein, refers to maintenance of
the tertiary structure of the globular head to thereby mimic the
tertiary structure of the globular head in the naturally occurring
influenza viral hemagglutinin, which can be assessed by the ability
to generate a sufficient immune response to stimulate a protective
immune response in a subject in vivo or viral neutralization in in
vitro assays, as described herein.
[0089] Influenza viruses are single-stranded RNA viruses that
belong to the viral family Orthomyxoviridae. Influenza viruses are
divided into three types (A, B, C) determined by the antigenic
differences in ribonucleoprotein (RNP) and matrix (M) antigens of
the viruses. Influenza A virus naturally infects humans and several
other mammalian species, including swine and horses, and a wide
variety of avian species, and causes epidemics and pandemics in the
human population. Influenza B virus appears to naturally infect
only humans and seals and can cause epidemics in humans. Influenza
C virus has been isolated from humans and swine, but generally does
not occur in epidemics and usually results in mild disease in
humans.
[0090] Mature influenza virions are enveloped with a pleomorphic
structure ranging in diameter from about 80 to about 120 nm. The
single-stranded RNA genome is closely associated with a helical
nucleoprotein and is present in seven (influenza C) or eight
(influenza A and B) separate segments of ribonucleoprotein (RNP),
each of which has to be present for successful replication of the
virus. The segmented genome is enclosed within an outer lipoprotein
envelope. Matrix protein 1 (MP1 or also referred to herein as "Ml")
lines the inside of the outer lipoprotein envelope and is bound to
the RNP.
[0091] Hemagglutinin (HA) is a surface glycoprotein on a virus
(e.g., an influenza virus) that is responsible for binding to
N-AcetylNeuraminic Acid (NeuNAc; also referred to herein as "sialic
acid") on host cells and subsequent fusion of viral and host
membranes. HA acquired its name by virtue of its ability to cause
red blood cells to clump, or agglutinate. Influenza HA consists of
the three monomeric (HA0) subunits. HA performs two critical
functions during the infection process: binding to a cell surface
sialyloligosaccharide receptor and fusion of virus and host cell
membrane. Following binding of the HA to the plasma membrane of a
host cell, the host cell membrane engulfs the virus in an endosome
and attempts to digest the contents of the endosome by acidifying
its interior and transferring it to a lysosome in the host cell.
However, the acidic environment of the lysosome destabilizes HA,
resulting in partial unfolding of HA0 which exposes a
protease-sensitive site (the maturational cleavage site) that is
cleaved by a host protease to form HA1 and HA2 subunits which are
connected by a single disulfide bond (Wiley, D. C., et al., Annu.
Rev. Biochem. 56:365-394 (1987)). Cleavage occurs at a specific
amino acid residue and generates a hydrophobic amino terminus for
the HA2 subunit. This hydrophobic terminus of HA2 mediates fusion
between the viral envelope and the endosomal membrane of the host
cell and releases the contents of the virion into the cytoplasm of
the cell, a process known as uncoating. Thus, cleavage of the HA
polypeptide is a requirement for infectivity.
[0092] The crystal structure of several viral hemagglutinins has
been determined (see, for example, Wilson, I. A., et al., Nature
289:366-373 (1981); Chen, J., et al., Cell 95:409-417 (1998); Ha,
Y., et al., The EMBO Journal 21: 865-875 (2002); Russell, R. J., et
al., Virology 325:287-296 (2004); and Cox, N. J., et al., In: Toply
and Wilson's Microbiology and Microbial Infections, eds. B. W. J.
Mathy, et al., Vol. 1 (9.sup.th ed.) New York, N.Y., Oxford Univ.
Press, Ch. 32, p. 634 (1998)). X-ray crystallographic structures
show that HA is folded into two structural components or domains--a
globular head and a fibrous stalk (see, for example, FIG. 1). The
globular head includes HA1, including that part of HA1 that binds
to sialic acid (also referred to as the "receptor binding site or
domain" or "sialic acid binding site or domain"), and antiparallel
.beta.-sheets. The fibrous stalk is more proximal to the viral
membrane and includes the HA2 subunit and part of HA1, including
the cleavage site between HA1 and HA2.
[0093] There are seventeen known subtypes of Influenza A HA
(H1-H17) that share between about 40 to about 60% sequence identity
(Tong, et al., Proc. Natl. Acad. Sci., 109:4269-4274 (2012)).
Influenza viruses containing all 17 HA subtypes have been isolated
from, for example, avian species (H5, H7, and H9), equine (H3 and
H7), seals (H3, H4 and H7), whales (H1 and H13) and swine (H1, H3,
and H9). Subtypes of influenza A virus are generally named
according to the particular antigenic determinants of HA (H, 17
major types) and neuraminidase (N, about 9 major types). For
example, subtypes include influenza A (H2N1), A(H3N2), A(H5N1),
A(H7N2), A(H9N2), A(H1/H0), A(H3/H0), A(H5/H0) and A(H7N9). In the
last century, three subtypes of influenza A resulted in pandemics:
H1 in 1918, 1977 and 2007; H2 in 1957 and H3 in 1968. In 1997, an
H5 avian virus and in 1999, an H9 virus resulted in outbreaks of
respiratory disease in Hong Kong.
[0094] HA from influenza type B viruses have been isolated from
humans and seals and are not divided into subtypes, although
influenza type B viruses are characterized by two antigenically
different lineages, Yamagata and Victoria. Influenza type B virus
strains from both the Yamagat and Victoria lineages typically
co-circulate. Compositions used in influenza vaccines can include
four antigens (i.e., "quadrivalent flu vaccines") that, in addition
to the two A strains, include both the circulating B strains.
Similar to influenza A HAs, X ray crystallagraphic studies of
influenza B HAs have revealed four major antigenic regions that are
located in the vicinity of the receptor binding site, which is in
the globular head of the influenza B HA (Wang, Q., et al., J.
Virol., March: 3011-3020 (2008)).
[0095] A host infected with influenza can mount an antibody
response to the globular head of HA that protects that host from
subsequent infection with the same strain of virus by blocking the
interaction between HA and the host cell, i.e., neutralizing the
infectivity of the virus. Due to the low fidelity and high rate of
influenza RNA replication, the virus is constantly experiencing
minor mutations in the HA gene that preserve the globular head
structure and host cell interaction, but may allow progeny virus to
escape immune surveillance. These point mutations are referred to
as "antigenic drift." In addition, if a single host is
simultaneously infected with two different strains of influenza A,
a new subtype of virus may emerge as a result of reassortment, or
the exchange of the RNA segments, or genes, between different
strains of influenza A viruses. The viruses emerging from
reassortment present the human immune system with a new antigenic
experience that usually results in high morbidity and mortality.
This type of drastic antigenic change is known as "antigenic
shift." Since type B influenza viruses circulate almost exclusively
in humans, these viruses cannot undergo reassortment with animal
strains and, thus, are changed only by antigenic drift.
[0096] Immunity to HA can reduce the likelihood of infection and
severity of disease if infection does occur. HA is an important
antigenic target and the efficacy of vaccines depends on the
antigenic match between the vaccine strain and the circulating
strain. Since the hemagglutinin protein readily undergoes antigenic
shift and drift in order to evade the host's immune defense,
traditional vaccines must be based on currently circulating
influenza strains and annually updated. Annual updates of influenza
vaccines are not only costly they also require significant amounts
of production time and manufacturing infrastructure. A vaccine
composition based on invariant regions of the virus may provide
broadly cross-reactive protection.
[0097] Hemagglutinin forms a trimer on the surface of the influenza
virus and infected cell. The monomeric subunits of the trimer are
depicted in FIG. 1, and include an HA1 subunit (membrane distal
globular head) and HA2 subunit (membrane proximal "stalk").
Portions of HA for use in the compositions of the inventor can
include at least a portion of the globular head, which includes the
cell surface receptor binding site and the majority of the
neutralizing antibody epitopes. Portions of HA, referred to as
HA1-1 and HA1-2 subunits, fused to flagellin (R3 and R32X
constructs of flagellin, see, for example, U.S. patent application
Ser. No. 12/905,584), have previously been employed in compositions
that provide protective immunity to viral challenge (see, for
example, U.S. Pat. No. 8,420,102). The HA1-1 and HA1-2 portions of
HA include a portion of the globular head and differ in amino acid
length and secondary structure. The HA1-1 and HA1-2 portions
include at least a portion of the globular head of HA and at least
one .beta.-strand at the bottom of the portion of the globular head
that maintains the portion of the globular head in its tertiary
structure.
[0098] When fused to R3 or R32x formats (also referred to herein as
"R3 constructs" or "R32x constructs") of flagellin, HA1-1 and HA1-2
portions of certain influenza viral antigens can provide protective
immunity against viral challenge (see, for example, U.S. Pat. No.
8,420,102). Fusion proteins that include flagellin lacking the
entirety of domain 3 and in which an HA antigen has been fused to
the flagellin in the region of flagellin that was domain 3 are
referred to as "R3 fusion proteins." "R32x fusion proteins (also
referred to "R3.2x")" are R3 fusion proteins that include a second
antigen, which is the same or distinct from the antigen in the
region of flagellin that was domain 3, fused to the most
carboxy-terminal amino acid of the carboxy-domain of flagellin
(see, for example, U.S. patent application Ser. No. 12/905,584).
For example, an HA1-1 portion can be fused in the region of
flagellin that was domain 3 and an HA1-2 portion can be fused to
the most carboxy-terminal amino acid of flagellin to form an R32x
fusion protein.
[0099] In contrast, in certain influenza A strains, R3 and R3.2x
fusion proteins that include portions of an influenza B HA, are
poorly immunogenic, as shown herein. For example, influenza B
fusion proteins that include an HA1-2 portion (SEQ ID NO: 45) of
the Yamagata lineage strain B/Florida/4/2006, fused to flagellin in
an R3 format (HL098, SEQ ID NO: 122) or an R2X format (HL118, SEQ
ID NO: 287) or Victoria lineage strain B/Brisbane/60/2008, SEQ ID
NO: 264 fused to flagellin in an R3format (HL169, SEQ ID NO: 147)
or an R32X format (HL171, SEQ ID NO: 288) failed to elicit
measurable levels of hemagglutination inhibition (HAI) titers, a
standard measure of protective immunity against influenza virus.
Every year, new multivalent blends of compositions for use as
vaccines are developed as treatments to prevent and manage disease
associated with viral influenza infection. For example, trivalent
influenza vaccine (TIV) treatments contain two different
inactivated influenza type A strains and one inactive influenza
type B strain, e.g. FLUVIRIN.RTM.. Quadrivalent vaccine (QIV)
treatments are available, e.g. FLUZONE.RTM. Quadrivalent vaccine,
which contains two different inactivated influenza type A strains
and two inactivated influenza B strains. Therefore, a need exists
to develop new and effective fusion proteins targeting influenza B
that can be included in multivalent compositions.
[0100] Influenza viral antigens fused to at least one loop of
domain 3 of flagellin can be a portion of an influenza B viral
antigen, such as a portion referred to herein as "HA1-1," "HA1-2"
and "HA1-1L." Compositions that include influenza viral
hemagglutinn antigens, such as HA1-1, HA1-2 and HA1-1L portions of
influenza hemagglutinin, can be employed in methods to treat or
prevent seasonal and pandemic influenza in subjects.
[0101] "A portion," as used herein with reference to an influenza
viral antigen, for example, an influenza viral hemagglutinin
antigen, refers to any part of the influenza viral hemagglutinin
that is less than the entirety of the influenza viral
hemagglutinin.
[0102] "HA1-1," as used herein, refers to a protein portion of a
viral hemagglutinin that includes at least about one
.beta.-sandwich that includes the substrate binding site, which
includes at least about two .beta.-sheets, at least about two to
about three short a-helixes, at least one small .beta.-sheet and at
least one additional small .beta.-sandwich at the bottom of the
molecule and at least about four disulfide bonds. The
.beta.-sandwich that includes the substrate binding site of the
HA1-1 includes at least about four .beta.-strands as the top sheet
and at least about three to about four .beta.-strands as the bottom
sheet. At least about one a-helix of the HA1-1 portion is located
by the side of .beta.-sandwich that includes the substrate binding
site and at least about one to about two are located at the bottom
of the .beta.-sandwich that includes the substrate binding site.
The small .beta.-sandwich of the HA1-1 can include at least about
two to about three .beta.-strands in each .beta.-sheet; or about
three to about four .beta.-strands. Exemplary HA1-1 protein
portions include SEQ ID NOs: 8-44. In a particular embodiment, the
HA1-1 portion of HA includes a portion of the globular head of HA
that has a .beta.-sheet and a .beta.-sandwich at the bottom of the
globular head that essentially retains the globular head in its
tertiary structure.
[0103] "HA1-2," as used herein, refers to a protein portion of a
viral hemagglutinin that includes at least about one
.beta.-sandwich that includes the substrate binding site, at least
about two to about three short a-helixes, at least about one small
.beta.-sheet at the bottom of the molecule and at least about two
disulfide bonds. A .beta.-strand in a viral hemagglutinin can
include between about two to about 15 amino acids. A small
.beta.-strand can include about two amino acids; or between about
two to about three amino acids; or between about two to four amino
acids or between about two to about five amino acids. A small
.beta.-sheet can include between about two to about three
.beta.-strands; or between about three to about four
.beta.-strands. The .beta.-sandwich that includes the substrate
binding site of HAI-2 can further include at least about four
.beta.-strands as the top sheet and at least about three to about
four .beta.-strands as the bottom sheet. At least about one a-helix
of the HA1-2 portion is located by the side of the .beta.-sandwich
that includes the substrate binding site and at least about one to
about two are located at the bottom of the .beta.-sandwich that
includes the substrate binding site. Exemplary HA1-2 protein
portions include SEQ ID NOs: 45-97 and 287. In a particular
embodiment, the HA1-2 portion of HA includes a .beta.-sheet at the
bottom of a portion of the globular head of hemagglutinin.
[0104] A single polypeptide can exhibit several types of secondary
structure. Without any stabilizing interactions, a polypeptide can
assume a random-coil conformation. However, secondary structures,
such as alpha(.alpha.)-helices and beta(.beta.)-strands, can
stabilize a protein or a portion of a protein. Lateral association
of .beta.-strands form .beta.-sheets (also referred to herein as
".beta.-pleated sheets"). Secondary structures can be located at
the surfaces of the antigen (e.g., portion of the viral
hemagglutinin) A tertiary structure of a protein is the
three-dimensional arrangement of amino acid residues. In contrast
to secondary structure, which is stabilized by, for example,
hydrogen bonds, .alpha.-helices, .beta.-strands, tertiary structure
results from hydrophobic interactions between non-polar side chains
of the antigen, such as a portion of an influenza viral
hemagglutinin. The hydrophobic interactions hold the helices
strands in random coils in a compact internal scaffold. The size
and shape of a protein can depend on its primary amino acid
sequence, as well as the number, size and arrangement of secondary
structures.
[0105] Portions of influenza viral hemagglutinins for fusion to at
least one loop of domain 3 of flagellin include at least a portion
of a globular head having secondary structures at the bottom of the
globular head that essentially retains the globular head in its
tertiary structure. Such secondary structures include at least one
.beta.-sheet; at least one .beta.-sheet and at least one
.beta.-sandwich; at least one .beta.-sheet, at least one
.beta.-sandwich and at least two .beta.-strands.
[0106] Portions of hemagglutinin employed in the composition of the
invention that are fused to at least one loop of domain 3 of
flagellin can lack a transmembrane domain and a cytoplasmic domain.
The portions of hemagglutinin employed in the compositions of the
invention can further lack at least a portion of the HA2 subunit or
the entirety of the HA2 subunit of hemagglutinin.
[0107] Antigens that have an isoelectric point greater than about
7.0 can be fused to at least one loop of domain 3 of flagellin to
form fusion proteins of the invention. The loops in domain 3, which
can be characterized as "extended regions," may accommodate amino
acid insertions of about 50 amino acids, about 100 amino acids,
about 150 amino acids, about 200 amino acids, about 250 amino
acids, about 300 amino acids, about 350 amino acids, about 400
amino acids, about 450 amino acids and about 500 amino acids in
length, including insertions between about 50 to about 100 amino
acids, between about 100 to about 250 amino acids and between about
250 amino acids to about 500 amino acids with minimal disruption of
the tertiary structure of flagellin. For example, the fusion
protein of SEQ ID NO: 126 (HL772) includes a portion of an
influenza B/WI1 that is 249 amino acids in length; the fusion
protein of SEQ ID NO: 128 (HL657) includes a portion of an
influenza B/BR60 that is 250 amino acids in length; the fusion
protein of SEQ ID NO: 129 (HL775) includes a portion of an
influenza A/PE16 that is 275 amino acids in length; and the fusion
protein of SEQ ID NO: 130 (HL1018) includes a portion of an
influenza A/AH1 that is 282 amino acids in length.
[0108] The antigen with an isoelectric point greater than about 7.0
includes secondary structures, such as at least one .beta.-sheet,
at least one .beta.-sandwich, at least one .beta.-pleat and at
least one .alpha.-helix, that maintain the antigen in its tertiary,
compact configuration when the antigen is fused to a loop of domain
3 of flagellin, thereby minimally disrupting the tertiary structure
of the loop of domain 3.
[0109] There are at least seventeen different HA antigens, the
different HA antigens are classified as subtypes and identified as
HA1 through H17. H1, H2 and H3 are found in human influenza viral
antigens. Hemagglutinin on the influenza virion is a trimer (three
copies of the HA polypeptides). The cleavage site for cell
proteases on the HA protein is located near the viral membrane. The
uncleaved form of hemagglutinin is referred to as HA0. After
cleavage by a cellular enzyme, two subunits of HA are produced,
specifically the HA1 subunit and the HA2 subunit. The two subunits
remain together on the surface of the virus particle. The HA2
subunit that is produced by cleavage contains a sequence of
hydrophobic acids referred to as a fusion peptide.
[0110] In an additional embodiment, the portion of hemagglutinin
that is fused to at least one loop of domain 3 of flagellin has at
least one .beta.-sheet at a bottom of the portion of the globular
head, such as HA1-2 portions of SEQ ID NOS: 45-97. In addition to
at least one .beta.-sheet at the bottom of a portion of the
globular head, the portion of hemagglutinin fused to at least one
loop of domain 3 of flagellin can further include at least one
.beta.-sandwich at the bottom of the globular head and, optionally,
at least two .beta.-strands at the bottom of the portion of the
globular head. Thus, the portion of influenza viral hemagglutinin
fused to at least one loop of domain 3 of flagellin can include at
least one .beta.-sheet, at least one .beta.-sandwich, and at least
two .beta.-strands at the bottom of a portion of a globular head of
influenza viral hemagglutinin, which is referred to herein a
"HA1-1L" portion of hemagglutinin. Exemplary HA1-1L portions of HA
are SEQ ID NOS: 98-121, 228 and 273-277.
[0111] Fusion proteins of the invention can include an amino acid
linker between at least one of an amino-terminus or a
carboxy-terminus of the antigen and the loop of domain 3 of the
flagellin. The amino acid linker can be between about 1 to about 10
amino acids in length, such as about 2, about 3, about 4, about 5,
about 8 or about 9 amino acids in length. Preferred amino acid
residues would include amino acid residues without side chains or
amino acid residues with small side chains, such as glycine,
alanine or serine, including combinations of glycine, serine and
alanine. For example, amino acid residues of a linker of at least
about 9 amino acids in length could be fused to the
carboxy-terminus of an antigen of SEQ ID NO: 125 (HL352) in a
fusion protein. Exemplary antigens fused to a linker are SEQ ID
NOS: 46, 48, 50, 52, 54, 56, 58, 60, 62, 89, 91, 93-97, 228,
273-277. Amino acid residues that comprise the amino acid linker
can have an isoelectric point of between about 3 and about 7 and
between about 4 and about 7. Amino acid linkers can include 2, 3, 4
or 5 negatively charged amino acid residues, such as aspartic acid
or glutamic acid. Exemplary amino acid linkers are described, for
example, in PCT/US2012/000099 (WO 2012/115715), by Song, L. et al.,
and PCT/US2012/000367 (WO 2013/066365), by Song, L., et al. It is
believed that negatively charged amino acids at or adjacent to
(about 1, 2, 3 or 4 amino acids) the amino- or carboxy-terminus of
the antigen at the site of fusion to a loop of domain 3 reduce
undesirable intramolecular interactions between the negatively
charged flagellin and a positively charged antigen thereby
tethering the antigen at the site of fusion. Amino acid linkers are
devoid of secondary structures, such as .alpha.-helices and
.beta.-sheets.
[0112] In an embodiment, the linker can include amino acid residues
that are native to the naturally occurring hemagglutinin and
adjacent to the portion of HA (e.g., HA1-1, HA1-2, HA1-1L) being
fused to a loop of domain 3, such as the 9 amino acid residues
(also referred to herein as "extension") of SEQ ID NO: 123, which
includes 2 negatively charged amino acid residues. For example, the
fusion protein of SEQ ID NO: 154 (HL719, which is an R3 construct)
includes a portion of influenza B Wisconsin construct (HA1-2 of SEQ
ID NO: 50) and 9 amino acids (SEQ ID NO: 123) of the naturally
occurring hemagglutinin, which has 2 negatively charged residues.
Exemplary antigens fused to the same linker are SEQ ID NOS: 46, 48,
52, 54, 56, 58, 60, 62, 89, 91, and 93. The fusion protein of SEQ
ID NO: 152 (HL724, which is an R3 construct) includes a portion of
an influenza B Wisconsin HA1-2 that has the 9 amino acids of SEQ ID
NO: 123 with an additional 3 negatively charged amino acid residues
in the portion of HA. Exemplary antigens fused with the same
modifications are SEQ ID NOS: 94-97.
[0113] An amino acid linker used to fuse the antigen to at least
one loop of domain 3 of flagellin can be about 1 to about 10 amino
acids in length, such as about 2 amino acids, about 3 amino acids,
about 4 amino acids, about 5 amino acids, about 6 amino acids,
about 7 amino acids, about 8 amino acids, about 9 amino acids and
about 10 amino acids in length. Preferably, amino acid linkers
include amino acid residues that have small or no side chains, such
as glycine, alanine and serine. In a particular embodiment, the
amino acid linker that would be fused to the carboxy-terminus of
the loop of domain 3 of flagellin has an overall negative charge,
such as an isoelectric point between about 2 to about 4.
[0114] In a further embodiment, the invention is a composition
comprising at least three fusion proteins that each activate a
Toll-like Receptor 5. The first fusion protein includes a first
flagellin and a first influenza A viral hemagglutinin antigen that
has an isoelectric point greater than about 6.0, such as an
isoelectric point between about 6.5 to about 7.0, fused to a
portion of the first flagellin. The second fusion protein includes
a second flagellin and a second influenza A viral hemagglutinin
antigen that is distinct from the first influenza A viral
hemagglutinin antigen and that has an isoelectric point greater
than about 7.0, such as an isoelectric point between about 7.5 to
about 8.5, about 7.8, about 8.0, about 8.5, about 9.0 and about
9.5, fused to a portion of the second flagellin. The third fusion
protein includes a third flagellin and a first influenza B viral
hemagglutinin antigen that has an isolectric point greater than
about 8.0, such as between about 8.0 to about 10.0, about 8.5,
about 9.0, about 9.5 and about 10.0, and is fused to at least one
loop of domain 3 of the third flagellin.
[0115] The composition that includes at least three fusion proteins
that activate TLR5 can further include a fourth fusion protein that
activates TLR 5. The fourth fusion protein includes a second
influenza B viral hemagglutinin antigen that is distinct from the
first influenza B antigen, and, that has an isolectric point
greater than about 8.0, such as between about 8.0 to about 10.0,
about 8.5, about 9.0, about 9.5 and about 10.0, fused to at least
one loop of domain 3 of a fourth flagellin.
[0116] In a particular embodiment, the first fusion protein of the
composition that includes at least three fusion proteins that each
activate TLR5 is a fusion protein that includes two similar
portions of an influenza A hemagglutinin that have a portion of a
globular head with a .beta.-sheet at the bottom of the globular
head (e.g., HA1-2) fused to an R32x construct of flagellin. This
first fusion protein has one HA portion fused to the portion of the
flagellin in the region where domain 3 was located and another
similar HA portion fused to the carboxy-terminus of the portion of
flagellin. An exemplary first fusion protein is SEQ ID NO: 268,
which is a portion of influenza A California/07/2009.
[0117] In a particular embodiment, the second fusion protein of the
composition that includes at least three fusion proteins that each
activate TLR5 is a fusion protein that includes a second influenza
A antigen that is distinct from the first influenza A antigen and
has an isoelectric point greater than about 7.0. The second
influenza A antigen is a portion of the globular head of HA that
has a .beta.-sheet, a one .beta.-sandwich and two .beta.-strands at
the bottom of a portion of a globular head of influenza viral
hemagglutinin (referred to herein a "HA1-1L") fused to an R3
construct of a portion of flagellin. The HA1-1L antigen replaces
domain 3 of the flagellin in an R3 construct. An exemplary second
fusion protein is SEQ ID NO: 269, which is a portion of influenza A
Perth/16/2009.
[0118] In a particular embodiment, the third fusion protein of the
composition that includes at least three fusion proteins that each
activate TLR5 is a fusion protein that includes a first influenza B
antigen that has an isoelectric point greater than about 8.0 and
that is fused to a loop of domain 3 of a portion of a third
flagellin, such as loop 6 between amino acid residues 277 and 278
of SEQ ID NO: 2. An exemplary third fusion protein is SEQ ID NO:
126 (HL772), which is a portion of influenza B
Wisconsin/1/2010.
[0119] In a particular embodiment, the fourth fusion protein of the
composition that includes at least three fusion proteins that each
activate TLR5 is a fusion protein that includes a second influenza
B antigen that has an isoelectric point greater than about 8.0 and
that is fused to a loop of domain 3 of a portion of a fourth
flagellin, such as loop 6 between amino acid residues 277 and 278
of SEQ ID NO: 2. The second influenza B antigen is distinct from
the first influenza B antigen. An exemplary fourth fusion protein
is SEQ ID NO: 158 (HL787), which is a portion of influenza B
Bangladesh/5495/2009.
[0120] "Distinct," as used herein in reference to an antigen,
protein or flagellin, means that the antigen, protein or flagellin
is different than a first or additional (second, third, fourth,
etc.) antigen, first or additional (second, third, fourth, etc.)
protein or first or additional (second, third, fourth, etc.)
flagellin. For example, an influenza A Perth/16/2009 hemagglutinin
(HA) antigen is distinct from an influenza A California/07/2009 HA
antigen. Likewise, an influenza B Wisconsin/1/2010 HA antigen is
distinct from an influenza B Bangladesh/5495/2009 HA antigen.
[0121] FIG. 31 depicts the domains (D0, D1, D2, D3) of a flagellin
construct (SEQ ID NO: 283) and a fusion protein that includes, in
sequence, the amino-domain 0, the amino-domain 1, the amino-domain
2, the carboxy-domain 2, the carboxy-domain 1 and the
carboxy-domain 0 of flagellin. An antigen (Ag) is fused between the
amino- and carboxy-domain 2 of the flagellin construct. The
flagellin construct of the fusion protein depicted in FIG. 31 lacks
the D3 domain of flagellin and is referred to herein as an "R3
construct" or the "R3 form of flagellin" or "R3 flagellin
construct" (SEQ ID NO: 283). "R3 (Replace Domain 3) construct," as
used herein, means that Domain 3 of the flagellin has been replaced
with an antigen, as previously described in U.S. patent application
Ser. No. 12/905,584. For example, an antigen can be fused between
amino acids 190 and 191 of SEQ ID NO: 283.
[0122] FIG. 32 depicts the domains (D0, D1, D2, D3) of a flagellin
construct (SEQ ID NO: 284) and a fusion protein that includes, in
sequence, the amino-domain 0, the amino-domain 1, the amino-domain
2, the carboxy-domain 2, the carboxy-domain 1 and the
carboxy-domain 0 of flagellin. For example, the fusion protein
depicted in SEQ ID NO: 284 has at least a portion of at least one
antigen and at least a portion of at least one other antigen fused
to two sites in a portion of flagellin. One antigen (also referred
to as "first antigen") is fused between the amino-domain 2 and the
carboxy-domain 2 of the flagellin construct. The other antigen,
which can be distinct from the first antigen, is fused to the
carboxy-terminal amino acid of the domain 0 of the flagellin
construct. The flagellin construct depicted in FIG. 32 lacks the D3
domain of flagellin and is referred to herein as an "R3-2xAg
construct" or the "R3-2xAg form of flagellin" or "R32x flagellin
construct" or "R32x" or the "R32x form of flagellin" or "2xR3" or
the "2xR3 form of flagellin" or the "R3/2x form of flagellin."
"R3-2xAg construct," as used herein, means that Domain 3 of the
flagellin is replaced with a one antigen and another antigen, such
as a distinct antigen, is fused to the carboxy-terminus of domain
0, as previously described in U.S. patent application Ser. No.
12/905,584. For example, one antigen can be fused between amino
acids 190 and 191 of SEQ ID NO: 284 and another antigen can be
fused to the terminal carboxy amino acid (e.g., SEQ ID NOs: 268,
287 and 288).
[0123] In still another embodiment, the first influenza A viral
hemagglutinin antigen of the compositions of the invention and for
use in the methods of the invention includes at least a portion or
the entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head; the second influenza A viral hemagglutinin
antigen of the compositions of the invention and for use in the
methods of the invention includes at least a portion or the
entirety of an HA1 subunit having at least a portion of a globular
head that includes at least one .beta.-sheet, at least one
.beta.-sandwich and at least two .beta.-strands at the bottom of
the portion of the globular head; and the first influenza B viral
hemagglutinin antigen of the compositions of the invention and for
use in the methods of the invention includes at least a portion or
the entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head. The second influenza B viral hemagglutinin
antigen of the compositions of the invention and for use in the
methods of the invention includes at least a portion or the
entirety of an HA1 subunit that has at least a portion of a
globular head that includes at least one .beta.-sheet at a bottom
of the globular head.
[0124] In a further embodiment, the first influenza A viral
hemagglutinin antigen of the compositions of the invention and for
use in the methods of the invention includes at least a portion of
an HA1 subunit that has at least a portion of a globular head that
includes at least one .beta.-sheet at a bottom of the globular
head; the second influenza A viral hemagglutinin antigen of the
compositions of the invention and for use in the methods of the
invention includes at least a portion of an HA1 subunit having at
least a portion of a globular head that includes at least one
.beta.-sheet, at least one .beta.-sandwich and at least two
.beta.-strands at the bottom of the portion of the globular head;
and the first influenza B viral hemagglutinin antigen of the
compositions of the invention and for use in the methods of the
invention includes at least a portion of an HA1 subunit that has at
least a portion of a globular head that includes at least one
.beta.-sheet at a bottom of the globular head. The second influenza
B viral hemagglutinin antigen of the compositions of the invention
and for use in the methods of the invention includes at least a
portion of an HA1 subunit that has at least a portion of a globular
head that includes at least one .beta.-sheet at a bottom of the
globular head.
[0125] In another embodiment, the invention is a method of
stimulating an immune response to an antigen in a subject. The
method includes the step of administering to the subject a
composition that includes a fusion protein that activates a
Toll-like Receptor 5, the fusion protein comprising a flagellin and
at least one antigen that has an isoelectric point greater than
about 7.0 and that is fused to at least one loop of domain 3 of the
flagellin. In a particular embodiment, the immune response that is
stimulated in the subject in response to administration of the
fusion protein provides protective immunity to a disease or an
infection consequent to exposure of an organism that includes the
antigens fused to the loop of domain 3 of flagellin. The method can
further include the administration of an adjuvant.
[0126] In yet another embodiment, the invention is a method of
stimulating an immune response in a subject, comprising the step of
administering to the subject a composition that includes at least
three fusion proteins each of which activates a Toll-like Receptor
5, wherein: (a) a first fusion protein includes a first flagellin
and a first influenza A viral hemagglutinin antigen that has an
isoelectric point greater than about 6.0 fused to a portion of the
first flagellin; (b) a second fusion protein includes a second
flagellin and a second influenza A viral hemagglutinin antigen that
is distinct from the first influenza A viral hemagglutinin antigen
and that has an isoelectric point greater than about 7.0 fused to a
portion of the second flagellin; and (c) a third fusion protein
includes a third flagellin and a first influenza B viral
hemagglutinin antigen that has an isolectric point greater than
about 8.0 and is fused to at least one loop of domain 3 of the
third flagellin.
[0127] In a further embodiment, the invention is a method of
stimulating an immune response in a subject, comprising the step of
administering to the subject a composition that includes at least
four fusion proteins each of which activates a Toll-like Receptor
5, wherein: (a) a first fusion protein includes a first flagellin
and a first influenza A viral hemagglutinin antigen that has an
isoelectric point greater than about 6.0 fused to a portion of the
first flagellin; (b) a second fusion protein includes a second
flagellin and a second influenza A viral hemagglutinin antigen that
is distinct from the first influenza A viral hemagglutinin antigen
and that has an isoelectric point greater than about 7.0 fused to a
portion of the second flagellin; (c) a third fusion protein
includes a third flagellin and a first influenza B viral
hemagglutinin antigen that has an isolectric point greater than
about 8.0 and is fused to at least one loop of domain 3 of the
third flagellin; and (d) a fourth fusion protein that activates a
Toll-like Receptor 5 includes a second influenza B viral
hemagglutinin antigen that is distinct from the first influenza B
viral hemagglutinin antigen and that has an isoelectric point
greater than about 8.0 fused to at least one loop of domain 3 in
the fourth flagellin.
[0128] In an embodiment, the method of the invention stimulates an
immune response by administering four different fusion proteins (2
fusion proteins that include influenza A antigens and two fusion
proteins that include influenza B antigens) each of which activate
TLR5 signaling. The four fusion proteins can be administered in
escalating doses. For example, an initial immunization can include
all four fusion proteins administered in the same dose (e.g., about
1 .mu.g or about 2 .mu.g), followed by a second dose that is twice
the first dose (e.g., about 2 .mu.g or about 4 .mu.g). Subsequent
doses (a third or a fourth dose) can include further increases in
the doses of fusion proteins that include influenza A to doses of
about 6 .mu.g to about 8 .mu.g, while maintaining the dose of the
fusion protein that includes influenza B at about 2 .mu.g or 4
.mu.g.
[0129] Doses of fusion proteins that include influenza A antigens
in multivalent compositions can be at least one member selected
from the group consisting of about 2 .mu.g, about 4 .mu.g, about 6
.mu.g, about 10 .mu.g, about 12 .mu.g, about 14 .mu.g, about 15
.mu.g and about 20 .mu.g doses. Doses of fusion proteins that
include influenza B antigens in multivalent compositions can be at
least one member selected from the group consisting of about 1
.mu.g, about 2 .mu.g, about 4 .mu.g, about 6 .mu.g, about 8 .mu.g
and about 10 .mu.g doses. Multivalent compositions include
compositions of the invention that have at least three fusion
proteins, such as a composition that includes two fusion proteins
having influenza A antigens and one or two fusion proteins having
influenza B antigens. The fusion protein of the composition
administered to the subject can be in a dose of at least one member
selected from the group consisting of about 1 .mu.g, about 2 .mu.g,
about 3 .mu.g, about 4 .mu.g, about 5 .mu.g, about 6 .mu.g, about 7
.mu.g, about 8 .mu.g, about 9 .mu.g, about 10 .mu.g dose, about 15
.mu.g dose, about 20 .mu.g dose, about 25 .mu.g dose, about 30
.mu.g dose, about 35 .mu.g dose, about 40 .mu.g dose, about 45
.mu.g dose and about 50 .mu.g dose.
[0130] In still another embodiment, the invention is a method of
stimulating an immune response to influenza A and influenza B
antigens in a subject. The method includes the step of
administering to the subject a composition that includes SEQ ID
NOs: 268, 269, 126, and 158.
[0131] The methods of the invention can further include the
administration of an adjuvant.
[0132] "Stimulating an immune response," as used herein, refers to
the generation of antibodies and/or T-cells to the antigen fused to
a loop of domain 3 of flagellin, such as the protein portions of
influenza B hemagglutinin (HA) (e.g., HA1-1, HA1-2, HA1-1L
proteins) described herein. Stimulating an immune response in a
subject can include the production of humoral and/or cellular
immune responses that are reactive against the antigen, such as a
viral protein, in particular, an influenza viral protein.
[0133] The compositions of the invention for use in methods to
stimulate immune responses in subjects, can be evaluated for the
ability to stimulate an immune response in a subject using
well-established methods. Exemplary methods to determine whether
the compositions of the invention stimulate an immune response in a
subject, include measuring the production of antibodies specific to
the antigen (e.g., IgG antibodies) by a suitable technique such as,
ELISA assays; the potential to induce antibody-dependent
enhancement (ADE) of a secondary infection; macrophage-like assays;
neutralization assessed by using the Plaque Reduction
Neutralization Test (PRNT.sub.80); and the ability to generate
serum antibodies in non-human models (e.g., mice, rabbits, monkeys)
(Putnak, et al., Vaccine 23:4442-4452 (2005)).
[0134] "Stimulates a protective immune response," as used herein,
means administration of the compositions of the invention that
include a fusion protein comprising, for example, an influenza B
antigen fused to at least one loop of domain 3 of flagellin that
result in production of antibodies to the protein to thereby cause
a subject to survive challenge by an otherwise lethal dose of a
viral protein, such as influenza B viral challenge. Techniques to
determine a lethal dose of a virus (e.g., an influenza B virus) are
known to one of skill in the art (see, for example,
WHO/CDS/CSR/NCS2002.5 "WHO Manual on Animal Influenza Diagnosis and
Surveillance" World Health Organization, Dept of Communicable
Disease Surveillance and Response, WHO Global Influenza Programme;
Harmon, M. W., et al., J. Clin. Microbiol. 26:333-337 (1988); Reed,
L. J., et al., Am. J. Hyg. 27:493-497 (1938); Rose, T., et al., J.
Clin. Microbiol. 37:937-943 (1999); Walls, H. H. et al., J. Clin.
Microbiol. 23:240-245 (1986); Current Protocols in Immunology,
19.11.1-19.11.32, Cottey, R., et al., John Wiley & Sons, Inc
(2001)). Exemplary techniques for determining a lethal dose can
include administration of varying doses of virus and a
determination of the percent of subjects that survive following
administration of the dose of virus (e.g., LD.sub.10, LD.sub.20,
LD.sub.40, LD.sub.50, LD.sub.60, LD.sub.70, LD.sub.80, LD.sub.90).
For example, a lethal dose of a virus that results in the death of
50% of a population of subjects is referred to as an "LD.sub.50"; a
lethal dose of a virus that results in the death of 80% of a
population of subjects is referred to herein as "LD.sub.80"; a
lethal dose of a virus that results in death of 90% of a population
of subjects is referred to herein as "LD.sub.90."
[0135] For example, determination of the LD.sub.90 can be conducted
in subjects (e.g., mice) by administering intranasally varying
doses (e.g., dilutions, such as log and half-log dilutions of
8.times.10.sup.3 egg-infectious doses (EID)) followed by an
assessment of the survival of the subjects about 14 days to about
21 days after infection with the virus. Protective immunity can be
assessed by physical appearance of the subject, general demeanor
(active), weight (initial loss of weight followed by return to a
weight about the weight of the subject prior to infection with the
virus) and survival after about 14 to about 21 days following
infection with the virus.
[0136] Assessment of stimulation of protective immunity can also be
made by employing assays that assess the ability of the antibodies
produced in response to the compositions of the invention (e.g., an
antigen, such as an influenza B viral antigen, such as a portion of
hemagglutinin) to neutralize binding of the viral protein (e.g.,
hemagglutinin protein) to a host cell (see, for example, Current
Protocols in Immunonology, 19.11.1-19.11.32, Cottey, R., et al.,
John Wiley & Sons, Inc (2001)). Assessment of stimulation of
protective immunity can also be made by employing assays that
measure the ability of antibodies to inhibit hemagglutinin binding
(see, for example, Burnett, F. M., et al., J. exp. Biol. Med. Sci.
25:227-233 (1947); Salk, J. E. J. Immunol. 49:87-98 (1944); Current
Protocols in Immunology, 19.11.1-19.11.32, Cottey, R., et al., John
Wiley & Sons, Inc (2001)).
[0137] It is believed that inhibition of hemagglutinin binding is
indicative of the ability of antibodies, formed from the
compositions and by the methods of the invention, to neutralize the
sialic acid binding sites of the naturally occurring viral
hemagglutinin ("neutralization of HA binding") and, thereby,
prevent infection of the host cell as a consequence of stimulating
a protective immune response. Inhibition or neutralization of
hemagglutinin binding is believed to correlate with an ability of
an immune response to protect against a lethal dose of virus.
[0138] Neutralization of HA binding can be assessed by in vitro
assays (See, for example, Current Protocols in Immunology
19.11.1-19.11.32, Cottey, R., et al., Suppl. 42, John Wiley &
Sons, Inc. (2001) and WHO Manual on Animal Influenza Diagnosis and
Surveillance, Webster, R., et al., pages 28-36, 48-54, 82-92
(2002)). Exemplary viral neutralization assays rely on the ability
of serum to specifically bind and prevent replication of influenza
virus in culture, such as in the Madin-Darby Canine Kidney (MDCK)
cell line. Briefly, cells are cultured in 96 well plates in the
presence of a previously titered virus and the cytopathic effect of
the replicating virus is observed under a microscope. To test
serum, serial dilutions of the serum are prepared and preincubated
with the viral stock for about 2 hours at 37.degree. C. prior to
infecting the MDCK cells. The mixture is incubated for an
additional 2 hours after which the virus/serum mixture is removed
and replaced with fresh media. The cells are grown for 4 days.
Wells are scored as positive for viral growth if at least about 50%
of the cells are dead in at least about half of the wells for a
given serum dilution. The reciprocal of the highest dilution of
serum which protects at least about half of the cells from death,
in at least about half of the wells, is considered the
neutralization titer.
[0139] Alternatively, a micro-neutralization in vitro assay can be
performed to assess neutralization of HA binding. For example,
serum is diluted and preincubated with a known titer of virus and
mixed with MDCK cells, as described above. After 2 days of
incubation, cells are washed and fixed with acetone. The plates are
developed as an ELISA using a monoclonal antibody to the influenza
nuclear antigen NP. A microneutralization titer is determined as
the reciprocal of the highest dilution which yields less than about
50% of the anti-NP reading of the virus-only control wells.
[0140] The Hemagglutination Inhibition (HAI) assay is based on the
HA antigen on the surface of the influenza virus agglutinating red
blood cells (RBC) and preventing red blood cells from
precipitating. Antibodies that specifically bind the sialic
acid-binding regions of HA prevent agglutination allowing
precipitation. The assay is performed in 96 well V bottom plates
with fresh chicken RBC. A stock of viral antigen is titered so that
about a 4-fold excess of antigen is present relative to the minimum
amount needed to prevent precipitation. The test serum, which can
be from several species including mouse, ferret, poultry or human,
is heated to about 56.degree. C. to inactivate complement. Serial
2-fold dilutions of the inactivated serum are performed and mixed
with the stock HA. After about 30 minutes at room temperature, the
RBCs are added and the plate is incubated for about 30 to about 45
minutes. Results are scored by observations: agglutination results
in cloudy wells while inhibition results in a "button" of red cells
precipitated at the bottom of the well. Controls include RBC with
no HA, which forms a button, and HA and RBC with no serum, which
remains cloudy. The HAI titer of a particular serum sample is the
reciprocal of the last dilution which prevents agglutination (i.e.,
forms a button). For example, if about a 1:128 dilution reads as a
button but the 1:256 dilution does not, the HAI titer is about
128.
[0141] Fusion proteins of the invention can be made employing
routine molecular biological techniques, as described herein. Host
cells can be transfected with nucleic acids encoding fusion
proteins of the invention. The host cells can be eukaryotic or
prokaryotic host cells. Suitable prokaryotic host cells include E.
coli, B. subtilis and Pseudomonas fluorescens.
[0142] The eukaryotic host cells employed in the methods of the
invention can include a Saccharomyces eukaryotic host cell, an
insect eukaryotic host cell (e.g., at least one member selected
from the group consisting of a Baculovirus infected insect cell,
such as Spodoptera frugiperda (Sf9) or Trichhoplusia ni (High5)
cells; and a Drosophila insect cell, such as Dme12 cells), a fungal
eukaryotic host cell, a parasite eukaryotic host cell (e.g., a
Leishmania tarentolae eukaryotic host cell), CHO cells, yeast cells
(e.g., Pichia) and a Kluyveromyces lactis host cell.
[0143] Suitable eukaryotic host cells and vectors can also include
plant cells (e.g., tomato; chloroplast; mono- and dicotyledonous
plant cells; Arabidopsis thaliana; Hordeum vulgare; Zea mays;
potato, such as Solanum tuberosum; carrot, such as Daucus carona
L.; and tobacco, such as Nicotiana tabacum, Nicotiana benthamiana
(Gils, M., et al., Plant Biotechnol J. 3:613-20 (2005); He, D. M.,
et al., Colloids Surf B Biointerfaces, (2006); Huang, Z., et al.,
Vaccine 19:2163-71 (2001); Khandelwal, A., et al., Virology.
308:207-15 (2003); Marquet-Blouin, E., et al., Plant Mol Biol
51:459-69 (2003); Sudarshana, M. R., et al. Plant Biotechnol J.
4:551-9 (2006); Varsani, A., et al., Virus Res, 120:91-6 (2006);
Kamarajugadda S., et al., Expert Rev Vaccines 5:839-49 (2006); Koya
V, et al., Infect Immun. 73:8266-74 (2005); Zhang, X., et al.,
Plant Biotechnol J. 4:419-32 (2006)).
[0144] The fusion proteins of the invention can be purified and
characterized employing well-known methods (e.g., gel
chromatography, cation exchange chromatography, SDS-PAGE), as
described herein.
[0145] For large scale production, fermentation techniques can be
employed. Exemplary fermentation techniques can include a proposed
cycle that can start with a culture inoculated into 6 L of MRBR
media, as described herein, held at about 30.degree. C., about pH
7, and DO controlled to greater than about 30%. A 6 liter feed can
then be started at least about 30 minutes after glucose exhaustion.
The proposed 6 liter feed media, when combined with 6 L of MRBR
media, can provide the necessary conditions for E. coli growth
based on about 52% utilization of carbon for growth. The feed may
or may not include IPTG. The batch can be induced with at least 2
mM IPTG, introduced as a bolus, shortly after the feed is started
to initiate production. The feed rate can start at about 20 mL feed
per hour per liter bioreactor volume and increase over time based
on the ability of the culture to accept more glucose without
glucose accumulation. The culture can be harvested when the feed is
complete. The 6 liter feed media, about pH 6.0, can include Glucose
180 g/L; KH.sub.2PO.sub.4 2 g/L; NaH.sub.2PO.sub.4 (H.sub.2O) 4
g/L; (NH.sub.4).sub.2HPO.sub.4 12 g/L; (NH.sub.4).sub.2HSO.sub.4 4
g/L; DL-Alanine 40 g/L; Citric Acid 4 g/L; MgSO.sub.4(7H.sub.2O)
5.5 g/L; Trace Metals 6 mL; CaCl.sub.2 2.5 g/L; FeSO.sub.4
7H.sub.2O 1 g/L.
[0146] Cell disruption and clarification in a large scale
production can include removal of Triton X-100 from the
resuspension buffer; dissolution of insolubles by the addition of
50 mM Tris, 25 mM NaCl, 8 M urea, about pH 8 to the lysate;
addition of PEI (polyethylamine) and subsequent removal by
centrifugation with one or more of the buffers to remove nucleic
acids and/or aid in filtration; the addition of flocullants, such
as AEROSIL 380, AEROSIL 200, ALKOXIDE ALU C, and CELPUR; and
subsequent removal by centrifugation to aid in filtration. Cation
exchange chromatography can include the use of a process resin,
adding a denaturing endotoxin removal step containing up to 8 M
urea and up to about 2% Triton X-100, and a step gradient elution.
The step elution gradient can include about 100 to about 200 mM
NaCl.
[0147] In an additional embodiment, the invention includes a
protein, polypeptide or peptide having at least about 70%, at least
about 75%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 98% and at least about 99%
sequence identity to the proteins, polypeptides and peptides of the
invention.
[0148] The percent identity of two amino acid sequences (or two
nucleic acid sequences) can be determined by aligning the sequences
for optimal comparison purposes (e.g., gaps can be introduced in
the sequence of a first sequence). The amino acid sequence or
nucleic acid sequences at corresponding positions are then
compared, and the percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % identity=# of identical positions/total # of
positions.times.100). The length of the protein or nucleic acid
encoding can be aligned for comparison purposes is at least about
70%, at least about 75%, at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 98%, at least
about 99% of the length of the reference sequence, for example, the
nucleic acid sequence of a flagellin, including a nucleic acid
sequence that encodes an R3 construct (e.g., SEQ ID NO: 283) or an
R32x construct (e.g., SEQ ID NO: 284, 285), flagellin (SEQ ID NOs:
2, 127), antigen (e.g., SEQ ID NOs: 50, 51, 52, 98 and 287)
employed in the fusion proteins of the invention of a fusion
protein of the invention and fusion proteins of the invention
(e.g., SEQ ID NOs:126, 158, 268 and 269).
[0149] The actual comparison of the two sequences can be
accomplished by well-known methods, for example, using a
mathematical algorithm. A preferred, non-limiting example of such a
mathematical algorithm is described in Karlin et al. (Proc. Natl.
Acad. Sci. USA, 90:5873-5877 (1993), the teachings of which are
hereby incorporated by reference in its entirety). Such an
algorithm is incorporated into the BLASTN and BLASTX programs as
described, for example, in Schaffer et al. (Nucleic Acids Res.,
29:2994-3005 (2001) and Oehmen, C. S. et al., Bioinformatics 29
(6): 797-798 (2013). When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
BLASTN; available at the Internet site for the National Center for
Biotechnology Information) can be used. In one embodiment, the
database searched is a non-redundant (NR) database, and parameters
for sequence comparison can be set at: no filters; Expect value of
10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an
Existence of 11 and an Extension of 1.
[0150] Another mathematical algorithm employed for the comparison
of sequences is the algorithm of Myers and Miller, CABIOS (1989).
Such an algorithm is incorporated into the ALIGN program (version
2.0), which is part of the GCG (Accelrys, San Diego, Calif.)
sequence alignment software package. When utilizing the ALIGN
program for comparing amino acid sequences, a PAM120 weight residue
table, a gap length penalty of 12, and a gap penalty of 4 is used.
Additional algorithms for sequence analysis are known in the art
and include ADVANCE and ADAM as described in Torellis, et al.,
Comput. Appl. Biosci., 10: 3-5 (1994) and FASTA described in
Pearson, et al., (Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988),
the teachings of which are hereby incorporated by reference in its
entirety).
[0151] The percent identity between two amino acid sequences can
also be accomplished using the GAP program in the GCG software
package (Accelrys, San Diego, Calif.) using either a Blossom 63
matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4
and a length weight of 2, 3, or 4. In yet another embodiment, the
percent identity between two nucleic acid sequences can be
accomplished using the GAP program in the GCG software package
(Accelrys, San Diego, Calif.), using a gap weight of 50 and a
length weight of 3.
[0152] The nucleic acid sequence encoding a protein portion of HA,
polypeptide or fusion proteins of the invention and polypeptides of
the invention can include nucleic acid sequences that hybridize to
nucleic acid sequences or complements of nucleic acid sequences of
the invention, for example, the nucleic acid sequence of a
flagellin, including an R3 construct or an R32x construct, antigen
employed in the fusion proteins of the invention of a fusion
protein of the invention under selective hybridization conditions
(e.g., highly stringent hybridization conditions). As used herein,
the terms "hybridizes under low stringency," "hybridizes under
medium stringency," "hybridizes under high stringency," or
"hybridizes under very high stringency conditions," describe
conditions for hybridization and washing of the nucleic acid
sequences. Guidance for performing hybridization reactions, which
can include aqueous and nonaqueous methods, can be found in
Aubusel, F. M., et al., Current Protocols in Molecular Biology,
John Wiley & Sons, N.Y. (2001).
[0153] For applications that require high selectivity, relatively
high stringency conditions to form hybrids can be employed. In
solutions used for some membrane based hybridizations, addition of
an organic solvent, such as formamide, allows the reaction to occur
at a lower temperature. High stringency conditions are, for
example, relatively low salt and/or high temperature conditions.
High stringency are provided by about 0.02 M to about 0.10 M NaCl
at temperatures of about 50.degree. C. to about 70.degree. C. High
stringency conditions allow for limited numbers of mismatches
between the two sequences. In order to achieve less stringent
conditions, the salt concentration may be increased and/or the
temperature may be decreased. Medium stringency conditions are
achieved at a salt concentration of about 0.1 to 0.25 M NaCl and a
temperature of about 37.degree. C. to about 55.degree. C., while
low stringency conditions are achieved at a salt concentration of
about 0.15 M to about 0.9 M NaCl, and a temperature ranging from
about 20.degree. C. to about 55.degree. C. Selection of components
and conditions for hybridization are well known to those skilled in
the art and are reviewed in Ausubel et al., Short Protocols in
Molecular Biology, John Wiley & Sons, New York N.Y., Units
2.8-2.11, 3.18-3.19 and 4-64.9, (1997).
[0154] A "subject," as used herein, can be a mammal, such as a
primate or rodent (e.g., rat, mouse). In a particular embodiment,
the subject is a human.
[0155] An "effective amount," when referring to the amount of a
composition and fusion protein of the invention, refers to that
amount or dose of the composition and fusion protein, that, when
administered to the subject is an amount sufficient for therapeutic
efficacy (e.g., an amount sufficient to stimulate an immune
response in the subject). The compositions and fusion proteins of
the invention can be administered in a single dose or in multiple
doses.
[0156] The methods of the present invention can be accomplished by
the administration of the compositions and fusion proteins of the
invention by enteral or parenteral means. Specifically, the route
of administration is by oral ingestion (e.g., drink, tablet,
capsule form) or intramuscular injection of the composition and
fusion protein. Other routes of administration as also encompassed
by the present invention including intravenous, intradermal,
intraarterial, intraperitoneal, or subcutaneous routes, and nasal
administration. Suppositories or transdermal patches can also be
employed.
[0157] The compositions and fusion proteins of the invention can be
administered ex vivo to a subject's autologous dendritic cells.
Following exposure of the dendritic cells to the composition and
fusion protein of the invention, the dendritic cells can be
administered to the subject.
[0158] The compositions and fusion proteins of the invention can be
administered alone or can be coadministered to the patient.
Coadministration is meant to include simultaneous or sequential
administration of the composition, fusion protein or polypeptide of
the invention individually or in combination. Where the composition
and fusion protein are administered individually, the mode of
administration can be conducted sufficiently close in time to each
other (for example, administration of the composition close in time
to administration of the fusion protein) so that the effects on
stimulating an immune response in a subject are maximal. It is also
envisioned that multiple routes of administration (e.g.,
intramuscular, oral, transdermal) can be used to administer the
compositions and fusion proteins of the invention.
[0159] The compositions and fusion proteins of the invention can be
administered alone or as admixtures with conventional excipients,
for example, pharmaceutically, or physiologically, acceptable
organic, or inorganic carrier substances suitable for enteral or
parenteral application which do not deleteriously react with the
extract. Suitable pharmaceutically acceptable carriers include
water, salt solutions (such as Ringer's solution), alcohols, oils,
gelatins and carbohydrates such as lactose, amylose or starch,
fatty acid esters, hydroxymethycellulose, and polyvinyl
pyrrolidine. Such preparations can be sterilized and, if desired,
mixed with auxillary agents such as lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring, and/or aromatic substances and
the like which do not deleteriously react with the compositions,
fusion proteins or polypeptides of the invention. The preparations
can also be combined, when desired, with other active substances to
reduce metabolic degradation. The compositions and fusion proteins
of the invention can be administered by is oral administration,
such as a drink, intramuscular or intraperitoneal injection or
intranasal delivery. The compositions and fusion proteins alone, or
when combined with an admixture, can be administered in a single or
in more than one dose over a period of time to confer the desired
effect (e.g., alleviate prevent viral infection, to alleviate
symptoms of virus infection, such as influenza or flaviviral
infection).
[0160] When parenteral application is needed or desired,
particularly suitable admixtures for the compositions and fusion
proteins are injectable, sterile solutions, preferably oily or
aqueous solutions, as well as suspensions, emulsions, or implants,
including suppositories. In particular, carriers for parenteral
administration include aqueous solutions of dextrose, saline, pure
water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil,
polyoxyethylene-block polymers, and the like. Ampules are
convenient unit dosages. The compositions, fusion proteins or
polypeptides can also be incorporated into liposomes or
administered via transdermal pumps or patches. Pharmaceutical
admixtures suitable for use in the present invention are well-known
to those of skill in the art and are described, for example, in
Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, Pa.) and
WO 96/05309 the teachings of which are hereby incorporated by
reference.
[0161] The compositions and fusion proteins of the invention can be
administered to a subject on a support that presents the
compositions, fusion proteins and polypeptides of the invention to
the immune system of the subject to generate an immune response in
the subject. The presentation of the compositions, fusion proteins
and polypeptides of the invention would preferably include exposure
of antigenic portions of the viral protein to generate antibodies.
The fusion proteins of the invention are in close physical
proximity to one another on the support. The fusion proteins of the
invention can be attached to the support by covalent or noncovalent
attachment. Preferably, the support is biocompatible.
"Biocompatible," as used herein, means that the support does not
generate an immune response in the subject (e.g., the production of
antibodies). The support can be a biodegradable substrate carrier,
such as a polymer bead or a liposome. The support can further
include alum or other suitable adjuvants. The support can be a
virus (e.g., adenovirus, poxvirus, alphavirus), bacteria (e.g.,
Salmonella) or a nucleic acid (e.g., plasmid DNA, CpG).
[0162] The dosage and frequency (single or multiple doses)
administered to a subject can vary depending upon a variety of
factors, including prior exposure to an antigen, a viral protein,
the duration of viral infection, prior treatment of the viral
infection, the route of administration of the composition or fusion
protein; size, age, sex, health, body weight, body mass index, and
diet of the subject; nature and extent of symptoms of viral
exposure, viral infection and the particular viral responsible for
the infection (e.g., influenza virus), or treatment or infection of
an other antigen, such as an influenza antigen, kind of concurrent
treatment, complications from the viral exposure, viral infection
or exposure or other health-related problems. Other therapeutic
regimens or agents can be used in conjunction with the methods and
compositions and fusion proteins of the present invention. For
example, the administration of the compositions and fusion proteins
can be accompanied by other viral therapeutics or use of agents to
treat the symptoms of a condition associated with or consequent to
exposure to the antigen, such as influenza infection. Adjustment
and manipulation of established dosages (e.g., frequency and
duration) are well within the ability of those skilled in the
art.
[0163] Exemplary portions of antigens, flagellin and fusion
proteins of the invention include those listed in Table 1.
TABLE-US-00001 TABLE 1 SEQ Fusion ID Protein HA Strain Name/Format
Insertion Site Portion of HA NO HL772 B/Wisconsin/1/2010 276-277 in
SEQ ID NO: 127 HA1-2 extension, 126 D3I-o1 SEQ ID NO: 50 HL657
B/Brisbane/60/2008 276-277 in SEQ ID NO: 127 HA1-2, SEQ ID 128
D3I-o1 NO: 48 HL775 A/Perth/16/2009 276-277 in SEQ ID NO: 127
HA1-1L modified, 129 D3I-o1 SEQ ID NO: 228 HL1018 A/Anhui/1/2013
276-277 in SEQ ID NO: 127 HA1-2, SEQ ID 130 D3I-o1 NO: 110 HL656
B/Florida/4/2006 276-277 in SEQ ID NO: 127 HA1-2 extension, 151
D3I-o1 SEQ ID NO: 50 HL774 B/Hong Kong/259/2010 276-277 in SEQ ID
NO: 127 HA1-2 extension, 157 D3I-o1 SEQ ID NO: 54 HL787
B/Bangladesh/5495/2009 276-277 in SEQ ID NO: 127 HA1-2 extension,
158 D3I-o1 SEQ ID NO: 52 HL849 B/Wisconsin/1/2010 259-260 in SEQ ID
NO: 2 HA1-2 extension, 159 D3I-i1 SEQ ID NO: 50 HL848
B/Wisconsin/1/2010 244-245 in SEQ ID NO: 2 HA1-2 extension, 160
D3I-s1 SEQ ID NO: 50 HL825 B/Wisconsin/1/2010 187-188 in SEQ ID NO:
2 HA1-2 extension, 161 D2I-o1 SEQ ID NO: 50 HL826
B/Wisconsin/1/2010 178-179 in SEQ ID NO: 2 HA1-2 extension, 162
D2I-o2 SEQ ID NO: 50 HL827 B/Wisconsin/1/2010 326-327 in SEQ ID NO:
2 HA1-2 extension, 163 D2I-o3 SEQ ID NO: 50 HL828
B/Wisconsin/1/2010 102-103 in SEQ ID NO: 2 HA1-2 extension, 164
D1I-o1 SEQ ID NO: 50 HL850 B/Wisconsin/1/2010 366-367 in SEQ ID NO:
2 HA1-2 extension, 165 D2I-i1 SEQ ID NO: 50 HL733
B/Brisbane/60/2008 187-188 in SEQ ID NO: 270 HA1-2 extension, 166
D2I-o1 SEQ ID NO: 48 HL856 B/Brisbane/60/2008 187-188 in SEQ ID NO:
2 HA1-2 extension, 167 D2I-o1 SEQ ID NO: 48 HL857
B/Brisbane/60/2008 178-179 in SEQ ID NO: 2 HA1-2 extension, 168
D2I-o2 SEQ ID NO: 48 HL888 B/Wisconsin/1/2010 230-231 in SEQ ID NO:
2 HA1-2 extension, 169 D3I-o2 SEQ ID NO: 50 HL890
B/Wisconsin/1/2010 308-309 in SEQ ID NO: 2 HA1-2 extension, 170
D2I-c1 SEQ ID NO: 50 HL892 B/Wisconsin/1/2010 388-389 in SEQ ID NO:
2 HA1-2 extension, 171 D2I-i2 SEQ ID NO: 50 HL858
B/Brisbane/60/2008 326-327 in SEQ ID NO: 2 HA1-2 extension, 172
D2I-o3, SEQ ID NO: 48 HL860 B/Brisbane/60/2008 244-245 in SEQ ID
NO: 2 HA1-2 extension, 173 D3I-sl, SEQ ID NO: 48 HL889
B/Brisbane/60/2008 230-231 in SEQ ID NO: 2 HA1-2 extension, 174
D3I-o2, SEQ ID NO: 48 HL891 B/Brisbane/60/2008 308-309 in SEQ ID
NO: 2, HA1-2 extension, 175 D2I-c1 SEQ ID NO: 48 HL893
B/Brisbane/60/2008 388-389 in SEQ ID NO: 2 HA1-2 extension, 176
D2I-i2 SEQ ID NO: 48 HL864 B/Wisconsin/1/2010 276-277 in SEQ ID NO:
178 HA1-2 extension, 177 D3I-o1 SEQ ID NO: 50 HL854
B/Wisconsin/1/2010 198-199 in SEQ ID NO: 271 HA1-2 extension, 179
R3 SEQ ID NO: 50 HL861 B/Brisbane/60/2008 259-260 in SEQ ID NO: 2
HA1-2 extension, 180 D3I-i1 SEQ ID NO: 48 HL862 B/Brisbane/60/2008
366-367 in SEQ ID NO: 2 HA1-2 extension, 181 D2I-i1 SEQ ID NO: 48
HL863 B/Sichuan/379/1999 276-277 in SEQ ID NO: 127 HA1-2 extension,
182 D3I-o1 SEQ ID NO: 56 HL903 B/Sichuan/379/1999 259-260 in SEQ ID
NO: 2 HA1-2 extension, 183 D3I-i1 SEQ ID NO: 56 HL869 B/Hubei-
276-277 in SEQ ID NO: 127 HA1-2 extension, 184 Wujiagang/158/2009
SEQ ID NO: 58 D3I-o1 HL871 B/Texas/6/2011 276-277 in SEQ ID NO: 127
HA1-2 extension, 185 D3I-o1 SEQ ID NO: 60 HL639 A/Perth/16/2009
276-277 in SEQ ID NO: 127 HA1-1L, SEQ ID 186 D3I-o1 NO: 98 HL836
A/Victoria/361/2011 276-277 in SEQ ID NO: 127 HA1-1L, SEQ ID 187
D3I-o1 NO: 98 HL982 A/Anhui/1/2005 276-277 in SEQ ID NO: 127
HA1-1L, SEQ ID 188 D3I-o1 NO: 104 HL960 A/Indonesia/5/2005 276-278
in SEQ ID NO: 127 HA1-1L, SEQ ID 189 D3I-o1 NO: 106 HL876 A/New
York/107/2003 276-277 in SEQ ID NO: 127 HA1-1L, SEQ ID 190 D3I-o1
NO: 112 HL926 A/Canada/Rv504/2004 276-277 in SEQ ID NO: 127 HA1-1L,
SEQ ID 191 D3I-o1 NO: 114 HL880 A/Netherlands/219/03 276-277 in SEQ
ID NO: 127 HA1-1L, SEQ ID 192 D3I-o1 NO: 115 HL958 B/Hong
Kong/259/2010 276-277 in SEQ ID NO: 127 HA1-2 extension, 193 D3I-o1
SEQ ID NO: 54
[0164] Additional portions of antigens, flagellin and fusion
proteins for use of the invention can include those listed in Table
2. For example, the fusion protein can include a portion of the
globular head of HA fused to loop 6 of domain 3 of S. typhimuium
FljB flagellin at the D3I-o1 insertion site between residues
276-277 in SEQ ID NO: 127.
TABLE-US-00002 TABLE 2 HA Strain Name Format Portion of HA SEQ ID
NO A/Perth/16/2009 D3I-o1 HA1-2, SEQ ID NO: 64 194 A/Perth/16/2009
D3I-o1 HA1-1, SEQ ID NO: 11 195 A/Wyoming/03/2003 D3I-o1 HA1-2, SEQ
ID NO: 65 196 A/Wyoming/03/2003 STF2.D3Ins.HA1- HA1-1, SEQ ID NO:
12 197 1 WY03 D3I-o1 A/Wyoming/03/2003 D3I-o1 HA1-1L, SEQ ID NO: 99
198 A/New York/2782/2004 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 66 199 2
NY2782 D3I-o1 A/New York/2782/2004 STF2.D3Ins.HA1- HA1-1, SEQ ID
NO: 13 200 1 NY2782 D3I-o1 A/New York/2782/2004 D3I-o1 HA1-1L, SEQ
ID NO: 201 100 A/Victoria/361/2011 STF2.D3Ins.HA1- HA1-2, SEQ ID
NO: 67 202 2 VT361 D3I-o1 A/Victoria/361/2011 STF2.D3Ins.HA1-
HA1-1, SEQ ID NO: 14 203 1 VT361 D3I-o1 A/Aichi/2/68
STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 68 204 2 Aichi2 D3I-o1
A/Aichi/2/68 STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 15 205 1 Aichi2
D3I-o1 A/Aichi/2/68 D3I-o1 HA1-1L, SEQ ID NO: 206 102
A/Wisconsin/67/2005 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 69 207 2 WI67
D3I-o1 A/Wisconsin/67/2005 STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 16 208
WI67 D3I-o1 A/Wisconsin/67/2005 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO:
209 1L WI67 103 D3I-o1 A/Anhui/1/2005 STF2.D3Ins.HA1- HA1-2, SEQ ID
NO: 70 210 2 AH1 D3I- o1 A/Anhui/1/2005 STF2.D3Ins.HA1- HA1-1, SEQ
ID NO: 17 211 1 AH1 D3I- o1 A/Bar headed STF2.D3Ins.HA1- HA1-2, SEQ
ID NO: 71 212 goose/Qinghai/1A/2005 2 QH1A D3I-o1 A/Bar headed
STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 18 213 goose/Qinghai/1A/2005 1
QH1A D3I-o1 A/Bar headed STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 214
goose/Qinghai/1A/2005 1L QH1A 105 D3I-o1 A/Indonesia/5/2005
STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 72 215 2 IND5 D3I-o1
A/Indonesia/5/2005 STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 19 216 1 IND5
D3I-o1 A/Vietnam/1203/2004 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 80 217
2 VN1203 D3I-o1 A/Vietnam/1203/2004 STF2.D3Ins.HA1- HA1-1, SEQ ID
NO: 20 218 1 VN1203 D3I-o1 A/Vietnam/1203/2004 STF2.D3Ins.HA1-
HA1-1L, SEQ ID NO: 219 1L VN1203 107 D3I-o1 A/Hubei/1/2010
STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 73 220 2 HB1 D3I- o1
A/Hubei/1/2010 STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 21 221 1 HB1 D3I-
o1 A/Hubei/1/2010 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 222 1L HB1 108
D3I-o1 A/Hong Kong/156/97 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 74 223
2 HK156 D3I-o1 A/Hong Kong/156/97 STF2.D3Ins.HA1- HA1-1, SEQ ID NO:
22 224 1 HK156 D3I-o1 A/Hong Kong/156/97 STF2.D3Ins.HA1- HA1-1L,
SEQ ID NO: 225 1L HK156 109 D3I-o1 A/Anhui/1/2013 STF2.D3Ins.HA1-
HA1-2, SEQ ID NO: 75 226 2 AH1 D3I- o1 A/Anhui/1/2013
STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 23 227 1 AH1 D3I- o1
A/Anhui/1/2013 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 228 1L AH1 110
D3I-o1 A/Turkey/Italy/214845/2002 STF2.D3Ins.HA1-2 HA1-2, SEQ ID
NO: 76 229 IT214845 D3I- o1 A/Turkey/Italy/214845/2002
STF2.D3Ins.HA1-1 HA1-1, SEQ ID NO: 24 230 IT214845 D3I- o1
A/Turkey/Italy/214845/2002 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 231
1L 111 IT214845 D3I- o1 A/New York/107/2003 STF2.D3Ins.HA1- HA1-2,
SEQ ID NO: 77 232 2 NY107 D3I-o1 A/New York/107/2003
STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 25 233 1 NY107 D3I-o1
A/Mallard/Netherlands/12/2000 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 78
234 2 NL12 D3I-o1 A/Mallard/Netherlands/12/2000 STF2.D3Ins.HA1-
HA1-1, SEQ ID NO: 26 235 1 NL12 D3I-o1
A/Mallard/Netherlands/12/2000 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO:
236 1L NL12 113 D3I-o1 A/Canada/Rv504/2004 STF2.D3Ins.HA1- HA1-2,
SEQ ID NO: 79 237 2 CA504 D3I-o1 A/Canada/Rv504/2004
STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 27 238 1 CA504 D3I-o1
A/Netherlands/219/03 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 81 239 2
NL219 D3I-o1 A/Netherlands/219/03 STF2.D3Ins.HA1- HA1-1, SEQ ID NO:
28 240 1 NL219 D3I-o1 A/Hong Kong/33982/2009 STF2.D3Ins.HA1- HA1-2,
SEQ ID NO: 82 241 2 HK33982 D3I-o1 A/Hong Kong/33982/2009
STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 29 242 1 HK33982 D3I-o1 A/Hong
Kong/33982/2009 STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 243 1L 116
HK33982 D3I- o1 A/Hong Kong/1073/99 STF2.D3Ins.HA1- HA1-2, SEQ ID
NO: 83 244 2 HK1073 D3I-o1 A/Hong Kong/1073/99 STF2.D3Ins.HA1-
HA1-1, SEQ ID NO: 30 245 1 HK1073 D3I-o1 A/Hong Kong/1073/99
STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 246 1L HK1073 117 D3I-o1
A/Chicken/Hong STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 84 247 Kong/G9/97
2 HKG9 D3I-o1 A/Chicken/Hong STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 31
248 Kong/G9/97 1 HKG9 D3I-o1 A/Chicken/Hong STF2.D3Ins.HA1- HA1-1L,
SEQ ID NO: 249 Kong/G9/97 1L HKG9 118 D3I-o1
A/chicken/Anhui/AH16/2008 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 85 250
2 AH16 D3I-o1 A/chicken/Anhui/AH16/2008 STF2.D3Ins.HA1- HA1-1, SEQ
ID NO: 32 251 1 AH16 D3I-o1 A/chicken/Anhui/AH16/2008
STF2.D3Ins.HA1- HA1-1L, SEQ ID NO: 252 1L AH16 119 D3I-o1
A/Swine/Hong Kong/9/98 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 86 253 2
HK9 D3I- o1 A/Swine/Hong Kong/9/98 STF2.D3Ins.HA1- HA1-1, SEQ ID
NO: 33 254 1 HK9 D3I- o1 A/Swine/Hong Kong/9/98 STF2.D3Ins.HA1-
HA1-1L, SEQ ID NO: 255 1L HK9 120 D3I-o1 A/swine/Guangxi/58/2005
STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 87 256 2 GX58 D3I-o1
A/swine/Guangxi/58/2005 STF2.D3Ins.HA1- HA1-1, SEQ ID NO: 34 257 1
GX58 D3I-o1 A/swine/Guangxi/58/2005 STF2.D3Ins.HA1- HA1-1L, SEQ ID
NO: 258 1L GX58 121 D3I-o1 B/Shanghai/361/2002 STF2.D3Ins.HA1-
HA1-2, SEQ ID NO: 89 259 2 D3I-o1 with extension
B/Malaysia/2506/2004 STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 62 260 2
D3I-o1 with extension B/Ohio/1/2005 STF2.D3Ins.HA1- HA1-2, SEQ ID
NO: 91 261 2 D3I-o1 with extension B/Hong Kong/330/2001
STF2.D3Ins.HA1- HA1-2, SEQ ID NO: 93 262 2 D3I-o1 with
extension
EXEMPLIFICATION
Example 1
Primary Sequence Analysis of Flagellin to Identify Antigen
Insertion Sites to Target Influenza B
[0165] Primary sequence analysis showed that the amino acid
sequence of portions of influenza viral hemagglutinin, such as
HA1-2 of the Flu B (Influenza B) globular head (SEQ ID NOs: 45, 47
and 61), included a higher content of positively charged residues
compared to most influenza A globular head portions, such as SEQ ID
NO: 63. At neutral pH, a portion of an influenza Flu B globular
head, such as SEQ ID NO: 45, has a net positive charge with a pI of
between about 8.5 to about 9.5. At neutral pH, flagellin has a net
negative charge due, in part, to a series of negatively charged
amino acids in Domain 1 (D1) of flagellin with a pI of about 4.5 to
about 5.0. Apposition of a positively charged portion of an
influenza B HA with the negatively charged D1 of flagellin, such as
in R3 and R3.2x fusion protein, may result in intra-molecular
interactions between the portion of HA and portion of flagellin
that may interfere with the ability of flagellin to trigger TLR5
signaling and with the presentation of the influenza B HA antigen
to immune cell receptors.
[0166] Immunogenicity of an R3 fusion protein of composition that
includes an influenza B, such as a B/Florida/4/2006 as in HL098
(SEQ ID NO: 122), may be improved by extending the portion of the
globular head amino acid sequence by an additional 9 amino acids
(SEQ ID NO: 123) at the C-terminus of HA1-2. For example, an
additional 9 amino acids of the HA (SLPLIGEAD (SEQ ID NO: 123),
which corresponds to native amino acid residues 300-308 of SEQ ID
NO: 124, can be added to the carboxy-terminus of HA1-2 (e.g., SEQ
ID NO: 45) to generate SEQ ID NO: 46. For example, the HA1-2 with
an extension can then be used to generate a fusion protein, such as
HL352 (SEQ ID NO: 125). The additional 9 amino acids to the portion
of HA (e.g., SEQ ID NO: 123) introduced two negatively charged
amino acids (E306 and D308) to the portion of the globular head,
which may serve to "repel" the portion of the globular head away
from the negatively charged flagellin. However, as described infra,
while improved, the activity (TLR5 activation and immunogenicity)
associated with fusion proteins having HA components with the
additional 9 amino acids was low, as discussed infra with respect
to, for example, FIGS. 3 and 5 and fusion proteins referred to as
"HL098" (SEQ ID NO: 122) and "HL352" (SEQ ID NO: 125).
[0167] Fusion of an antigen with an isoelectric point greater than
about 7.0 to a loop of Domain 3 of flagellin is believed to
position the HA antigen component of the fusion protein distant
(i.e., away) from domain (D1) (the TLR5-binding domain) of
flagellin. Portions of an influenza viral hemagglutinin that
include at least a portion of a globular head having at least one
.beta.-sheet at the bottom of the globular head and that have an
isoelectric point greater than about 7.0, such as an influenza B
viral hemagglutinin (e.g., SEQ ID NO: 45, 61), can be fused within
at least one loop of domain 3 of flagellin to form a "D3Ins
format," also referred to as a "D3I" or "D3I construct." Likewise,
portions of an influenza viral hemagglutinin that include at least
a portion of a globular head having at least one .beta.-sheet at
the bottom of the globular head and that have an isoelectric point
greater than about 7.0, such as an influenza A viral hemagglutinin
of SEQ ID NO: 98, and A/Perth/16/2009 strains, can be fused within
at least one loop of domain 3 of flagellin to form a "D3Ins
format." Fusion proteins that include both influenza A and
influenza B antigens can be combined to form multivalent
compositions that target both influenza A and influenza B antigens
to thereby prevent or minimize disease associated with influenza A
and influenza B infection.
[0168] The crystal structure of domain 3 of Salmonella typhimurium
flagellin (FliC, Protein Data Bank ID (PDB): 1UCU) has been
reported (Yonekura, K., et al., Nature, 424: 643-650 (2003)) and
includes the identification of 6 loops in domain 3 of SEQ ID NO: 1.
Secondary structure prediction of Salmonella typhimurium flagellin
FliC using PHD is depicted below and, consistent with the known
tertiary structure, predicts 6 loops in domain 3. Designations
below are AA: Primary amino acid sequence; PROF_sec: Secondary
structure prediction where "H" stands for .alpha.-Helix and "E"
stands for .beta.-strand (Rost, B., et al., Proteins, 19: 55-72
(1994)). The predicted loops in domain 3 of SEQ ID NO: 1 are
indicated in boxes below. The secondary structures adjacent to the
predicted loops are essentially similar to the known secondary
structures for Salmonella typhimurium flagellin (FliC, Protein Data
Bank ID (PDB): 1UCU) (Yonekura, K., et al., Nature, 424: 643-650
(2003)).
[0169] The predicted secondary structure of FliC (S. typhimurium,
SEQ ID NO: 1) is substantially similar to the known high resolution
atomic model determined by combination of X-ray crystallography and
electron microscopy (1UCU, Yonekura, K., et al. Nature, 424:
643-650 (2003)). The substantial similarity between the predicted
secondary structure and the known secondary structure of S.
typhimurium FliC (SEQ ID NO: 1) indicates that secondary structures
predicted employing CLUSTALW and PHD adjacent to loops of domain 3
of other flagellins, including S. typhimurium FljB (SEQ ID NO: 2),
can be employed to select insertion sites that correspond to known
loops in domain 3 in other flagellin, such as of S. typhimurium
FliC.
[0170] Secondary structure prediction using PHD of Flagellin FliC
(Salmonella typhimurium, Protein Data Bank ID: 1UCU, SEQ ID NO: 1)
is depicted below. Predicted loops in domain 3 are indicated by
boxed text.
TABLE-US-00003
....,....1....,....2....,....3....,....4....,....5....,....6 AA
AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGL
PROF_sec EEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHHH
....,....7....,....8....,....9....,....10...,....11...,....12 AA
TQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLN
PROF_sec HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH
....,....13...,....14...,....15...,....16...,....17...,....18 AA
EIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKV
PROF_sec HHHHHHH EEEEE EEEEEE EEEEEEEEEE EEEEEEEEEEE
....,....19...,....20...,....21...,....22...,....23...,....24 AA
##STR00001## PROF_sec EEEE EEEEE EEE EEE EEEEEEEEEEE
....,....25...,....26...,....27...,....28...,....29...,....30 AA
##STR00002## PROF_sec EEEEEEE EEEEEEEE EEEE EEE
....,....31...,....32...,....33...,....34...,....35...,....36 AA
TGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTAL
PROF_sec EEEE EEEE EEE EEEE EE EEE
....,....37...,....38...,....39...,....40...,....41...,....42 AA
NKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDT
PROF_sec EE EEEE EEE HHHHHHHHHHHHHHHHHHHH
....,....43...,....44...,....45...,....46...,....47...,....48 AA
LRSDLGAVQNRFNSAITNLGNTVNNLTSVRSRIEDSDYATEVSNMSRAQILQQAGTSVLA
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHH
....,....49... AA QANQVPQNVLSLLR (SEQ ID NO: 1) PROF_sec HH HHHHHHH
(predicted secondary structure)
[0171] Secondary structure prediction using PHD of Flagellin FljB
(S. typhimurium, SEQ ID NO: 2). Predicted loops in domain 3 are
indicated by boxed text.
TABLE-US-00004
....,....1....,....2....,....3....,....4....,....5....,....6 AA
MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
....,....7....,....8....,....9....,....10...,....11...,....12 AA
LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
....,....13...,....14...,....15...,....16...,....17...,....18 AA
NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDSLNVQKAYD
PROF_sec HHHHHHHH EEEE EEEEE EEEEEEEEE EEEEEEEEEEEEE
....,....19...,....20...,....21...,....22...,....23...,....24 AA
##STR00003## PROF_sec EEE EEEEEEE EEEEE EEE EEEEE EEEEEEE
....,....25...,....26...,....27...,....28...,....29...,....30 AA
##STR00004## PROF_sec E EEEEE EEEE EEE
....,....31...,....32...,....33...,....34...,....35...,....36 AA
ALIAGGVDATDANGAELVKMSYTDKNGKTIEGGYALKAGDKYYAADYDEATGAIKAKTTS
PROF_sec EEEE EEEEEE EE EEE EEE
....,....37...,....38...,....39...,....40...,....41...,....42 AA
YTAADGTTKTAANQLGGVDGKTEVVTIDGKTYNASKAAGHDFKAQPELAEAAAKTTENPL
PROF_sec EEEEEEEEE EEEE EE HHHHHHHH
....,....43...,....44...,....45...,....46...,....47...,....48 AA
QKIDAALAQVDALRSDLGAVQNRFNSAITNLGNTVNNLSEARSRIEDSDYATEVSNMSRA
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHH
....,....49...,....50...,. AA QILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID
NO: 2) PROF_sec HHHHHHHHHHHHHH HHHHHHH (predicted secondary
structure)
[0172] The predictions of loops in domain 3 employing PHD compared
to known loops identified by X-ray crystallography show that the
PHD computer program can be employed to predict amino acid
sequences and loops for sites of insertion of antigens within
domain 3 of a flagellin.
[0173] Sequence alignment between S. typhimurium FliC (SEQ ID NO:
1) and S. typhimurium FljB (SEQ ID NO: 2) was performed using the
multiple sequence alignment tool CLUSTALW and secondary structure
prediction by PHD with PROF_sec: structure prediction where "H"
stands for alpha-helix and "E" stands for beta-strand (Thompson, J.
D., et al., Nucleic Acids Res. 22: 4673-4680 (1994); Rost, B., et
al., Proteins 19: 55-72 (1994)). The sequence alignment below
showed about 74.75% identity of amino acid residues denoted with
(*) and about 10.26% difference with about 7.30% strongly similar
(:) and about 7.69% (.) weakly similar amino acid residues. Domain
boundaries of D0, D1, D2 and D3 are underlined differently and
three D3 insertion sites were marked.
[0174] The objective of this alignment was to predict loop regions
in Domain 3 of S. typhimurium FljB (SEQ ID NO: 2) that correspond
to known loop regions of Domain 3 of S. typhimurium FliC (SEQ ID
NO: 1) for points of insertion of portions of HA that have an
isoelectric point greater than about 7.0 to generate fusion
proteins of the inventions.
[0175] Primary amino acid sequence alignment between FliC (PDB:
1UCU (Yonekura, K., et al., Nature, 424: 643-650 (2003)), SEQ ID
NO: 1) and FljB (SEQ ID NO: 2) of Salmonella typhimurium indicated
that the two flagellins shared highly conserved D0 (domain 0) and
D1 (domain 1) domains, but varied in the D2 (domain 2) and D3
(domain 3) domains. However, secondary structure prediction, using
PHD (Rost, B., et al., Proteins 19: 55-72 (1994)), showed that both
D2 and D3 in the FliC flagellin FliC (SEQ ID NO: 1) and FljB
flagellin (SEQ ID NO: 2) of Salmonella typhimurium share similar
secondary structures, despite the differences in primary amino acid
sequence.
[0176] Sequence alignment and secondary structure prediction using
CLUSTALW and PHD of FliC (SEQ ID NO: 1) and FljB (SEQ ID NO: 2) of
S. typhimurium is depicted below. Predicted loops in domain 3 are
indicated by boxed text.
TABLE-US-00005 10 20 30 40 50 60 | | | | | | FliCxx0
MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec EEE
HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH FlijBx1 ##STR00005##
PROF_sec EE HHHHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
************************************************************ 70 80
90 100 110 120 | | | | | | FliCxx0
LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec
HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH FlijBx1
##STR00006## PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH
HHHHHHHHHHHHHHH
************************************************************ 130
140 150 160 170 180 | | | | | | FliCxx0
NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYK 1UCU
HHHHHHHHHH EEEEEE EEEEEE E PROF_sec HHHHHHHH EEEEE EEEEEE
EEEEEEEEEE EEEEEEEEEEE FlijBx1 ##STR00007## PROF_sec HHHHHHHH EEEE
EEEEE EEEEEEEEE EEEEEEEEEEEEE
***************************************************:****: *. 190
200 210 220 230 240 | | | | | | FliCxx0 ##STR00008## 1UCU EEEEE
EEEE EEEE HHHH HHHHH EEEE EEE EEEEEE PROF_sec EEEE EEEEE EEE EEE
EEEEEE FlijBx1 ##STR00009## PROF_sec EEE EEEEEEE EEEEE EEE EEEEE
EEEEEEE *.***.*...**:. . .**::::**:: * .** . .* :*** ..**:..: 250
260 270 280 290 300 | | | | | | FliCxx0 ##STR00010## 1UCU EEEEEE
EEE EEE EEEEE HHHHH PROF_sec EEEE E EEEEEEE EEEEEEEE EEEE FlijBx1
##STR00011## PROF_sec E EEEEE EEEE EEE .**. :.*:* ***.* *:*
****.***.. . .:* ** : :.. . ::** D3I site .uparw. .uparw. .uparw.
310 320 330 340 350 360 | | | | | | FliCxx0
AALTAAGVTGTAS----VVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKD-GSISINTT 1UCU
HHHHHH EE EEEEEEE EEEEEEEEEE EEEE EEE PROF_sec EEE E EEE EEEE EEE
EEEE FlijBx1 ##STR00012## PROF_sec EEEE EEEEEE EE EEE EEE ** *.**
.* : :*******:*****:** *:*.**.**:* :: *:*. :** 370 380 390 400 410
420 | | | | | | FliCxx0
KYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENP 1UCU
EEE EEEEEE EEEE HHHH HH PROF_sec EE EEEEE EEEE EEE HHHHHHH FlijBx1
##STR00013## PROF_sec EEEEEEEEE EEEE EE HHHHHHH .*** ***:***
*:***.*******:*.**** **** **:*****:******.***** 430 440 450 460 470
480 | | | | | | FliCxx0
LQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSVRSRIEDSDYATEVSNMSR 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHH PROF_sec
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHH FlijBx1
##STR00014## PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH
HHHHHHHHHHHH
************:**************************:..****************** 490
500 | | FliCxx0 AQILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1) 1UCU
HHHHHHHHHHHHHHHHHHHHHHHH (empirical structure) PROF_sec
HHHHHHHHHHHHHHH HHHHHHH (predicted secondary structure) FlijBx1
##STR00015## (SEQ ID NO: 2) PROF_sec HHHHHHHHHHHHHHH HHHHHHH
(predicted secondary structure) **************************
[0177] An insertion site in a predicted loop in domain 3 of S.
typhimurium FljB, referred to herein as "D3I-o1," for fusion of an
antigen that has an isoelectric point greater than about 7.0, is
between amino acid residues G277 and A278 of SEQ ID NO: 2. The
insertion site D3I-o1 is between predicted .beta.-strands VTLA (SEQ
ID NO: 131), which is amino acid residues 263-266 of SEQ ID NO: 2,
and VVS, which is amino acid residues 293-295 of SEQ ID NO: 2.
Another insertion site in a second predicted loop in domain 3 of S.
typhimurium FljB, referred to herein as "D3I-i1," is between
residues T259 and D260 of SEQ ID NO: 2. The insertion site D3I-i1
is between .beta.-strands EVNVA (SEQ ID NO: 132), which corresponds
to amino acid residues 254-258 of SEQ ID NO: 2, and VTLA (SEQ ID
NO: 131), which corresponds to amino acid residues 263-266 of SEQ
ID NO: 2. An additional insertion site in a third predicted loop in
domain 3 of S. typhimurium FljB, referred to herein as "D3I-s1," is
between residue G244 and A245 of SEQ ID NO: 2. The insertion site
of D3I-s1 is between .beta.-strands KYFVTIGG (SEQ ID NO: 133),
which corresponds to amino acid residues 234-241 of SEQ ID NO: 2,
and EVNVA (SEQ ID NO: 132), which corresponds to amino acid
residues 254-258 of SEQ ID NO: 2. The secondary structures adjacent
to the predicted loops of S. typhimurium FliC are substantially
similar to the predicted secondary structures adjacent to loops of
domain 3 of S. typhimurium FljB and are depicted in FIG. 29.
[0178] In an embodiment, fusion of the antigen with an isoelectric
point greater than about 7.0 can be about 2 to about 10 amino acids
towards the carboxy- or amino-terminus of flagellin from the
designated insertion site, based on the proximity of the adjacent
secondary structural elements. For example, the D3I-o1 site can be
from amino acid residues G268 through D289 of SEQ ID NO: 2, which
does not invade adjacent .beta.-strands, and are predicted at VTLA
(SEQ ID NO: 131) at amino acid residues 263-266 of SEQ ID NO: 2 and
VVS at amino acid residues 293-295 of SEQ ID NO: 2. In addition,
for example, the D3I-i1 site can only accommodate a shift of 1 or 2
amino acids towards the carboxy-terminus of flagellin of SEQ ID NO:
2 at amino acid residues D260 or G261 of SEQ ID NO: 2 before
disrupting the neighboring .beta.-strands, which are predicted at
EVNVA (SEQ ID NO:132) at amino acid residues 254-258 of SEQ ID NO:
2) and VTLA (SEQ ID NO: 131) at amino acid residues 263-266 of SEQ
ID NO: 2.
[0179] Based on predicted secondary structure analysis, loops of
domain 3 of E. coli FliC flagellin (SEQ ID NO: 3) and Pseudomonas
aeruginosa PAO1flagellin (SEQ ID NO: 4) can be predicted. Based on
this analysis, E. coli FliC primary amino acid sequence (SEQ ID NO:
3) was aligned with the primary amino acid sequence of Salmonella
typhimurium FliC (SEQ ID NO: 1). The secondary structures were
assigned either directly from known structure of 1UCU for
Salmonella typhimurium FliC. (SEQ ID NO: 1) or by prediction using
PHD. The secondary structures of E. coli flagellin (SEQ ID NO: 3)
or Pseudomonas aeruginosa PAO1 flagellin (SEQ ID NO: 4) were
assigned using the PHD program. Designations depicted below are AA:
Primary amino acid sequence; PROF_sec: Secondary structure
prediction where "H" stands for .alpha.-Helix and "E" stands for
beta-strand (Rost, B., et al., Proteins 19: 55-72 (1994)).
[0180] Secondary structure prediction using PHD of Flagellin FliC
(E. coli, SEQ ID NO: 3) is depicted below. Predicted loops in
domain 3 are in boxed text.
TABLE-US-00006
....,....1....,....2....,....3....,....4....,....5....,....6 AA
MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTANIKG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
....,....7....,....8....,....9....,....10...,....11...,....12 AA
LTQASRNANDGISVAQTTEGALNEINNNLQRIRELSVQATNGTNSDSDLSSIQAEITQRL
PROF_sec HHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
....,....13...,....14...,....15...,....16...,....17...,....18 AA
EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK
PROF_sec HHHHHHHH EEEEE EEEEE EEEEEEEEE EEEEEEEEEE
....,....19...,....20...,....21...,....22...,....23...,....24 AA
##STR00016## PROF_sec EEEEEE EEEEE EEEEEEE EEEEEE EEEEEE EE
....,....25...,....26...,....27...,....28...,....29...,....30 AA
##STR00017## PROF_sec EEE EEEEEEEEE EEEE EEE EEEEEE EEEEEE
....,....31...,....32...,....33...,....34...,....35...,....36 AA
NAPTGAGATITTDTAVYKNSAGQFTTTKVENKAATLSDLDLNAAKKTGSTLVVNGATYNV
PROF_sec EEE EEEEE EEEEE EEE EEEEEEEEEEEE
....,....37...,....38...,....39...,....40...,....41...,....42 AA
SADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETIDKALAKVDNLR
PROF_sec E EEE EEEEE EEE HHHHHHHHHHHHHHHHHHHHH
....,....43...,....44...,....45...,....46...,....47...,....48 AA
SDLGAVQNRFDSAITNLGNTVNNLSSARSRIEDADYATEVSNMSRAQILQQAGTSVLAQA
PROF_sec HHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHH
....,....49. AA NQTTQNVLSLLR (SEQ ID NO: 3) PROF_sec HHHHHHH
(predicted secondary structure)
[0181] Secondary structure prediction using PHD of flagellin (P.
aeruginosa PAO1, SEQ ID NO: 4) is depicted below. Predicted loops
in domain 3 are indicated by boxed text.
TABLE-US-00007
....,....1....,....2....,....3....,....4....,....5....,....6 AA
MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
....,....7....,....8....,....9....,....10...,....11...,....12 AA
LNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQ
PROF_sec HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
....,....13...,....14...,....15...,....16...,....17...,....18 AA
AELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAG
PROF_sec HHHHHHHH EEEEE EEEE EEEEEEEEE EEE EEEEE
....,....19...,....20...,....21...,....22...,....23...,....24 AA
##STR00018## PROF_sec EEEEE EEEEEEEEEE EEEEE EEEEEE EEE
....,....25...,....26...,....27...,....28...,....29...,....30 AA
##STR00019## PROF_sec E EEE EEEEEEEEE EEEEE EEEEEEE EEEEE
....,....31...,....32...,....33...,....34...,....35...,....36 AA
SATGENVKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTTIVTGYVQLNSP
PROF_sec EE EE EEEEEEEE EEEE EEEE EEEEEEEEEE
....,....37...,....38...,....39...,....40...,....41...,....42 AA
TAYSVSGTGTQASQVFGNASAAQKSSVASVDISTADGAQNAIAVVDBALAAIDAQRADLG
PROF_sec EEEEE EEEE EE EE HHHHHHHHHHHHHHHHHHHHHHHHHH
....,....43...,....44...,....45...,....46...,....47...,....48 AA
AVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQVLQQAGTAILAQANQLP
PROF_sec HHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHH
....,....4 AA QAVLSLLR (SEQ ID NO: 4) PROF_sec HHHHHHH (predicted
secondary structure)
[0182] The E. coli FliC primary amino acid sequence (SEQ ID NO: 3)
was initially aligned with the primary amino acid sequence of
Salmonella typhimurium FliC (SEQ ID NO: 1). Sequence alignment was
performed employing the multiple sequence alignment tool CLUSTALW
and secondary structure prediction by PHD with PROF_sec: denoting
secondary structure prediction where "H" stands for alpha-helix and
"E" stands for .beta.-strand (Thompson, J. D., et al., Nucleic
Acids Res. 22: 4673-4680 (1994); Rost, B., et al., Proteins 19:
55-72 (1994)). The sequence alignment showed about 53.98% identity
of amino acid residues denoted with (*) and about 21.91% difference
with about 12.95% strongly similar (:) and about 11.16% (.) weakly
similar amino acid residues. The predicted secondary structure of
E. coli FliC (ECFlic, SEQ ID NO: 3) was substantially similar to
that of S. typhimurium FliC(STFliC, Protein Data Bank ID: 1UCU
(Yonekura, K., et al., Nature 424:643-650 (2003)), SEQ ID NO: 1)
despite differences in primary amino acid sequences.
[0183] Sequence alignment and secondary structure prediction using
CLUSTALW and PHD of S. typhimurium FliC (SEQ ID NO: 1) and E. coli
FliC (SEQ ID NO: 3) to select loops in domain 3 for fusion for to
antigens is depicted below. The predicted loops in domain 3 are
indicated by boxed text.
TABLE-US-00008 10 20 30 40 50 60 | | | | | | STFlic
MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec EEE
HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH ECFlic
MAQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTANIKG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH
***********************:*.:********************************* 70 80
90 100 110 120 | | | | | | STFlic
LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec
HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH ECFlic
LTQASRNANDGISVAQTTEGALNEINNNLQRIRELSVQATNGTNSDSDLSSIQAEITQRL
PROF_sec HHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
*************:*****************:***:**::*.***:***.********** 130
140 150 160 170 180 | | | | | | STFlic
NEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQ--QK 1UCU
HHHHHHHHHH EEEEEE EEEEEE PROF_sec HHHHHHHH EEEEE EEEEEE EEEEEEEEEE
EEEEEEEEEEE ECFlic
EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK AA
EEIDRVSEQTQFNGVKVLAENNEMKIQVGANDGETITINLAKIDAKTLGLDGFNIDGAQK
PROF_sec HHHHHHHH EEEEE EEEEE EEEEEEEEE EEEEEEEEEE :******
***********::* :.*********** *:* :*:::***** :*:: ** 190 200 210 220
230 240 | | | | | | STFlic ##STR00020## 1UCU EEEEEE EEEEEEEE HHHH
HH HHH EEEE EEE EEEEEE PROF_sec EEEE EEEEE EEE EEE EEEEEEEE ECFlic
##STR00021## PROF_sec EEEEEE EEEEE EEEEEEE EEEEEE EEEEEE EE . **
:.... . . :...*:.... * .* : * :. * : . :...*: 250 260 270 280 290
300 | | | | | | STFlic ##STR00022## 1UCU EEEEEE EEE EEE EEEEE
HHHHHHH PROF_sec EE E EEEEEEE EEEEEEEE EEEEEE ECFlic ##STR00023##
PROF_sec EEE EEEEEEEEE EEEE EEE EEEEEE EEEEE * . . ::::. :: *.: * :
: ** * : *.* .** * :.: : D3I site .uparw. .uparw. .uparw. 310 320
330 340 350 360 | | | | | | STFlic
LTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDG 1UCU
HHHH EEEEEEEEE EEEEEEEEEE EEEE EEE PROF_sec E EEEE EEEE EEE EEEE EE
ECFlic IDSNAPTGAGATIT---TDTAVYKNSAGQFTTTLVENKAATLS---DLDLNAAKKTGSTL
PROF_sec E EEE EEEEE EEEEE EEE EEE : : . **:.:.:. **. . ..* ... :
:** . .:.:*::* *.. 370 380 390 400 410 420 | | | | | | STFlic
TSKTALNKLGGADGKTEVVSIGG--KTYAASKAEGHNFKAQPDLAEAAATTTENPLQKID 1UCU
EEE EEEEEE EEEE HHHH HH HHH PROF_sec EEEEE EEEE EEE HHHHHHH HHH
ECFlic VVNGATYNVS-ADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETID
VVNGATYNVS ADGKTVTDTTPGAPKVMYLSKSEGGSPILVNEDAAKSLQSTTNPLETID
PROF_sec EEEEEEEEEE EEE EEEEE EEE HHHHHHHHHHH . : * ::. ***** . : *
*. **:** . : * : :* ***:.** 430 440 450 460 470 480 | | | | | |
STFlic AALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQ
1UCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHH
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH
ECFlic KALAKVDNLRSDLGAVQNRFDSAITNLGNTVNNLSSARSRIEDADYATEVSNMSRAQILQ
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHHHHH
***:**.************:*************:********:**************** 490 500
| | STFlic QAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1) 1UCU
HHHHHHHHHHHHHHHHHHH (empirical structure) PROF_sec HHHHHHHHHH
HHHHHHH (predicted secondary structure) ECFlic
QAGTSVLAQANQTTQNVLSLLR (SEQ ID NO: 3) PROF_sec HHHHHHHHHH HHHHHHH
(predicted secondary structure) ************..********
[0184] Sequence alignment of Salmonella typhimurium FliC(STFlic,
Protein Data Bank ID: 1UCU, SEQ ID NO: 1) and Pseudomonas
aeruginosa PAO1 flagellin (SaFlix1, SEQ ID NO: 4) was performed by
using the multiple sequence alignment tool CLUSTALW and secondary
structure prediction by PHD with PROF_sec: denoting secondary
structure prediction where "H" stands for alpha-helix and "E"
stands for beta-strand (Thompson, J. D., et al., Nucleic Acids Res.
22: 4673-4680 (1994); Rost, B., et al., Proteins 19: 55-72 (1994)).
The sequence alignment showed about 36.24% identity of amino acid
residues denoted with (*) and about 30.10% difference with about
20.59% strongly similar (:) and about 13.07% (.) weakly similar
amino acid residues.
[0185] Sequence alignment and secondary structure prediction using
CLUSTALW and PHD of S. typhimurium FliC (SEQ ID NO: 1) and P.
aeruginosa PAO1 flagellin (SEQ ID NO: 4) to select loops in domain
3 of the flagellin for fusion to antigens is depicted below. The
predicted loops in domain 3 are indicated by boxed text.
TABLE-US-00009 10 20 30 40 50 60 | | | | | | STFlic
MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKG 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec EEE
HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH SaFlix1
MALTVNTNIASLNTQRNLNASSNDLNTSLQRLTTGYRINSAKDDAAGLQISNRLSNQISG
PROF_sec EEEE HHHHHHHHHHHHHHHHHHHHHHHH EE HHHHHHHHHHHHHHHH ** .:***
** **.*** *.. *.*:::**::* *********** *:**:: :*.* 70 80 90 100 110
120 | | | | | | STFlic
LTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRL 1UCU
HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHH PROF_sec
HHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH SaFlix1
LNVATRNANDGISLAQTAEGALQQSTNILQRIRDLALQSANGSNSDADRAALQKEVAAQQ
PROF_sec HHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHH *.
*:********:***:****:: .* ***:*:**:****.:**::* ::* *:: : 130 140 150
160 170 180 | | | | | | STFlic
NEIDRVSGQTQFNGVKVLAQDN-TLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKY 1UCU
HHHHHHHHHH E EEEEE EEEEEE PROF_sec HHHHHHHH EEEEE EEEEEE EEEEEEEEEE
EEEEEEEEEEE SaFlix1
AELTRISDTTTFGGRKLLDGSFGTTSFQVGSNAYETIDISLQNASASAIGSYQVGSNGAG
PROF_sec HHHHHHHH EEEEE EEEE EEEEEEEEE EEE EEEEE *: *:*. * *.* *:*
. * ::***:* *****.*:: .:.::* :. : 190 200 210 220 230 240 | | | | |
| STFlic ##STR00024## 1UCU EEEEEE EEEEEEEE H HHH HHHHH EEEE EEE EEE
PROF_sec EEEE EEEEE EEE EEE EEE SaFlix1 ##STR00025## PROF_sec EEEEE
EEEEEEEEEE EEEEE EEEEEE EEE .*:..* **.:* *. *: * . . *:. . . :*:**
: .: .: Prim.cons.
2V222AGTAT22G2A22T22L22GGQVK2222A2222222222K2DG2222222222222 250
260 270 280 290 300 | | | | | | STFlic ##STR00026## 1UCU EEE EEEEEE
EEE EEE EEEEE HHHH PROF_sec EEEEEEEE EEEEEEE EEEEEEEE EEEE SaFlix1
##STR00027## PROF_sec E EEE EEEEEEEEE EEEEE EEEEEEE .. *:* ** .
::*:*... *:*** :*. : : :. ::. : : D3I site .uparw. .uparw. .uparw.
310 320 330 340 350 360 | | | | | | STFlic
KAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVG--DDYYSATQNKDGSISINTTKY 1UCU
HHHHHHH EEEEEEEEE EEEEEEEEEE EEEE EEE PROF_sec EEE EEEE EEEE EEE
EEEE E SaFlix1
KGVLTITSATGEN----VKFGAQTGTATAGQVAVKVQGSDGKFEAAAKNVVAAGTAATTT
PROF_sec EEEEEEE EE EEEEEEEE EEEE EEEE E *..** :..** :.: :.*.: *
:**** *. :.*: :: : . :*. 370 380 390 400 410 420 | | | | | | STFlic
TADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQ
FliCxx0
TADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQ 1UCU
EEE EEEEEE EEEE HHHH HHHH PROF_sec E EEEEE EEEE EEE HHHHHHHHH
SaFlix1
IVTGYVQLNSPTAYSVSGTGTQASQVFGNASAAQKSS------VASVDISTADGAQNAIA
PROF_sec EEEEEEEEE EEEEE EEEE EE EE HHHHHHHHH . . .. .: . . :. *:.
.: *:: **.*:. ..: ::* ::*.: 430 440 450 460 470 480 | | | | | |
STFlic KIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQ
1UCU HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHH
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHH
SaFlix1
VVDNALAAIDAQRADLGAVQNRFKNTIDNLTNISENATNARSRIKDTDFAAETAALSKNQ
PROF_sec HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHH HHHHHHHHHHHHHH
:* *** :*: *:*********:.:* ** * :* *.*****:*:*:*:*.: :*: * 490 500
| | STFlic ILQQAGTSVLAQANQVPQNVLSLLR (SEQ ID NO: 1) 1UCU
HHHHHHHHHHHHHHHHHHHHHH (empirical structure) PROF_sec HHHHHHHHHHHHH
HHHHHHH (predicted secondary structure) SaFlix1
VLQQAGTAILAQANQLPQAVLSLLR (SEQ ID NO: 4) PROF_sec HHHHHHHHHHHHH
HHHHHHH (predicted secondary structure) structure)
:******::******:** ******
[0186] The insertion site, referred to herein as "D3I-o1," in a
loop of domain 3 of S. typhimurium FljB (SEQ ID NO: 2) for fusion
to an antigen having an isoelectric point greater than about 7.5,
is between predicted .beta.-strands VTLA (SEQ ID NO: 131), which is
amino acid residues 263-266 of SEQ ID NO: 2, and VVS, which is
amino acid residues 293-295 of SEQ ID NO: 2. The insertion site,
referred to herein as "D3I-i1," in a loop of domain 3 is between
.beta.-strands EVNVA (SEQ ID NO: 132), which corresponds to amino
acid residues 254-258 of SEQ ID NO: 2, and VTLA (SEQ ID NO: 131,
which corresponds to amino acid residues 263-266 of SEQ ID NO: 2).
The insertion site, referred to herein as "D3I-s1," in a loop of
domain 3, is between .beta.-strands KYFVTIGG (SEQ ID NO: 133),
which corresponds to amino acid residues 234-241 of SEQ ID NO: 2,
and EVNVA (SEQ ID NO: 132), which corresponds to amino acid
residues 254-258 of SEQ ID NO: 2 in FljB (S. typhimurium, SEQ ID
NO: 2). The D3I-o1, D3I-i1 and D3I-s1 insertion sites predicted for
S. typhimurium FljB (SEQ ID NO: 2) were loop conformations in the
predicted secondary structures of E. coli (SEQ ID NO: 3) and P.
aeruginosa (SEQ ID NO: 4) flagellin. Therefore, with reference to
the loops in domain 3 of SEQ ID NO: 2, predicted loops in domain 3
and insertion sites, such as D3I-i1 and D3I-o1, can be selected for
E. coli (SEQ ID NO: 3) and P. aeruginosa (SEQ ID NO: 4)
flagellin.
[0187] Insertion sites in loops in domain 3 for fusion to antigens
with an isoelectric point greater than about 7.0 in E. coli FliC
flagellin (SEQ ID NO: 3), can include an D3I-o1 site between G274
and A275 of SEQ ID NO: 3 in the loop between .beta.-strand ITF
(amino acid residues 267-269 of SEQ ID NO: 3) and .beta.-strand
VLTANI (SEQ ID NO: 134, amino acid residues 284-289 of SEQ ID NO:
3). The D3I-i1 site is between A257 and D258 of SEQ ID NO: 3 in the
loop between .beta.-strand ELAKLAIKL (SEQ ID NO: 135, amino acid
residues 248-256 of SEQ ID NO: 3) and IEYK (SEQ ID NO: 136, amino
acid residues 262-265 of SEQ ID NO: 3). The D3I-s1 site was
selected between S240 and G241 of SEQ ID NO: 3 in the loop between
.beta.-strand KV (residues 238-239 of SEQ ID NO: 3) and
.beta.-strand SID (residues 243-245 of SEQ ID NO: 3).
[0188] For P. aeruginosa flagellin (PAO1, SEQ ID NO: 4), the D3I-o1
site was selected between Q272 and D273 in the loop between
.beta.-strand TVSLA (SEQ ID NO: 137, amino acid residues 262-266 of
SEQ ID NO: 4) and (3-strand LGITASI (SEQ ID NO: 138, amino acid
residues 285-291 of SEQ ID NO: 4). The D3I-i1 site was between S260
and N261 of SEQ ID NO: 4 in the loop between .beta.-strand
SLNFDVTVG (SEQ ID NO: 139, amino acid residues 251-259 of SEQ ID
NO: 4) and TVSLA (SEQ ID NO: 137, amino acid residues 262-266 of
SEQ ID NO: 4). The D3I-s1 site was selected between 5245 and G246
of SEQ ID NO: 4 in the loop between .beta.-strand TVFT (SEQ ID NO:
140, residues 238-241 of SEQ ID NO: 4) and .beta.-strand GVT
(residues 246-248 of SEQ ID NO: 4).
Example 2
Fusion Proteins of an Influenza B HA Antigen that Replaces Domain 3
of Flagellin
[0189] Compositions that include R3 constructs of flagellin fused
to portions of HA have been described (U.S. patent application Ser.
No. 12/905,584). Previously described R3 fusion proteins include
Influenza A HA antigen from PR8 (H1, A/Puerto Rico/8/34, SEQ ID NO:
141), seasonal SI03 (H1, A/Solomon Island/3/2006, SEQ ID NO: 142),
pandemic CA07 (H1, A/California/07/2009, SEQ ID NO: 143), and IN05
(H5, A/Indonesia/5/2005, SEQ ID NO: 144), which are immunologically
potent with minimal side-effects (U.S. patent application Ser. No.
12/905,584).
[0190] However, R3 constructs fused to Flu B HA1-2 portions having
a fl-sheet at the bottom of the globular head, such as
B/Florida/4/2006 (B/FL) of Yamagata lineage (HL098, SEQ ID NO:
122), were poorly immunogenic in a mouse model (FIG. 2A). In this
study, groups of 5-10 BALB/c mice (female, 6-8 weeks old) were
immunized twice (day 0 and day 21) subcutaneously (s.c.) or
intramuscularly (i.m.) with 10 .mu.g ( ) or 3 .mu.g (.box-solid.)
of HL098 (SEQ ID NO: 122) or with 10 .mu.g (.tangle-solidup.) or 3
.mu.g () of HL199 (SEQ ID NO: 145). The HL098 fusion protein
includes a flagellin that lacks domain 3 and replaces it with an
HA1-2 (SEQ ID NO: 45), which is referred to herein as an "R3
construct." The HL199 fusion protein includes a flagellin that
lacks domains 2 and 3 and replaces domains 2 and 3 with an HA1-2
(SEQ ID NO: 45), which are referred to herein as "R23 constructs."
Mice were bled on day 35 after the booster immunization. Serum
samples were prepared and stored at -20.degree. C. until use. Serum
antibody titers were measured by hemaglutination inhibition (HAI)
assay or microneutralization (MN) assay.
[0191] The HAI test was used to measure the influenza HA specific
antibodies in serum samples (Liu, G., et al., PLoS ONE 6(6):e20928
(2011); Kendal, A. P., et al., J. Clin. Microbiol. 18(4):930-934
(1983)). In this assay, immune serum samples, collected at various
times after immunization, were treated with receptor destroying
enzyme (RDE) II overnight to eliminate non-specific
hemagglutination inhibitors. The samples were then heat inactivated
(56.degree. C., about 30-45 min), serially diluted starting at
about a 1:5 ratio, and incubated with about 4 HA units (HAU) of
influenza B virus for about 30 minutes at room temperature. Turkey
red blood cells (0.5%) were incubated with the samples for about 30
to about 60 min at room temperature. HAI titers were measured and
reported as the reciprocal of the highest dilution at which
hemagglutination is completely inhibited. Ferret post infection
anti-influenza B virus (from the Center for Disease Control) is
used as reference serum. Serum HAI antibody titers were measured by
HAI test against B/Florida/4/06, and plotted as individual values
with geometric mean titers (GMTs). HL098 (SEQ ID NO: 122) elicits
no HAI titers.
[0192] F147 buffer, the negative control, contains 10 mM
L-histidine, 150 mM NaCl, 5% trehalose, 0.02% polysorbate 80, 0.1
mM EDTA, 0.5% ethanol, and 10 mM Tris at pH 7.2. F147 is a buffer
control (.diamond-solid.) and BV (baculovirus) expressed HA0
(.largecircle., full-length HA) is used as a positive control. As
shown in FIG. 2A, none of the mice generated neutralizing
antibodies to the original R3 format composition. A modest
improvement in immunogenicity was observed with the alternative
fusion protein format in which the HA1-2 portion (SEQ ID NO: 45)
replaced both D2 and D3 domain, termed R23 (HL199, SEQ ID NO: 145).
However, the activity was not sufficient (too low) to advance the
composition for use in methods for clinical development. In
subsequent testing, R23 (HL199; SEQ ID NO: 145) was used as a base
line for comparison to generate new, improved fusion proteins.
[0193] The predicted isoelectric point (pI) of the portion of HA of
influenza B (Flu B), for example, the HA1-2 portion of the globular
head of Flu B (SEQ ID NO: 45), is between about 8.5 to about 9.5,
which is relatively high compared that of H1 Influenza A that has a
pI of about 7.0, or those of H5 Influenza A that has a pI of about
8.0. The positively charged amino acids in a portion of a globular
head of Influenza B HA (e.g., SEQ ID NOs: 8, 9, 45, and 61) may
interact with the negatively charged flagellin at neutral pH. As a
result, an undesirable charge-charge interaction may distort the
fusion protein conformation, which could interfere with TLR5
signaling by the flagellin component of the fusion protein and
presentation of the antigen component of the fusion protein.
Various efforts to lower the pI of the Flu B globular head, such as
introducing negative amino acid residues in non-antigenic regions
were unsuccessful. The addition of negatively charged residues in
the linking region between HA head and flagellin improved TLR5
signaling and the fusion protein immunogenicity. For example, if 9
amino acid residues (SEQ ID NO: 123) having a negative charge were
fused to the C-terminus of the of an HA1-2 portion of influenza B
(HL352, SEQ ID NO: 125), which naturally contains two negatively
charged residues (E306 and D308), immunogenicity of the Flu B
fusion protein was moderately improved (FIG. 2B). This 9 amino acid
residue of SEQ ID NO: 123 is native to the naturally occurring HA.
In another embodiment, additional negatively charged amino acid
residues can be added to the portion of HA or negatively charged
amino acid residues can replace naturally occurring amino acid
residues at the carboxy-terminus of the HA.
[0194] In this study, serum was evaluated in the
microneutralization (MN) test (Rowe, T., et al., J. Clin.
Microbiol. 37:937-943 (1999); Song, L., et al. PLoS ONE 3:e2257
(2008)). Briefly, groups of 10 BALB/c mice were treated s.c. with
fusion protein compositions containing HL199 ( , SEQ ID NO: 145) or
HL352 (, SEQ ID NO: 126) on days 0 and 21, and bled on day 35.
Serum samples were treated with receptor destroying enzyme II,
serially diluted in duplicate, starting at a ratio of about 1:10
and co-cultivated with 100 TC.sub.ID50 (tissue culture infective
dose, TCID) of influenza B virus for about 1.5 hr. Madin-Darby
Canine Kidney (MDCK) cells (about 4.times..sup.104/well) were then
added and incubated for about 20 hours at about 37.degree. C. Cells
were washed, fixed, air-dried and incubated with a monoclonal
anti-influenza B nucleoprotein antibody (primary) and goat
anti-mouse Fc.gamma. specific IgG:HRP (secondary). Serum antibody
titers were measured by MN test against B/Florida/4/06 virus, and
plotted individually with GMTs (horizontal lines, FIG. 2B). Control
groups include the formulation buffer (+, F147, negative) and BV
(baculovirus) expressed HA0 (*, full-length HA, positive). The
negative charges in the linker fusing the HA portion to flagellin
may alleviate the charge-charge interactions and allow flagellin to
bind to TLR5 more efficiently, which results in adaptive immune
responses.
[0195] The ability of fusion proteins described herein to stimulate
the innate arm of the immune system, i.e., the TLR5 bioactivity,
was assessed by an in vivo TLR5 assay The assay consists of an
in-life phase and an in vitro serum cytokine rise readout. In
general, groups of BALB/c mice (5 per group) were injected with
vehicle control (naive) or test fusion protein compositions at
about 1 .mu.g each. Three hours later, mice were bled and the serum
was prepared and analyzed for inflammatory cytokines using a mouse
cytometric bead array (CBA) from BD (BD Biosciences, San Jose,
Calif.), and run on a flow cytometer.
[0196] For the cytokine assessment, only IL-6 and TNF-.alpha.
displayed a range of responses that are useful for characterizing
fusion protein composition candidates while others either produced
only baseline levels at the dose and time point chosen, or produced
plateau levels which are not useful for differentiation of
composition potency to stimulate TLR5 activation (data not shown).
Using naive serum levels, as well as those from low potency fusion
protein (STF2.HA1-2, B/Florida/4/2006, SEQ ID NO: 146) that
generated low micro-neutralization (MN) titers, thus the minimum
innate immune stimulation required for an immune response, 4 zones
of activity were established. These 4 zones of activity levels are
depicted in FIGS. 3A and 3B. The lowest level of cytokine activity,
which is designated as inactive, consists of levels less than about
or equal to the mean plus 3 standard deviations (SD) of that
detected in naive mice. The next level, which is designated as low,
consists of levels from greater than about the mean plus 3 SD of
the naive and less than about or equal to the mean plus 1 SD of low
potency fusion protein (SEQ ID NO: 146). The next zone, which is
designated as moderate, consists of values greater than about the
mean plus 1 SD of low potency fusion protein composition but less
than about or equal to the mean plus 3 SD of high potency fusion
protein composition (HL185, STF2R3.HA1-2, an R3 construct)
(A/California/07/2009, CA07), SEQ ID NO: 143). Finally, the fourth
zone, designated as high, is for values greater than about the mean
plus 3 Standard deviations (SD) of high potency composition.
[0197] In vivo TLR5 bioassays were employed to evaluate
charge-charge interactions that may influence TLR5 binding
activity. The addition of a 9 amino acid negatively charged linker
(SEQ ID NO: 123) to a fusion protein that includes an HA1-2 portion
of Influenza B/Florida/4/2006 to generate the fusion protein
referred to as HL352 (SEQ ID NO: 125) improved cytokine responses,
as shown by elevated IL6 and TNF-.alpha. responses (FIGS. 3A and
3B).
[0198] Similar results were obtained for a fusion protein employing
sequences from B/Brisbane/60/2008 (B/BR) as a prototypical
composition for the Victoria lineage (FIGS. 4A and 4B). FIGS. 4A
and 4B show the Balb/c mice were left naive (.diamond-solid.) or
injected with the indicated fusion protein composition for the B/BR
(Victoria lineage), at a 1 .mu.g dose: R3.HA1-2 B (, HL169, SEQ ID
NO: 147) and R3.HA1-2 B with a 9 amino acid linker B (.box-solid.,
HL483, SEQ ID NO: 148). The R3.HA1 ( , HL185, SEQ ID NO: 143)
fusion protein was included as a positive control. At 3 hours, mice
were bled to generate serum. Cytokine levels were quantified using
a mouse inflammation cytometric bead array (BD Biosciences, San
Jose, Calif.). Dotted lines in FIGS. 4A and 4B indicate the
activity levels discussed above.
[0199] An additional three negatively charged amino acid residues
were added to the carboxy-terminus of the portion of HA in the
fusion proteins HL610 (SEQ ID NO: 149) for B/FL and HL611 for B/BR
(SEQ ID NO: 150) for a total of 5 negatively charged amino acid
residue extension. The three negatively charged residues are native
to the naturally occurring HA sequence. For example, the three
negatively charged amino acid residues substitutions can be K298E,
S300D and 1304D in HA B/FL of SEQ ID NO: 124 or K299E, S301D and
1305D in HA B/BR60 of SEQ ID NO: 264. The three negatively charged
amino acid residues are not necessary a continuous stretch of amino
acid residues in the naturally occurring HA protein. The negatively
charged amino acid residues may reduce charge-charge interaction
between the positively charged portion of the HA globular head and
the negatively charged flagellin, to thereby minimize charge-charge
intramolecular interactions between the antigen and flagellin
component of the fusion proteins.
[0200] The in vivo TLR5 bioassay was performed as above with Balb/c
mice were left naive (.box-solid.) or injected with the indicated
fusion protein composition at a 1 .mu.g dose: B/FL (Yamagata
lineage) R3.HA1-2 B with a 9 amino acid linker of SEQ ID NO: 123
(.box-solid., HL352, SEQ ID NO: 125) and the same format with
additional negative charges (described supra, K298E, S300D and
1304D in HA B/FL in SEQ ID NO: 125 and K299E, S301D and 1305 in HA
B/BR60 in SEQ ID NO: 264 introduced in the linker ( , HL610, SEQ ID
NO: 149) and B/BR (Yamagata lineage) R3.HA1-2 B with a 9 amino acid
linker (SEQ ID NO: 123) (.tangle-solidup., HL169, SEQ ID NO: 147)
and the same format with additional negatively charged amino acids
introduced in the linker (.diamond-solid., HL611, SEQ ID NO: 150).
R3.HA1 ( , HL185, SEQ ID NO: 143) was included as a positive
control. Dotted lines in FIGS. 5A and 5B indicate thresholds of
cytokines levels correlated with immunogenicity as, described
above. Constructs HL610 (SEQ ID NO: 149) and HL611 (SEQ ID NO: 150)
improved the cytokine profiles compared to constructs HL352 (SEQ ID
NO: 125) and HL169 (SEQ ID NO: 147) (FIGS. 5A and 5B).
[0201] In addition to the in vivo TLR5 cytokine assay that was used
to assess the functionality of flagellin moiety, a neutralization
inhibition assay (NIA) was employed to evaluate the potency of the
HA moiety. NIA was used to measure the ability of a fusion protein
composition to compete with virus for binding to neutralizing
antibodies present in sheep hyper-immune serum (available from
CBER, Center for Biologics Evaluation and Research in FDA), similar
to that used for the potency release assay (SRID, Single Radial
Immunodiffusion) of a commercial influenza composition (TIV,
Trivalent Influenza Vaccine (TIV) containing two different
inactivated influenza type A strains and one inactive influenza
type B strain, e.g. FLUVORIN.RTM.), thereby assessing the integrity
of neutralizing epitopes on the fusion proteins described
herein.
[0202] In the NIA assay, higher potency of the fusion proteins was
demonstrated by increased depletion of the neutralizing antibodies
in the sheep hyper-immune serum, which allows the test virus to
infect MDCK cells. The sheep hyper-immune serum raised against the
test Flu B influenza virus (sheep anti-HA of B/Florida/4/06 serum
(FDA) or sheep anti-HA of B/Brisbane/60/08 serum (FDA)) was
pre-incubated with serially diluted test articles at about a 1:1
ratio for about 90 minutes. 50 TCID.sub.50 B/Florida/4/06 virus or
50 TCID.sub.50 B/Brisbane/60/08 virus was then added and allowed to
incubate for about 1 hour at 37.degree. C. prior to addition of
MDCK cells. Following an incubation of about 18 hours to about 20
hours of the mixture with MDCK cells at about 37.degree. C., the
cells were fixed with a cold solution of 80% acetone in DPBS (1.47
mM KH2PO4, 2.67 mM KCl, 138 mM NaCl, 8.06 mM Na2HPD4.7H.sub.2O,
Invitrogen Cat. No. 14190-250).
[0203] Intracellular influenza virus was quantified in an ELISA
(enzyme-linked immunosorbent assay) format using influenza B
NP-specific (nuclear protein-specific) mAbs (monoclonal antibodies)
as primary antibodies and horseradish peroxidase (HRP) conjugated
goat anti-mouse IgG (Jackson ImmunoResearch, Cat. no. 115-035-008).
After color development with TMB (3,3'-5,5'-tetramethylbenzidine)
substrate (ThermoScientific, Cat. no. 34028), the virus was
quantified by measuring OD.sub.450. NIA curves were generated by
Log-Logit fit model using Softmax software 5.2. The greater the
integrity of the neutralizing epitopes in the composition the
higher infectivity of the virus and the higher the signal in the
NIA assay.
[0204] To complement the TLR5 assay, NIA assays were performed to
determine the integrity of HA antigens in the Flu B compositions
that contain negative linkers (SEQ ID NOs: 149 for HL610 and 150
for HL611), described supra. As shown in FIG. 6, B/Florida/4/06 R3
compositions of the fusion proteins HL352 (SEQ ID NO: 125) or HL610
(SEQ ID NO: 149) exhibit comparable ability to inhibit antibody
neutralization. As shown in FIG. 7, B/Brisbane/60/08 R3
compositions of the fusion proteins HL169 (SEQ ID NO: 147), HL483
(SEQ ID NO: 148), and HL611 (SEQ ID NO: 150) also show similar
integrity of the neutralizing epitopes. These results indicate that
influenza B portions of HA globular heads fused to a loop of domain
3 of flagellin are folded similarly and present epitopes in a
conformation similar to that of the wild type virus. Given this
similarity, the difference in the ability of fusion proteins
described herein, such as FluB antigens fused to a loop of domain 3
with or without a 9 amino acid extension (SEQ ID NO: 123) or with
or without an additional three negatively charged amino acid
residues corresponding to amino acid residues of the naturally
occurring HA, to trigger TLR5 signaling becomes critical to
determine the fusion protein potency.
Example 3
Fusion Proteins with HA Antigen Inserted into Loop Regions of
Domains 1, 2, or 3 of Flagellin to Shift pI to Target Influenza
B
[0205] Although negative charges in the linker regions improved the
TLR5 signaling, the immunogenicity of these fusion proteins (SEQ ID
NOs: 149 and 150) remained suboptimal, especially for Victoria
lineages, such as B/BR. The negative linker may only locally adjust
the orientation of HA head with respect to flagellin, which may not
be sufficient to allow the formation of 2:2 heterodimers that are
critical to initiate a TLR5 signaling cascade. As described below,
a portion of an HA globular head was positioned further away from
domain 1 of flagellin, the TLR5 binding domain. For example, as
depicted in FIG. 14, domain 1 of S. typhurium FliC flagellin is
between amino acid residues 47-176 of SEQ ID NO: 2 (amino-domain 0)
and amino acid residues 415-464 of SEQ ID NO: 2 (carboxy-domain 0).
Fusion protein constructs (HL656, SEQ ID NO: 151, D3Ins of B/FL and
HL657, SEQ ID NO: 128, D3Ins of B/BR) were created by fusion of a
portion of the globular head of HA antigen into domain 3 of
flagellin, which are referred to as domain 3 insertions
("D3Ins").
[0206] As shown in FIGS. 8A and 8B, more favorable cytokine
profiles were observed with the D3Ins fusion protein as assessed by
in vivo TLR5 assays. As described above, Balb/c mice were left
naive (.diamond-solid.) or injected with the indicated composition
candidate, at about a 1 .mu.g dose: B/FL (Yamagata lineage) R3.HA B
(.box-solid., HL352, SEQ ID NO: 125) and D3Ins.HA B
(.tangle-solidup., HL656, SEQ ID NO: 151), B/BR (Victoria lineage)
R3.HA B (.tangle-solidup., HL169, SEQ ID NO: 147) and D3Ins.HA B (,
HL657, SEQ ID NO: 128). R3.HA1 ( , HL185, SEQ ID NO: 143) was
included as a positive control. Fusion proteins generated by
removal of domain 3 of flagellin and replacement with a portion of
an HA globular head, referred to as "R3" fusion proteins (e.g.,
R3.HAB) were made employing previously described methods (see, for
example, U.S. application Ser. No. 12/905,584). NIA assay did
detect an appreciable difference between D3Ins fusion protein and
the R3 fusion protein, indicating the antigen retained similar
integrity in both formats (FIGS. 9A-9C). The NIA assay assesses the
functionality of the portion of the globular head of HA.
[0207] The NIA assay was performed as described above using about a
1:1 ratio of serially diluted test articles B/Florida/4/06
(.largecircle., HL352, SEQ ID NO: 125 and .quadrature., HL656, SEQ
ID NO: 151) (FIG. 9A); B/Brisbane/60/08 (.quadrature., HL611, SEQ
ID NO: 150 and .largecircle., HL657, SEQ ID NO: 128) (FIG. 9B); or
B/Wisconsin/1/10 (.tangle-solidup., HL724, SEQ ID NO: 152 and
.largecircle., HL772, SEQ ID NO: 126) (FIG. 9C) mixed with sheep
anti-HA of B/Florida/4/06 serum (A), B/Brisbane/60/08 (B), or
B/Wisconsin/1/10 (C). It is believed that TLR5 signaling plays a
more critical role than neutralization inhibition in eliciting
protective immune responses. When mice were immunized with a D3Ins
fusion protein, D3Ins format compositions including HL610 (SEQ ID
NO: 149) (.largecircle., R3 fusion protein with 9 amino acid
extension and the addition of negatively charged amino acids (SEQ
ID NO: 153)) and HL656 (SEQ ID NO: 151) (.largecircle., D3Ins with
9 amino acid extension (SEQ ID NO: 123)) elicited stronger immune
responses than other fusion proteins, such as HL352 (SEQ ID NO:
125) ( , R3 with the 9 amino acid extension of SEQ ID NO: 123),
(FIG. 10). HA352 (SEQ ID NO: 125) is a R3 fusion protein that
includes an HA1-2 portion of the globular head of influenza B FL4.
Serum antibody titers in FIG. 10 were measured by MN test using
B/Florida/4/06 virus antigen. Data are shown as titers of
individual mice with GMTs and seroconversion rates (percent (%) of
mice with a MN titer.gtoreq.80) above each group. These data show
that the D3Ins format (HL656, SEQ ID NO: 151) was superior to the
R3 format for Flu B (HL352, SEQ ID NO: 125).
[0208] The immunogenicity of D3Ins fusion proteins listed in Table
3 were evaluated using B/Wisconsin/1/2010, a recently circulating
Yamagata lineage strain. In an immunogenicity study, groups of 6
BALB/c mice were immunized with 3 different formats of fusion
proteins that include flagellin and portions of the globular head
of the HA of Influenza B at a 6 .mu.g dose delivered s.c. in two
injections on day 0 and day 21 with the Flu B compositions: R3 B/WI
( , HL719, SEQ ID NO: 154, R3 with negative linker), R3 B/WI
(.box-solid., HL724, SEQ ID NO: 152, R3 with additional negative
charges in the linker), D3Ins B WI (.tangle-solidup., HL772, SEQ ID
NO: 126), or F147 buffer ( ). Mice were bled on day 35 for HAI
titers. Controls included a group immunized with F147 buffer (10 mM
L-histidine, 150 mM NaCl, 5% trehalose, 0.02% polysorbate 80, 0.1
mM EDTA, 0.5% ethanol, 10 mM Tris, pH 7.2). All fusion proteins
were prepared in F147 buffer. Sera were tested with HAI assays
using extracted B/Wisconsin/1/2010 virus antigen. Data are shown as
titers of individual mice with GMTs. Statistical differences were
determined in 1-way ANOVA/Tukey test with *, p<0.05, ***,
p<0.001.
[0209] As illustrated in FIG. 11, the highest titers in the
monovalent groups were seen with D3Ins fusion protein (HL772,
.tangle-solidup., SEQ ID NO: 126) followed by an R3 fusion protein
HL724 (.quadrature., SEQ ID NO: 152, B/WI counterpart of B/FL
HL610, SEQ ID NO: 149) and HL719 ( , SEQ ID NO: 154, B/WI
counterpart of B/FL HL352, SEQ ID NO: 125). Titers of both HL724
(R3 format) and HL772 (D3Ins format) groups were significantly
higher than the F147 control. This is consistent with the same
formats of B Florida and B Brisbane where the D3Ins was highest,
followed by R3 formats (FIG. 10 for B/FL, FIG. 12 for B Victoria
lineage, including Brisbane, Hong Kong and Bangladesh). Thus, as
predicted by TLR5 data, the D3Ins format improved immunogenicity to
the B/WI strain compared to the R3 formats for fusion proteins in
mice. A positive immunization control was not included, because the
immunogen for B/Wisconsin/1/10 was unavailable at the time of the
analysis.
[0210] HAI testing of immune sera from a mouse immunogenicity study
comparing R3 and D3Ins formats of B Brisbane-like strains was
performed. The same modifications were made to the HA antigen (SEQ
ID NOS: 95-97), and these antigens were employed in the R3 and
D3Ins formats. Groups of 6 BALB/c were immunized s.c. on days 0 and
21 with 6 .mu.g of fusion protein R3 compositions (HL611, SEQ ID
NO: 150; HL753, SEQ ID NO: 155; HL742, SEQ ID NO: 156) or fusion
protein D3Ins compositions of B/Brisbane/60/08-like viruses
(B/Brisbane/60/08 or B/BR, HL657, SEQ ID NO: 128; B/Hong
Kong/259/10 or B/HK, HL774, SEQ ID NO: 157; and
B/Bangladesh/5945/09 or B/BD, HL787, SEQ ID NO: 158), and bled on
day 35. Mice in control groups received 15 .mu.g FLUVIRIN.RTM. or
F147 buffer. Neutralizing antibody titers of the serum samples were
measured by HAI test using ether extracted B/Brisbane/60/08, B/Hong
Kong/259/10, or B/Bangladesh/5945/09 virus, and expressed as GMTs.
Statistical differences were determined in 2-way ANOVA/Tukey tests
with *, p<0.05, **, p<0.01.
[0211] Results of this study indicated that the D3Ins format of
B/Brisbane/60/2008-like fusion protein (B/Brisbane/60/08, B/BR SEQ
ID NO: 128; B/Hong Kong/259/10, B/HK SEQ ID NO: 157 and
B/Bangladesh/5495/09, B/BD SEQ ID NO: 158) elicited significantly
higher HAI titers than the strain-matched R3 format measured using
3 different B/Brisbane-like viruses (FIG. 12). D3Ins B/BD (HL787,
SEQ ID NO: 158) elicited HAI geometric mean titers comparable to
FLUVIRIN.RTM. containing B Brisbane, particularly when B/Hong Kong
and B/Bangladesh viruses were used as HAI antigens. These data show
that the D3Ins fusion proteins consistently have higher
hemagglutination inhibition than R3 fusion proteins across multiple
influenza B strains and within a cluster of antigenically similar
viruses. Fusion proteins that include portions of the globular head
of influenza B fused to a loop of domain 3 of flagellin improved
immunogenicity compared to HA portions fused to R3 constructs.
Example 4
Screening the Insertion Variant as a Flu B Fusion Protein
[0212] The initial design of the insertion fusion proteins targeted
the D3 domain as the insertion site. Three insertion sites (D3I-o1,
D3I-i1 and D3I-s1) in loops of domain 3 were selected for fusion of
portions of HA antigens (see, for example, FIGS. 29 and 30). In
addition to the BNFL fusion protein, B/Wisconsin/1/2010 (B/WI, SEQ
ID NO: 263), a currently circulating B Yamagata lineage was chosen
to develop seasonal Flu B fusion protein for use in methods of
stimulating immune responses. B/BR remains as another candidate in
fusion protein development representing another circulating B
Victoria lineage Flu B strain. The three constructs of B/WI (HL772,
D3I-o1, SEQ ID NO: 126; HL849, D3I-i1, SEQ ID NO: 159; and HL848,
D3I-s1, SEQ ID NO: 160) were cloned into the PET24a expression
vector, and proteins were purified from E. coli cell culture using
standard fermentation as previously described (Song, L., et al.,
PLoS One 3:e2257 (2008); Song, L., et al., Vaccine 27:5875-5884
(2009)).
[0213] The TNF-.alpha. and IL-6 cytokine levels of B/WI constructs
are depicted in FIGS. 13A and 13B. The R3 format of H1 CA07 R3.HA1
(HL185, also referred to as "VAX128," SEQ ID NO: 143) fusion
protein was used as a positive control. The CA07 vaccine R3.HA1
(HL185, SEQ ID NO: 143) fusion protein has been clinically proven
to be safe and immunogenic in humans (Taylor, D., et al., Vaccine
30:5761-5769 (2012) and a strong stimulator of TLR5. The positive
controls elicited high levels of TNF-.alpha. and IL-6.
[0214] The fusion proteins that include an HA antigen fused to a
loop of domain 3, B Wisconsin (Yamagata lineage), including HL772
(D3I-o1, SEQ ID NO: 126) and HL849 (D3I-i1, SEQ ID NO: 159),
elicited comparable IL-6 levels in the "High" zone with HL848
(D3I-s1, SEQ ID NO: 160) the lowest in the "Moderate" zone. The
TNF-.alpha. levels for HL772 (D3I-o1, SEQ ID NO: 126) and HL849
(D3I-i1, SEQ ID NO: 159) were lower compared to a positive control
but were well above the minimum threshold required to achieve
proper immune responses. The fusion protein HL848 (D3I-s1, SEQ ID
NO: 160) had TLR5 activity in the "Inactive" zone, indicating the
D3I-s1 format may not elicit a strong immune response (FIGS. 13A
and 13B). These data show that fusion proteins that include an
antigen fused to a loop of domain 3 of flagellin have fewer side
effects (are less reactogenic) than fusion proteins that include
antigens fused to R3 constructs of flagellin, yet elicit suitable
cytokine activity that is critical for immune responses. See Tables
1, 3 and 4 for a list of fusion proteins and sites of insertion in
flagellin.
[0215] To assess for a fusion protein that maximizes TLR5 signaling
and immunogenicity with minimal side effects, loops in domain 2 and
domain 1 flagellin, in addition to loops in domain 3 of flagellin,
were selected as possible sites of fusion to antigens. The
insertion (fusion) sites in flagellin were located in the loop
regions so the HA antigen could be inserted without disturbing the
overall flagellin structure conformation, including the tertiary
structure of domain 3 of flagellin as depicted in FIGS. 14 and 29.
The primary amino acid sequence of flagellin indicating the amino
acid residues of flagellin between which portions of the globular
head of HA from influenza B were fused is depicted in FIGS. 29 and
30. The predicted loops in domains of flagellin is based on a
comparison of the known tertiary structure of S. typhimuium FliC
flagellin, as discussed supra. Exemplary fusion proteins of the
invention include the Yamagata lineage B/Wisconsin/1/2010 listed in
Table 3, and Victoria lineage B/Brisbane/60/2008 listed in Table
4.
[0216] Table 3. Flu B Yamagata Lineage (B/Wisconsin/)/2010) Fusion
Proteins. The fusion protein includes a portion of the globular
head of HA (SEQ ID NO: 50) fused to a loop of a domain of S.
typhimuium FljB flagellin (SEQ ID NO: 2 or 127).
TABLE-US-00010 TABLE 3 Fusion Protein SEQ ID NO: Format Insertion
Site HL772 126 D3I-o1 276-277 in SEQ ID NO: 127 HL656 151 D3I-o1
276-277 in SEQ ID NO: 127 HL849 159 D3I-i1 259-260 in SEQ ID NO: 2
HL848 160 D3I-s1 244-245 in SEQ ID NO: 2 HL825 161 D2I-o1 187-188
in SEQ ID NO: 2 HL826 162 D2I-o2 178-179 in SEQ ID NO: 2 HL827 163
D2I-o3 326-327 in SEQ ID NO: 2 HL828 164 D1I-o1 102-103 in SEQ ID
NO: 2 HL850 165 D2I-i1 366-367 in SEQ ID NO: 2 HL888 169 D3I-o2
230-231 in SEQ ID NO: 2 HL890 170 D2I-c1 308-309 in SEQ ID NO: 2
HL892 171 D2I-i2 388-389 in SEQ ID NO: 2
[0217] Table 4. Flu B Victoria Lineage (B/Brisbane60/2008) Fusion
Proteins. The fusion proteins include a portion of the globular
head of HA (SEQ ID NO: 48) fused to a domain of S. typhimuium FljB
flagellin (SEQ ID NO: 2, 127, or 270).
TABLE-US-00011 TABLE 4 SEQ ID Fusion Protein NO: Format Insertion
Site HL657 128 D3I-o1 276-277 in SEQ ID NO: 127 HL733 166 D2I-o1
187-188 in SEQ ID NO: 270 HL856 167 D2I-o1 187-188 in SEQ ID NO: 2
HL857 168 D2I-o2 178-179 in SEQ ID NO: 2 HL858 172 D2I-o3 326-327
in SEQ ID NO: 2 HL860 173 D3I-s1 244-245 in SEQ ID NO: 2 HL889 174
D3I-o2 230-231 in SEQ ID NO: 2 HL891 175 D2I-c1 308-309 in SEQ ID
NO: 2 HL893 176 D2I-i2 388-389 in SEQ ID NO: 2 HL861 180 D3I-i1
259-260 in SEQ ID NO: 2 HL862 181 D2I-i1 366-367 in SEQ ID NO:
2
[0218] The fusion protein listed in Tables 3 and 4 were evaluated
using a TLR5 assay, as described supra. Comparison of fusion
proteins with insertions into domain 3 of flagellin (D3I) to
insertions into domain 2 of flagellin (D2I) are shown in FIGS.
15A-15B and 16A-16B. The fusion protein constructs used were B
Wisconsin (Yamagata lineage) D3I-o1 (HL772, SEQ ID NO: 126), D2I-o1
(HL825, SEQ ID NO: 161), D2I-o2 (HL826, SEQ ID NO: 162), D2I-o3
(HL827, SEQ ID NO: 163), D1I-o1 (HL828, SEQ ID NO: 164) and D2I-i1
(HL850, SEQ ID NO: 165) and B Brisbane (Victoria lineage) D3I-o1
(HL657, SEQ ID NO: 128), D2I-o1 (HL733, SEQ ID NO: 166), D2I-o1
(HL856, SEQ ID NO: 167) and D2I-o2 (HL857, SEQ ID NO: 168) (FIGS.
15A-15B). The fusion protein constructs used were B Wisconsin
(Yamagata lineage, FIG. 16A) D3I-o1 (HL772, SEQ ID NO: 126), D3I-o2
(HL888, SEQ ID NO: 169), D2I-c1 (HL890, SEQ ID NO: 170), D2I-i2
(HL892, SEQ ID NO: 171) and B Brisbane (Victoria lineage, FIG. 16B)
D3I-o1 (HL657, SEQ ID NO: 128), D2I-o3 (HL858, SEQ ID NO: 172),
D3I-s1 (HL860, SEQ ID NO: 173), D3I-o2 (HL889, SEQ ID NO: 174),
D2I-c1 (HL891, SEQ ID NO: 175) and D2I-i2 (HL893, SEQ ID NO: 176).
R3.HA1 (HL185, SEQ ID NO: 143) was included as a positive
control.
[0219] These data show that fusion proteins that include antigens
fused to loops in domain 3 of flagellin ("D3 insertion constructs,"
"D3I," "D3Ins," "D3Ins fusion proteins") had enhanced TLR5 activity
compared to fusion proteins that include an antigen fused to loops
in domain 2 of flagellin ("D2 insertion constructs"). Fusion
proteins that include an antigen fused to a loop in domain 1 of
flagellin ("D1 insertion constructs") were not active. Among the
D3Ins fusion proteins, D3I-o1 (HL772, SEQ ID NO: 21) and D3I-i1
(HL849, SEQ ID NO: 22) were more active than D3I-s1 (HL848, SEQ ID
NO: 23) (FIGS. 14-16).
[0220] Antibodies generated to fusion proteins having portions of
the globular head fused to domain 3, domain 2, or domain 1 were
compared for their ability to inhibit virus neutralization in an
NIA assay. The NIA assay data for antibodies generated to fusion
proteins include (B/Wisconsin/1/2010): D3Ins (.largecircle., HL772,
D3I-o1, SEQ ID NO: 126), various D2Ins (.DELTA., HL825, D2I-o1, SEQ
ID NO: 161; .diamond., HL826, D2I-o2, SEQ ID NO: 162; , HL827,
D2I-o3, SEQ ID NO: 163), and D1Ins (.box-solid., HL828, D1I-o1, SEQ
ID NO: 164), in comparison to R3 format (.tangle-solidup., HL724,
SEQ ID NO: 152) and are shown in FIG. 17. The NIA assay data for
D3I insertion fusion proteins in comparison to the D2I insertion
fusion proteins are shown in FIG. 18. The portion of the globular
head fused to a loop of domain 2 or a loop of domain 3 is has the
same amino acid sequence (SEQ ID NO: 50).
[0221] FIG. 18A shows NIA assay data for B/Wisconsin/1/2010 D3I
insertion variants: D3I-o1 (.largecircle., HL772, SEQ ID NO: 126),
D3I-s1 (.DELTA., HL848, SEQ ID NO: 160), D3I-i1 (.diamond., HL849,
SEQ ID NO: 159), and D3I (.tangle-solidup., HL864, SEQ ID NO: 177),
in comparison to the D2I-i1 ( , HL850, SEQ ID NO: 165) and D2I
(.box-solid., HL854, SEQ ID NO: 179) fusion proteins. FIG. 18A
shows the NIA assay data for B/Wisconsin/1/2010 D3I variants:
D3I-o1 (.largecircle., HL772, SEQ ID NO: 126), D3I-o2
(.quadrature., HL888, SEQ ID NO: 169), and D2I-c1 (.DELTA., HL890,
SEQ ID NO: 170), in comparison to D2I-i2 (.diamond., HL892, SEQ ID
NO: 171).
[0222] Relatively higher NIA titers were observed with D3 insertion
fusion proteins compared to the R3 format as demonstrated by the
comparison of HL772 (.largecircle., D3Ins, SEQ ID NO: 126) to HL724
(.tangle-solidup., R3, SEQ ID NO: 152) (FIG. 17), indicative of
slightly better antigen presentation for D3 insertion format than
R3 format. NIA titers indicate that fusion of an antigen to
flagellin can deplete serum neutralizing antibodies, which may not
correlate with the ability of the fusion protein to generate a
protective immune response. The ability to generate a protective
immune response includes a balance of the ability of the fusion
protein to activate TLR5 and stimulate a sufficient immune response
with minimum side effects.
[0223] Advantages of fusing antigens to a loop of domain 3 of
flagellin by inducing key cytokine secretion in response to
immunization is depicted in FIGS. 9A-9C. Taken together, the
cytokine and NIA results indicated that the D3 Insertion format of
flagellin was a superior format for fusing portion of the globular
head of influenza B compared to the R3 format. Furthermore, D3
insertion fusion proteins exhibited higher neutralizating antibody
titers compared to D2 insertion fusion proteins. Consistent with
the cytokine assay, D1 insertion fusion proteins were inactive in
NIA assay. Among three D3 insertion fusion proteins, D3I-o1 and
D3I-i1 again demonstrated a greater ability to neutralize viral
infection than D3I-s1. The NIA data is consistent with the cytokine
activation to assess TLR5 activation.
[0224] The NIA was employed to evaluate fusion proteins that
include portions of the globular head of influenza B for the Flu B
Victoria lineage using B/Brisbane/60/2008 as a prototype. The NIA
assay data for D3I fusion proteins: D3I-o1 (.largecircle., HL657,
SEQ ID NO: 128) and D3I-i1 (.box-solid., HL861, SEQ ID NO: 180),
compared to the R3 (.quadrature., HL611, SEQ ID NO: 150) fusion
protein are shown in FIG. 19A. FIG. 19B shows the NIA assay data
for B/Brisbane/60/2008 R3 (.quadrature., HL611, SEQ ID NO: 150)
fusion protein in comparison to other fusion proteins in which
portions of the globular head are fused to other regions of domain
3, or domain 2: D3I-o1 (.largecircle., HL657, SEQ ID NO: 128),
D2I-o1 (.DELTA., HL733, SEQ ID NO: 166), D2I-o1 (.box-solid.,
HL856, SEQ ID NO: 167), D2I-o2 (.tangle-solidup., HL857, SEQ ID NO:
168), and D2I-i1 (.diamond-solid., HL862, SEQ ID NO: 181). The NIA
assay data for D3I fusion proteins: D3I-o1 (.largecircle., HL657,
SEQ ID NO: 128) and D2I-o3 (.quadrature., HL858, SEQ ID NO: 172),
in comparison to D2I fusion proteins: D3I-s1 (.DELTA., HL860, SEQ
ID NO: 173), D3I-o2 (HL889, SEQ ID NO: 174), D2I-c1 (HL891, SEQ ID
NO: 175), D2I-i2 (HL893, SEQ ID NO: 176), and D2I-i1 (HL862, SEQ ID
NO: 181), are shown in FIG. 20.
[0225] As shown in FIG. 19A, the D3Ins fusion proteins were
comparable to the R3 fusion protein in neutralization inhibition,
which indicated similar integrity in both fusion protein formats.
When the in vivo cytokine data for the TLR5 test was evaluated, it
was determined that the R3 format fusion protein of
B/Brisbane/60/2008 was a poor trigger of cytokine release, which is
believed to be a key event to elicit an adaptive immune response.
Therefore, the D3Ins fusion protein is an exemplary format for Flu
B Victoria lineage.
[0226] As shown in FIG. 19B, the D2Ins fusion proteins was similar,
generating activity slightly lower as compared to D3Ins fusion
proteins. However, the TLR5 activity of the D2Ins fusion protein
constructs was significantly lower than D3Ins fusion proteins
(FIGS. 15A, 15B, and 16B). Consequently, D3Ins fusion proteins,
such as HL657 (D3I-o1, SEQ ID NO: 128) and HL861 (D3I-i1, SEQ ID
NO: 180), are believed to be better for methods of stimulating an
immune response.
[0227] In conclusion, fusion of an antigen with an isoelectric
point greater than about 7.0 to a loop of domain 3 of flagellin
("D3Ins fusion proteins") can significantly improve TLR5
bioactivity compared to the R3 format. In the NIA functional assay,
D3Ins fusion proteins also demonstrated equivalent or greater
activity to compete with virus for neutralizing antibodies. D3
insertion format is suitable, for example, as a fusion protein
format for use in fusing portions of the globular head of the HA
influenza B to flagellin, and is superior to either R3 or D2Ins
formats for use with antigens of HA of influenza B, or influenza A
antigens that have an isoelecric point greater than about 7.0, such
as about 7.5, about 8.0 or about 8.5., about 9.0, about 9.5, about
10.5 or about 11.0.
Example 5
Immunogenicity of Fusion Proteins with HA Antigen Inserted into
Loop Regions of Domain 3 of Flagellin to Shift pI to Target
Influenza B in a Mouse Model
[0228] The immunogenicity of three different fusion proteins
inserted into three different regions of loops of domain 3 was
evaluated in mice. These fusion proteins were D3I-o1, HL772, (SEQ
ID NO: 126), D3I-i1, HL849 (SEQ ID NO: 159), and D2I-i1, HL850 (SEQ
ID NO: 165) of B/Wisconsin/1/2010 (B/WI). Groups of 8 BALB/c mice
were treated i.m. with 2 dose levels (3 .mu.g and 12 .mu.g) of
D3I-o1 B/WI (HL772, SEQ ID NO: 126), D3I-i1 B/WI (HL849, SEQ ID NO:
159), and D2I-i1B/WI (HL850, SEQ ID NO: 165) fusion protein on days
0 and 21. All animals were bled on day 35. Serum HAI antibodies
were measured by HAI test using ether-extracted B/Wisconsin/1/2010
virus, and horizontal lines represented geometric mean titers
(GMTs) (GMT values above the lines above) in FIG. 21A.
Seroconversion rates (% of mice showing an HAI.gtoreq.40) were also
given in FIG. 21A as percentages. Statistical differences were
determined against F147 buffer control group in ANOVA tests with
***p<0.001.
[0229] As shown in FIG. 21A, D3I-o1 B/WI elicited about 2-fold
higher HAI antibody titers compared to D3I-i1 B/WI. Inactivated
virus antigen of B/Hubei-Wujiagang/158/2009 (a
B/Wisconsin/1/2010-like virus obtained from CBER, HA HU at 6 .mu.g
in FIG. 21A) was used as a positive control and also elicited
significant HAI titers. In contrast, D2I-i1 B/WI (HL850) and
baculovirus expressed rHA0 of Protein Sciences induced
significantly lower HAI titers. Buffer F147 was used as a negative
control (FIG. 21A). Therefore, the immunogenicity of three
B/Wisconsin/1/2010 fusion proteins ranked as
D3I-o1>D3I-i1>D2I-i1, consistent with results from cytokine
tests and NIA assays.
[0230] The D3Ins fusion proteins of B/Sichuan/379/1999 (B Yamagata
lineage included in 2001/2002 TIV and B/Florida/4/2006 (in
2008/2009 TIV) were also evaluated for immunogenicity as shown in
FIG. 21B. Groups of 10 mice were immunized s.c. with 3 different
doses (0.05 .mu.g, 0.5 .mu.g and 5 .mu.g) of B/Sichuan/379/1999
D3I-o1 (HL863, SEQ ID NO: 182) or D3I-i1(HL903, SEQ ID NO: 183)
fusion protein on days 0 and 21. As shown in FIG. 21B, the D3Ins
fusion proteins of B/Sichuan/379/1999 were immunogenic with no
statistically significant difference between of or it formats. In
general, the D3Ins is more immunogenic than the D2Ins
constructs.
Example 6
D3Ins Fusion Protein Format
[0231] In order to confirm the general applicability of the D3Ins
format of flagellin for use in generating fusion proteins of
portions of the globular head of influenza B, fusion proteins for
three currently circulating strains in the Yamagata lineage were
evaluated in a mouse immunogenicity study. In this study, treatment
of groups of 8 BALB/c mice immunized with 2 doses (3 .mu.g and 12
.mu.g) of fusion protein prepared with HA sequence from
B/Wisconsin/1/2010 (D3Ins B/WI, HL772, SEQ ID NO: 126);
B/Hubei-Wujiagang/158/2009 (D3Ins B/HU HL869, SEQ ID NO: 184); and
B/Texas/6/2011 (D3Ins B/TX, HL871, SEQ ID NO: 185). HL869 includes
a portion of HA of SEQ ID NO: 58. HL871 includes a portion of HA of
SEQ ID NO: 60.
[0232] As shown in FIG. 22, the D3Ins fusion proteins of the three
strains elicited significant HAI titers (GMTs=71-283 in 3 .mu.g
dose groups; 273-719 in 12 .mu.g dose groups). Furthermore, D3Ins
B/WI at a 3 .mu.g dose elicited a 12-fold higher geometric mean HAI
titer (GMT=283) and a higher seroconversion rate (100%) than those
of the baculovirus expressed full length Wisconsin HA (rHA, HA0 WI,
Protein Sciences Corporation, Meriden, Conn.) at a 6 .mu.g dose
(GMT=23, SC=38%). Therefore, the D3 insertion format fusion protein
that includes a portion of the globular head of influenza B
Wisconsin (SEQ ID NO: 126) was more immunogenic in mice than the
rHA WI (Protein Sciences) and was comparable to the inactivated
B/HB virus antigen (obtained from CBER).
[0233] Multiple Victoria lineage strains were also evaluated. In
particular, the Brisbane-like strains that were recommended for
2009-2011 trivalent influenza vaccines and are still recommended
for a quadrivalent influenza vaccine in 2013-14 were evaluated.
Having previously demonstrated improved potency of the B
Brisbane-like strain B/Bangladesh/5945/2009 in mice, the
immunogenicity of the Victoria prototypic fusion protein was
evaluated in the rabbit model. Six NZW (New Zealand White) rabbits
(3 of each gender) were treated i.m. with the indicated doses (6
.mu.g, 12 .mu.g, and 15 .mu.g) of fusion proteins that include an
antigen fused to a loop of domain 3 for either B/Brisbane/60/2008
(SEQ ID NO: 264) or B/Bangladesh/5945/2009 (SEQ ID NO: 265).
FLUVIRIN.RTM. 2011-12 (TIV, 15 .mu.g total) was included as a
positive immunogenicity control, and F147 buffer was included as a
negative control. HAI titers were performed with ether-extracted
virus matched to the composition: CBER anti B/Brisbane/60/2008
1:5,120 or ferret anti B/Bangladesh/5945/2009 1:10,240. Data were
shown as titers of individual rabbits with bars.
[0234] Horizontal lines represented geometric mean titers (GMT
values above), and seroconversion percentage was provided in FIG.
23. As shown in FIG. 23, the Bangladesh fusion protein (HL787, SEQ
ID NO: 158) was more immunogenic than the Brisbane fusion protein
(HL657, SEQ ID NO: 128), with 100% seroconversion, when tested with
the matched virus. The Brisbane-like Bangladesh strain, (D3Ins
B/BR-like) as the Victoria lineage candidate may be useful in
methods of providing protective immunity.
[0235] Fusion proteins of antigens fused to a loop of domain 3 were
further evaluated for suitability across at least three recent and
historical strains. Consistent with this strategy, testing of
historical B candidates was carried out using D3Ins format of
B/Sichuan/379/1999, a historical flu B virus (Yamagata lineage,
STF2D3Ins B/SI, HL863, SEQ ID NO: 182). The portion of HA fused to
the D3-o1 site (see loop 6 of FIGS. 29 and 30) is SEQ ID NO: 56.
HAI testing of mouse immune sera raised against B/Sichuan/379/1999
virus was performed. In this experiment, groups of 10 BALB/c mice
were treated s.c. twice on days 0 and 21 with the STF2D3InsB/SI
(HL903, SEQ ID NO: 183) fusion protein at a dose of 6 .mu.g or with
formulation buffer (F147). Mice were bled on day 35. Serum HAI
antibodies were measured using ether-extracted B/Sichuan/379/1999
virus, and values were plotted individually with GMTs and
seroconversion indicated above. The immunogenicity results in FIG.
24 showed that the D3Ins composition of B Sichuan (SEQ ID NO: 182)
at a 6 .mu.g dose elicited a GMT of 132 with a seroconversion rate
of 100%. These data show that the D3Ins is suitable for use in
generating compositions that include fusion protein of flagellin
and portions of the globular head of influenza B for use in methods
to treat seasonal exposure to influenza.
Example 7
Efficacy of Fusion Proteins with HA Antigen Inserted into Loop
Regions of Domain 3 of Flagellin to Shift pI to Target Influenza B
in Mice
[0236] In addition to immunogenicity testing of the D3Ins fusion
proteins of the historical B virus, B/Sichuan/379/99 (D3Ins B/SI,
HL863, SEQ ID NO: 182, Yamagata lineage) described above, efficacy
testing in a lethal mouse challenge model was evaluated. Groups of
10 BALB/c mice were treated s.c. with the indicated doses (0.05
.mu.g, 0.5 .mu.g, and 5 .mu.g) of D3Ins B/SI 99 (HL863, SEQ ID NO:
182) fusion protein or F147 buffer control on days 0 and 21, and
bled on day 35. On day 42, mice were challenged intranasally with
5.times.LD.sub.50 of B/Sichuan/379/1999 10.sup.5 pfu per mouse and
monitored daily for mortality for 21 days and weight change for 14
days.
[0237] Survival rates and weights (mean percentage of initial
weight) were plotted in FIGS. 25A-25B. Doses of about 5 .mu.g and
about 0.5 .mu.g fusion protein D3Ins B/SI composition (two
immunizations each) resulted in a 100% survival rate and about less
than 12% weight loss in mice challenged with a lethal dose of
B/Sichuan/379/99 virus (FIGS. 25A-25B). In contrast, the placebo
group showed about a 20% survival rate and greater than or equal to
about a 75% weight loss. These results indicated that the D3Ins
fusion protein composition of B/Sichuan/379/99 was efficacious at a
submicrogram dose. The efficacy of D3Ins B/SI in this study thus
supported the general suitability of D3Ins format for use in
compositions that include portions of influenza B.
Example 8
Immunogenicity and Safety of Fusion Proteins with HA Antigen
Inserted into Loop Regions of Domain 3 of Flagellin to Shift pI to
Target Influenza B in a Rabbit Model
[0238] The immunogenicity of D3Ins fusion proteins was also
evaluated in a rabbit model. A rabbit study designed to evaluate
the immunogenicity of a range of doses of the R3 and D3Ins formats
of B/WI was performed. HAI titers elicited by R3 and D3Ins formats
of B Wisconsin in rabbits were measured. Fusion protein R3 B
Wisconsin (HL724, SEQ ID NO: 152) and D3Ins B Wisconsin (HL772, SEQ
ID NO: 126, a D3I-o1 insertion, see Table 1) doses of 6, 12 and 18
.mu.g were evaluated in groups of 6 New Zealand White rabbits, 3 of
each gender (Covance Research Products, Denver, Pa.). For this
study, fusion proteins were injected i.m. on days 0 and 21, and
serum was collected on day 35 for HAI analysis. A blend of
baculovirus produced HA or egg-produced inactivated virus for
matching strains (5 .mu.g each) was included as a positive control,
and F147 buffer was included as a negative control. HAI titers were
performed with ether-extracted B/Wisconsin/1/210 virus.
[0239] Data are shown as titers of individual rabbits, values
plotted individually with GMTs and seroconversion indicated above
(FIG. 26). One of the aims of the study was to confirm that the
D3Ins fusion protein format of B/WI (HL772, SEQ ID NO: 126) was
superior to the R3 format (HL724, SEQ ID NO: 152). As shown in FIG.
26, the fusion protein R3 format elicited low HAI titers and only
achieved a high seroconversion rate (about 67%) at 18 .mu.g with a
GMT of about 22. By contrast, the fusion protein D3Ins format
generated serconversion rates greater than or equal to about 50% at
all three doses with a peak response at 12 .mu.g (about 83%). This
result confirmed previous mouse results demonstrating the
superiority of the D3Ins format. The HL772 (SEQ ID NO: 126) may be
useful in methods of treating subjects to provide protective
immunity.
[0240] In order to establish a suitable dose range for the rabbit
model in terms of safety and immunogenicity, a dose range study
with D3Ins B/WI (HL772, SEQ ID NO: 126) was performed. Groups of 10
NZW rabbits (5 of each gender) were treated i.m. with doses (3
.mu.g, 6 .mu.g, 9 .mu.g, 12 .mu.g, 15 .mu.g, and 18 .mu.g) of the
STF2D3Ins B/WI (HL772, SEQ ID NO: 126) fusion protein, inactivated
B/Hubei-Wujiagang/158/2009 virus (5 .mu.g Hubei virus, CBER), or
formulation buffer (F147) on days 0 and 21, and bled on day 35.
Serum HAI antibodies were measured by HAI test using
ether-extracted B/Wisconsin/1/2010 virus, and plotted individually
with GMTs and seroconversion.
[0241] The immunopotency results are shown in FIG. 27. Similar to
the results with mice, strong HAI titers were elicited by doses in
the low to mid microgram range, in this case with seroconversion of
100% occurring at doses as low as about 6 .mu.g. The higher titers
observed in this study were likely the result of a higher purity
protein preparation. These results indicated that strong influenza
B titers can be observed with low doses of the D3 insertion fusion
protein that includes an influenza B antigen in multivalent mixes,
thereby leading to a low total dose and a suitable safety window
for multivalent blends for compositions that can be employed in
methods of stimulating an immune response to the antigen, in
particular providing protective immunity to a disease causing
organism that includes the antigen.
[0242] The reactogenicity of D3Ins fusion proteins was examined in
a rabbit safety model employing techniques previously been
described (Taylor, D. N., et al., Vaccine 30:5761-5769 (2012)). In
this model, three measures are found to be predictive of the
appearance of adverse events in humans: poor appetite (low food
consumption) within 24 hours of prime, increase in body temperature
6 hours after prime, and elevated serum CRP 24 hours
post-prime.
[0243] Rabbits were treated with either formula buffer control
(F147) as a negative control, or a fusion protein in which an
antigen with an isoelectric point at least about 7.5
(B/Wisconsin/2010) is fused to a loop of domain 3 (HL772, SEQ ID
NO: 126) at varying doses (3 .mu.g, 6 .mu.g, 9 .mu.g, 12 .mu.g, 15
.mu.g, and 18 .mu.g). Food consumption was measured about 24 hours
after immunization (FIG. 28A). Temperature was measured rectally 6
hours post-immunization (FIG. 28B). CRP was measured from serum
taken 24 hours after prime (FIG. 28C). Data for all measures were
shown as results of individual rabbits with lines representing
means and standard error of the mean. Dotted lines represent the
safety threshold calculated using the data from formula control
rabbits. Using the mean of the formulation control animals from
several studies and using confidence intervals or multiples of
standard deviations, safety thresholds were developed for each of
the three measures. In this study a range of doses of fusion
proteins in which antigens of influenza B of Wisconsin have been
fused to loop 6 of domain 3 of flagellin (FIGS. 29 and 30) were
evaluated
[0244] The results show that the highest doses of B Wisconsin were
safe to a dose of about 18 .mu.g (D3Ins B Wisconsin, HL772, SEQ ID
NO: 126). Doses from about 3 to about 18 .mu.g resulted in little
decrease in food consumption (FIG. 28A), with all group means
falling within the safe threshold. Similarly, elevation in
temperature at about 6 hours was minimal over the tested dose range
(FIG. 28B), with all values below the safety thresholds. Finally
serum CRP at about 24 hours showed only modest elevation up to
about 18 .mu.g (FIG. 28C). It is believed that doses as high as
about 18 .mu.g of D3Ins of B Wisconsin may be suitable (i.e., safe)
as an influenza B component in a multivalent blend that can be
employed in a method to stimulate an immune response to influenza
B, in particular a protective immunity response to disease
consequent or associated with influenza B infection.
[0245] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0246] While this invention has been particularly shown and
described with references to example embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140255438A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20140255438A9).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References