U.S. patent application number 15/573701 was filed with the patent office on 2018-12-06 for methods and compositions for inducing an immune response using conserved element constructs.
This patent application is currently assigned to THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND. The applicant listed for this patent is THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND, UNIVERSITY OF WASHINGTON. Invention is credited to Barbara K. Felber, Siriphan Manocheewa, James I. Mullins, George N. Pavlakis, Antonio Valentin.
Application Number | 20180346520 15/573701 |
Document ID | / |
Family ID | 56081603 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180346520 |
Kind Code |
A1 |
Felber; Barbara K. ; et
al. |
December 6, 2018 |
METHODS AND COMPOSITIONS FOR INDUCING AN IMMUNE RESPONSE USING
CONSERVED ELEMENT CONSTRUCTS
Abstract
The invention provides methods and compositions for eliciting
broad immune responses to HIV envelope proteins. The methods
include use of nucleic acids constructs that encode conserved
elements of HIV Env to induce immune responses.
Inventors: |
Felber; Barbara K.;
(Rockville, MD) ; Pavlakis; George N.; (Rockville,
MD) ; Mullins; James I.; (Seattle, WA) ;
Valentin; Antonio; (Frederick, MD) ; Manocheewa;
Siriphan; (Seattle, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE
DEPARTMENT OF HEALTH AND
UNIVERSITY OF WASHINGTON |
Rockville
Seattle |
MD
WA |
US
US |
|
|
Assignee: |
THE UNITED STATES OF AMERICA AS
REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH
AND
Rockville
MD
HUMAN SERVICES
Seattle
WA
UNIVERSITY OF WASHINGTON
|
Family ID: |
56081603 |
Appl. No.: |
15/573701 |
Filed: |
May 13, 2016 |
PCT Filed: |
May 13, 2016 |
PCT NO: |
PCT/US2016/032317 |
371 Date: |
November 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62161123 |
May 13, 2015 |
|
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62241599 |
Oct 14, 2015 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/02 20130101;
A61K 2039/545 20130101; A61K 39/21 20130101; A61K 2039/572
20130101; A61K 9/0019 20130101; A61K 2039/575 20130101; C12N
2740/16234 20130101; A61K 2039/53 20130101; A61K 2039/55522
20130101; A61K 2039/54 20130101; C12N 2740/16222 20130101; C12N
2740/16122 20130101; C07K 14/161 20130101; A61K 9/0009 20130101;
A61K 39/39 20130101; C07K 14/535 20130101; C12N 2740/16271
20130101; C12N 2740/16134 20130101; C07K 14/005 20130101; C12N
2740/16171 20130101; A61K 2039/55561 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61K 9/00 20060101 A61K009/00; A61K 39/21 20060101
A61K039/21; A61K 39/39 20060101 A61K039/39; C07K 14/535 20060101
C07K014/535 |
Claims
1. A method of inducing an immune response to a protein of
interest, the method comprising: priming an immune response to the
protein of interest, wherein priming comprises administering at
least one nucleic acid construct encoding a conserved element
polypeptide, wherein the conserved elements are from the protein of
interest and the polypeptide comprises at least three conserved
elements, each from 8 to 50 amino acids in length where the
conserved elements are joined by non-naturally occurring linkers,
with the proviso that a nucleic acid construct encoding the
fill-length protein, or a substantially full-length region thereof,
is not administered in the priming step; boosting the immune
response to the protein of interest, wherein bolting comprises
co-administering (i) a nucleic acid construct encoding the
full-length protein, or substantially full-length protein, and (ii)
the nucleic acid construct encoding the conserved element
polypeptide.
2. The method of claim 1, wherein the boosting step comprises
administering the nucleic acid of (i) and the nucleic acid of (ii)
at the same time.
3. The method of claim 1or 2, wherein the nucleic acid constructs
are administered intramuscularly by in vivo electroporation.
4. The method of any one of claims 1 to 3, wherein the priming and
boosting steps further comprise administering a second conserved
element nucleic acid construct that encodes a second conserved
element polypeptide that comprises at least one variant of a
conserved element contained in the first conserved element
polypeptide, wherein the variant in the second polypeptide differs
from the variant in the first polypeptide by 1, 2, 3, 4, or 5 amino
acids.
5. The method of claim 4, the first and second conserved element
nucleic acid constructs are administered sequentially.
6. The method of claim 4. the first and second conserved element
nucleic acid constructs are administered concurrently.
7. The method of any one of claims 1 to 6, wherein the protein of
interest is HIV-1 Gag and the conserved elements are from HIV-1
p24.sup.gag.
8. The method of claim 7, wherein one conserved element polypeptide
comprises at least 7 conserved elements from different regions of
p24.sup.gag; and each of the conserved elements has a conserved
element sequence shown in Table 2.
9. The method of claim 8, wherein one conserved element polypeptide
comprises conserved elements that each have a sequence set forth in
SEQ ID NOS:26, 27, 28, 29, 30, 31, and 32; and a second conserved
element polypeptide comprises conserved elements that each have a
sequence set forth in SEQ ID NOS: 33, 34, 35, 36, 37, 38, and
39.
10. The method of claim 9, wherein one Gag conserved element
polypeptide comprises the amino acid sequence of SEQ ID NO:40 and
the second Gag conserved element polypeptide comprises the amino
acid sequence of SEQ ID NO:41.
11. The method of claim 9 or 10, wherein the conserved element
polypeptides encoded by the first and second nucleic acid Gag
conserved element nucleic acid constructs are each fused to a
GM-CSF signal peptide.
12. The method of any one of claims 1 to 6, wherein the protein of
interest is an HIV-1 Env sequence.
13. The method of claim 12, wherein one conserved element
polypeptide comprises at least 12 conserved elements from different
conserved regions of Env of HIV-1 Group M and each of the conserved
elements has a conserved element sequence shown in Table 1.
14. The method of claim 13, wherein one conserved element
polypeptide comprises conserved elements that each have a sequence
set forth in SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and
22; and a second conserved element polypeptide comprises conserved
elements that each have a sequence set forth in SEQ ID NOS:1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, and 23.
15. The method of claim 14. wherein one Env conserved element
polypeptide comprises the amino acid sequence of SEQ ID NO 24 and
the second Gag conserved element polypeptide comprises the amino
acid sequence of SEQ ID NO:25.
16. The method of claim 14 or 15, wherein the conserved element
polypeptides encoded by the first and second nucleic acid Env
conserved element nucleic acid constructs are each fused to a
GM-CSF signal peptide.
17. The method of any one of claims 4 to 16, wherein the first and
second nucleic acid conserved element nucleic acid constructs are
contained in the same vector.
18. The method of claims 4 to 16, wherein the first and second
nucleic acid constructs are contained in different vectors.
19. The method of any one of claims 1 to 18, wherein boosting is
performed at least about two weeks after the priming step.
20. The method of any one of claim 1 to 19, wherein the priming
step comprises two or three administrations of the one or more
conserved element polypeptides, each separated by at least about 2
weeks; and the boosting step comprises two co-administrations, each
separated by at least about 2 weeks.
21. A method of inducing an immune response to HIV Gag in a
subject, the method comprising: priming an immune response to HIV
Gag, wherein priming comprises administering to the subject a
CE-encoding expression vector that comprises a nucleic acid
sequence encoding p24.sup.GagCE1 as set forth in Table 2 fused at
the N-terminus to a human GM-CSF signal peptide and a nucleic acid
sequence encoding p24.sup.GagCE2 as set forth in Table 2 fused at
the N-terminus to a human GM-CSF signal peptide; wherein the
subject is not co-administered or has not previously been
administered a recombinant nucleic acid construct encoding a
full-length or substantiallyl full-length HIV Gag polypeptide;
boosting an immune response to HIV Gag, wherein boosting comprises
co-delivering the CE-encoding expression vector with an expression
vector that encodes full-length HIV-1 p55.sup.gag or substantially
full-length HIV-1 p55.sup.gag.
22. The method of claim 21, wherein the boosting step is performed
about two months after the priming step.
23. The method of claim 21 or 22, wherein the method comprises two
or three priming steps, each separated by about two months; and two
boost steps, each separated by about two months.
24. A nucleic acid encoding an immunogenic HIV Env conserved
element polypeptide, wherein the HIV Env conserved element
polypeptide comprises at least five conserved elements selected
from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4; SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the conserved elements
are separated by linkers of 1 to 5 amino acids in length.
25. The nucleic acid of claim 24, wherein the HIV Env conserved
element polypeptide comprises at least six, seven, eight, nine,
ten, or eleven conserved elements selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and
SEQ ID NO:22
26. The nucleic acid of claim 24, wherein the HIV Env conserved
element polypeptide comprises the conserved elements of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ NO:8, SEQ ID
NO:10, SEQ NO:12, SEQ NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, and SEQ ID NO:22.
27. The nucleic acid of claim 24, 25, or 26, wherein the HIV Env
conserved element polypeptide further comprises a V1V2 variable
region sequence.
28. The nucleic acid of claim 24, wherein the HIV Env conserved
element polypeptide comprises SEQ ID NO:24.
29. A nucleic acid encoding an immunogenic HIV Env conserved
element polypeptide, wherein the HIV Env conserved element
polypeptide comprises at least five conserved elements selected
from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, and SEQ ID NO:23; and the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
30. The nucleic acid of claim 29, wherein the HIV Env conserved
element polypeptide comprises at least six, seven, eight, nine,
ten, or eleven conserved elements selected from SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and
SEQ ID NO:23.
31. The nucleic acid of claim 29, wherein the HIV Env conserved
element polypeptide comprises the conserved elements of SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, and SEQ ID NO:23.
32. The nucleic acid of claim 29, 30, or 31, further comprising a
V1V2 variable region sequence.
33. The nucleic acid of claim 29, wherein the HIV Env conserved
element polypeptide comprises SEQ ID NO:25.
34. The nucleic acid of any one of claims 24 to 33, wherein the HIV
Env conserved element polypeptide comprises a signal peptide.
35. The nucleic acid of claim 34, wherein the signal peptide is the
signal peptide of GM-CSF.
36. An immunogenic HIV Env conserved element polypeptide comprising
at least five conserved elements selected from SEQ ID NO: 1, SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ NO:20, and SEQ
ID NO:22, wherein the conserved elements are separated by linkers
of 1 to 5 amino acids in length
37. The immunogenic HIV Env conserved element polypeptide of claim
36, wherein the polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ NO:1, SEQ
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ NO:8, SEQ NO:10, SEQ ID NO:12,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID
NO:22
38. The immunogenic HIV Env conserved element polypeptide of claim
36, wherein the polypeptide comprises the conserved elements of SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO: 10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, and SEQ ID NO:22.
39. The immunogenic HIV Env conserved element polypeptide of claim
36, 37, or 38, wherein the polypeptide further comprises a V1V2
variable region sequence.
40. The immunogenic HIV Env conserved element polypeptide of claim
36, wherein the polypeptide comprises SEQ ID NO:24.
41. An immunogenic HIV Env conserved element polypeptide comprising
at least five conserved elements selected from SEQ ID NO: 1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and
SEQ ID NO:23; wherein the conserved elements are separated by
linkers of 1 to 5 amino acids in length.
42. The immunogenic HIV Env conserved element polypeptide of claim
41, wherein the polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, and SEQ ID NO:23.
43. The immunogenic HIV Env conserved element polypeptide of claim
41, wherein the polypeptide comprises the conserved elements of SEQ
ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, and SEQ ID NO:23.
44. The immunogenic HIV Env conserved element polypeptide of claim
41, 42, or 43, wherein the polypeptide further comprises a V1V2
variable region sequence.
45. The immunogenic HIV Env conserved element polypeptide of claim
41, wherein the polypeptide comprises SEQ ID NO:25.
46. A composition comprising: a first nucleic acid construct
encoding a first immunogenic HIV Env conserved element polypeptide
that comprises at least five conserved elements selected from SEQ
ID NO:1, SEQ NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, and SEQ ID NO:22, wherein the conserved elements are
separated by linkers of 1 to 5 amino acids in length; and a second
nucleic acid construct encoding a second immunogenic HIV Env
conserved element polypeptide comprises at least five conserved
elements selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length.
47. The composition of claim 46, wherein the first immunogenic HIV
Env conserved element polypeptide comprises at least six, seven,
eight, nine, ten, or eleven conserved elements selected from SEQ ID
NO:1. SEQ NO:2, SEQ ID NO:4, SEQ NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, and SEQ ID NO:22; and the second immunogenic HIV Env
conserved element polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ 1D NO:15, SEQ SEQ ID NO:19, SEQ ID NO:21, and SEQ
ID NO:23.
48. The composition of claim 46, wherein the first immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22.
49. The composition of claim 46, wherein the second immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, and SEQ ID NO:23.
50. The composition of claim 46, wherein the first immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second immunogenic
HIV Env conserved element polypeptide comprises the conserved
elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, and SEQ NO:23.
51. The compositions of any one of claims 46 to 50, wherein the
first immunogenic HIV Env conserved element polypeptide or the
second immunogenic HIV Env conserved element polypeptide; or both
the first immunogenic HIV Env conserved element polypeptide and the
second immunogenic HIV Env conserved element polypeptide further
comprise a V1V2 variable region sequence.
52. The composition of claim 46, wherein the first immunogenic HIV
Env conserved element comprises the amino acid sequence of SEQ ID
NO:24 and the second immunogenic HIV Env conserved element
polypeptide comprises the amino acid sequence of SEQ ID NO:25.
53. The composition of any one of claims 46 to 52, wherein the
first nucleic acid and second nucleic acid are contained in
separate expression vectors,
54. The composition of any one of claims 46 to 52, wherein the
first nucleic acid and second nucleic acid are contained in the
same expression vector.
55. A composition comprising: a first immunogenic HIV Env conserved
element polypeptide comprising at least five conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ NO:18, SEQ ID NO:20, and SEQ ID NO:22, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length; and a second immunogenic HIV Env conserved element
polypeptide comprises at least five conserved elements selected
from SEQ NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
56. The composition of claim 55, wherein the first immunogenic HIV
Env conserved element polypeptide comprises at least six, seven,
eight, nine, ten, or eleven conserved elements selected from SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, and SEQ ID NO:22; and the second immunogenic HIV Env
conserved element polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,
SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID
NO:21, and SEQ ID NO:23.
57. The composition of claim 55, wherein the first immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22.
58. The composition of claim 55, wherein the second immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, and SEQ ID NO:23.
59. The composition of claim 55, wherein the first immunogenic HIV
Env conserved element polypeptide comprises the conserved elements
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second immunogenic
HIV Env conserved element polypeptide comprises the conserved
elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ NO:17, SEQ
ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
60. The composition of any one of claims 55 to 59, wherein the
first immunogenic HIV Env conserved element polypeptide or the
second immunogenic HIV Env conserved element polypeptide; or both
the first immunogenic HIV Env conserved element polypeptide and the
second immunogenic HIV Env conserved element polypeptide further
comprise a V1V2 variable region sequence.
61. The composition of claim 55, wherein the first immunogenic HIV
Env conserved element comprises the amino acid sequence of SEQ ID
NO:24 and the second immunogenic HIV Env conserved element
polypeptide comprises the amino acid sequence of SEQ NO:25.
62. A method of inducing an immune response to HIV Env, the method
comprising administering a nucleic acid construct of any one of
claims 24 to 35 to a subject or an HIV Env conserved element
polypeptide of any one of claims 36 to 45 to the subject.
63. A method of inducing an immune response to HIV Env, the method
comprising administering: a first nucleic acid encoding a first
immunogenic HIV Env conserved element polypeptide that comprises at
least five conserved elements selected from SEQ ID NO:1, SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID
NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and
SEQ ID NO:22, wherein the conserved elements are separated by
linkers of 1 to 5 amino acids in length; and a second nucleic acid
encoding a second immunogenic HIV Env conserved element polypeptide
comprises at least five conserved elements selected from SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID
NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, and SEQ ID NO:23, wherein the conserved elements are
separated by linkers of 1 to 5 amino acids in length.
64. The method of claim 63, wherein: the first immunogenic HIV Env
conserved element polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, and SEQ ID NO:22; and the second immunogenic HIV Env
conserved element polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:3, SEQ ID NO:5, SEQ NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ
ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,
and SEQ ID NO:23.
65. The method of claim 63, wherein: the first immunogenic HIV Env
conserved element polypeptide comprises the conserved elements of
SEQ NO:1, SEQ NO:2, SEQ ID NO:4, SEQ NO:6, SEQ ID NO:8, SEQ ID
NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ
ID NO:20, and SEQ NO:22; and the second immunogenic HIV Env
conserved element polypeptide comprises the conserved elements of
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,
SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID
NO:19, SEQ ID NO:21, and SEQ ID NO:23.
66. The method of any one of claims 63 to 65, wherein the first
immunogenic HIV Env conserved element polypeptide or the second
immunogenic HIV Env conserved element polypeptide; or both the
first immunogenic HIV Env conserved element polypeptide and the
second immunogenic HIV Env conserved element polypeptide further
comprise a V1V2 variable region sequence.
67. The method of claim 63, wherein the first immunogenic HIV Env
conserved element comprises the amino acid sequence of SEQ ID NO:24
and the second immunogenic HIV Env conserved element polypeptide
comprises the amino acid sequence of SEQ ID NO:25.
68. The method of any one of claims 63 to 67, wherein the first
nucleic acid and second nucleic acid are contained in separate
expression vectors.
69. The method of any one of claims 63 to 67, wherein the first
nucleic acid and second nucleic acid are contained in the same
expression vector.
70. The method of any one of claims 63 to 67, wherein the first and
the second nucleic acids are administered sequentially.
71. The method of any one of claims 63 to 67, wherein the first and
second nucleic acids are administered concurrently.
72. The method of any one of claims 63 to 71, wherein a nucleic
acid encoding a full-length Env polypeptide, or substantially
full-length Env polypeptide, is administered as a boost after the
first and the second nucleic acid encoding the first and the second
HIV conserved element polypeptides.
73. The method of any one of claims 63 to 72, wherein the nucleic
acid constructs are administered intramuscularly by in vivo
electroporation.
74. A method of inducing an immune response to HIV Env, the method
comprising administering: a first immunogenic HIV Env conserved
element polypeptide that comprises at least five conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ NO:22, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length; and a second immunogenic HIV Env conserved element
polypeptide comprises at least five conserved elements selected
from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
75. The method of claim 74, wherein the first immunogenic Env
conserved element polypeptide comprises at least six, seven, eight,
nine, ten, or eleven conserved elements selected from SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:4, SEQ NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ
ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20,
and SEQ ID NO:22; and the second immunogenic HIV Env conserved
element polypeptide comprises at least six, seven, eight, nine,
ten, or eleven conserved elements selected from SEQ NO:1, SEQ ID
NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID
NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO.21, and
SEQ ID NO:23.
76. The method of claim 74, wherein the first immunogenic HIV Env
conserved element polypeptide comprises the conserved elements of
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second immunogenic
HIV Env conserved element polypeptide comprises the conserved
elements of SEQ ID NO:1, SEQ ID NO:3, SEQ NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ NO:21, and SEQ ID NO:23.
77. The method of any one of claims 74 to 76, wherein the first
immunogenic HIV Env conserved element polypeptide or the second
immunogenic HIV Env conserved element polypeptide; or both the
first immunogenic My Env conserved element polypeptide and the
second immunogenic HIV Env conserved element polypeptide further
comprise a V1V2 variable region sequence.
78. The method of claim 74, wherein the first immunogenic HIV Env
conserved element comprises the amino acid sequence of SEQ ID NO:24
and the second immunogenic HIV Env conserved element polypeptide
comprises the amino acid sequence of SEQ ID NO:25.
79. The method of any one of claims 74 to 78, wherein the first and
the second immunogenic HIV Env conserved element polypeptides are
administered sequentially.
80. The method of any one of claims 74 to 78, wherein the first and
the second immunogenic HIV Env conserved element polypeptides are
administered concurrently.
81. The method of any one of claims 74 to 80, wherein a full-length
Env polypeptide, or substantially fill-length Env polypeptide, is
administered after the first and the second second HIV conserved
element polypeptides.
82. An HIV Gag CE polypeptide comprising: (a) SEQ ID NO:27, SEQ ID
NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, and/or SEQ NO:37;
or (b) SEQ ID NO:28, SEQ NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ
NO:36, and/or SEQ ID NO:38.
83. An HIV Gag CE polypeptide comprising: P24CE8-1, p24CE9-1,
p24CE3-1, p24CE10-1, p24CE5-1, p24CE11-1, p24CE6-1, p24CE12-1,
p24CE13-1 as shown in FIG. 23; or P24CE8-1, p24CE9-2, p24CE3-2,
p24CE10-21, p24CE5-2, p24CE11-2, p24CE6-2, p24CE12-2, p24CE13-2 as
shown in FIG. 23.
84. The method of any one of claims 62 to 81, further comprising
administering an immunogenic composition to induce an immune
response to HIV Gag, wherein the immunogenic composition is an HIV
Gag CE polypeptide comprising p24 CE1, CE2, CE3, CE4, CE5, CE6, and
CE7 as shown in FIG. 23; or an HIV Gag CE polypeptide of claim 82
or 83.
85. The method of any one of claims 62 to 81, further comprising
administering an immunogenic composition to induce an immune
response to HIV Gag, wherein the method comprises administering an
HIV Gag CE polypeptide comprising P24CE8-1, p24CE9-1, p24CE3-1,
p24CE10-1, p24CE5-1, p24CE11-1, p24CE6-1, p24CE12-1, p24CE13-1 as
shown in FIGS. 15 and P24CE8-1, p24CE9-2, p24CE3-2, p24CE10-21,
p24CE5-2, p24CE11-2, p24CE6-2, p24CE12-2, p24CE13-2 as shown in
FIG. 23.
86. A nucleic acid construct encoding an HIV Gag CE polypeptide of
claim 82 or 83.
87. The method of claim 84 or 85, wherein the HIV Gag CE
polypeptide is administered as a nucleic acid that encodes the HIV
Gag CE polypeptide.
Description
CROSS-REFERENCE TO RELATING APPLICATIONS
[0001] This application claims priority benefit of U.S. Provisional
Application No. 62/161,123, filed May 13, 2015; and U.S.
Provisional Application No. 62/241,599, filed Oct. 14, 2015; each
of which applications is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Sequence diversity and immunodominance are major obstacles
in the design of an effective vaccine against HIV. A vaccine that
can induce cross-clade specific immune responses is sought. One
approach to achieving robust cross-clade immune responses is the
use of conserved element (CE) vaccines (see, e.g., WO 2012/062873
and WO 2013/131099) that can serve as a "universal" vaccine in that
it is able to induce immune responses to most or all circulating
strains of a virus. It was additionally determined that improved
immune responses to highly conserved regions were obtained using a
prime-boost vaccine regimen that included CE prime followed by
boost with a vector expressing the full-length immunogen. This
method led to the induction of immune responses with altered
breadth and greatly improved cytotoxic responses (see, e.g.,
Kulkami, et al., PLoS One;9:c86254, 2014; Kulkarni, et al., PLos
One 9:c111085, 2014; U.S. Patent Application Publication No.
20150056237, WO 2012/062873 and WO 2013/131099). The present
invention addresses the need for an improved protocol for inducing
an immune response using a conserved element vaccine and
substantially full-length antigen. Further, the invention provides
surprisingly effective HIV Env conserved element immunological
composition that generates a robust immune response to the
conserved regions of HIV Env proteins.
BRIEF SUMMARY OF SOME EMBODIMENTS OF THE DISCLOSURE
[0003] In one aspect, the present disclosure provides an
improvement to methods for inducing an immune response using a
nucleic acid CE immunogenic composition, which comprises one or
more nucleic acids encoding one or more CE polypeptides, where the
method comprises administering one or more CE nucleic acid
constructs to a patient as a prime follow by co-administration of
the CE nucleic acid immunogen(s) with an immunogenic composition
comprising a nucleic acid construct encoding a substantially
full-length form of the antigen from which the CEs are derived.
Boosting by co-administering the CE immunogenic composition(s) and
full-length, or substantially full-length, antigen improves immune
responses, e.g., by further enhancing the levels of cytotoxic T
cells, focusing the immune response to the highly conserved
epitopes, compared to boosting with only fill-length antigen.
[0004] Thus, the disclosure provides an improved prime-boost
immunization regimen employing CE immunogens in combination with
fill-length immunogens that result in superior breadth, magnitude
and quality of immune responses. This is accomplished with a
priming administration of a CE immunogenic compositions(s) focusing
the immune responses to highly conserved epitopes of the virus or
immunogen of interest. The boosting step is accomplished by
co-administration of a vaccine expressing CEs together with
full-length molecule. Although this method may find use for
inducing immune responses to an HIV antigen, such as Gag or Env,
the method is not limited to HIV but can be employed to improve the
induction of immune responses to any subdominant epitopes (cellular
and or humoral) to increase breadth, magnitude and quality of the
immune responses.
[0005] In a further aspect, the present disclosure provides
immunogenic conserved element (CE) vaccine compositions and methods
of using such compositions to induce an immune response to HIV
envelope polypeptides. An immunogenic composition of the invention
can induce immune responses to most or all circulating strains of
HIV. This disclosure includes a description of 12 regions of the
HIV envelope, which are highly conserved throughout the known
universe of HIV M Group sequences. The invention provides CE
polypeptides that comprise multiple CEs from the conserved regions
of HIV. In typical embodiments, two CE polypeptides that differ
from one another by only a few amino acids are administered in
order to elicit an immune response across most strains of HIV.
[0006] In some embodiments, an immunogenic composition of the
invention comprises multiple CE sequences, each having an amino
acid sequence of SEQ NOS:1-23. In illustrative embodiments, the
conserved element regions employed in the immunogens are 11, 14,
21, 15, 23, 21, 13, 12, 14, 43, 20, and 13 amino acids in length.
In typical embodiments, each CE segment of the immunogen is
separated by linkers that facilitate processing of the protein.
[0007] In some embodiments, an immunogenic composition of the
invention comprises multiple CE sequences that include CE from each
of the conserved regions of HIV Env, where each CE has a sequence
shown in FIG. 24,
[0008] In some embodiments, the invention provides immunogenic
compositions comprising multiple HIV Gag CE elements (e.g., FIG. 23
or Gag CE elements listed under Table 2) and methods of using such
immunogenic compositions to induce an immune response to Gag. The
HIV Gag CE element immunogenic compositions may be administered
with HIV Env immunogenic compositions as described herein. In such
embodiments, the CE constructs (HIV Gag and Env CE constructs) may
be administered concurrently or sequentially.
[0009] In some embodiments, an immunogenic polypeptide comprising
Env conserved elements in accordance with the invention further
comprises variable region sequences, e.g., a V1V2 variable region
sequence.
[0010] The conserved element immunogens are typically administered
as nucleic acid vaccines in which DNA comprising one ore more
polynucleotide sequences encoding one or more polypeptides
comprising the CEs is administered to a subject.
[0011] In some embodiments, DNA vectors are engineered to express
the CE immunogens (conserved element polypeptides comprising
multiple conserved elements, such as Env-CE1 and Env-CE2 as shown
in FIG. 10 and as further described herein) only, to express
secreted Env-CE proteins having the N-terminal Env signal peptide
or an N-terminal GM-CSF signal peptide, to express the CE
polypeptide(s) as a core fusion to the monocyte chemoattractant
protein 3 (MCP3) chemokine to stabilize the protein expression and
enhance secretion of the proteins, to express the CE polypeptide(s)
as a fusion to lysosomal associated membrane protein 1 (LAMP-1) to
direct the proteins to the lysosomal compartment including access
to the MHC class II pathway, or to target the protein to the
degradation pathway using a sequence such as a .beta.-catenin
degradation signal.
[0012] Illustrative embodiments of the disclosure, include, but are
not limited to, the following:
[0013] Embodiment 1: :A method of inducing an immune response to a
protein of interest, the method comprising: priming an immune
response to the protein of interest, wherein priming comprises
administering at least one nucleic acid construct encoding a
conserved element polypeptide, wherein the conserved elements are
from the protein of interest and the polypeptide comprises at least
three conserved elements, each from 8 to 50 amino acids in length
where the conserved elements are joined by non-naturally occurring
linkers, with the proviso that a nucleic acid construct encoding
the full-length protein, or a substantially full-length region
thereof, is not administered in the priming step; boosting the
immune response to the protein of interest, wherein boosting
comprises co-administering (i) a nucleic acid construct encoding
the full-length protein, or substantially full-length protein and
(ii) the nucleic acid construct encoding the conserved element
polypeptide.
[0014] Embodiment 2: The method of Embodiment 1, wherein the
boosting step comprises administering the nucleic acid of (i) and
the nucleic acid of (ii) at the same time.
[0015] Embodiment 3: The method of Embodiment 1 or 2, wherein the
nucleic acid constructs are administered intramuscularly by in vivo
electroporation.
[0016] Embodiment 4: The method of any one of Embodiments 1 to 3,
wherein the priming and boosting steps further comprise
administering a second conserved element nucleic acid construct
that encodes a second conserved element polypeptide that comprises
at least one variant of a conserved element contained in the first
conserved element polypeptide, wherein the variant in the second
polypeptide differs from the variant in the first polypeptide by 1,
2, 3, 4, or 5 amino acids.
[0017] Embodiment 5: The method of Embodiment 4, the first and
second conserved element nucleic acid constructs are administered
sequentially.
[0018] Embodiment 6: The method of Embodiment 4, the first and
second conserved element nucleic acid constructs are administered
concurrently,
[0019] Embodiment 7: The method of any one of Embodiments 1 to 6,
wherein the protein of interest is Gag and the conserved elements
are from HIV-1 p24gag.
[0020] Embodiment 8: The method of Embodiment 7, wherein one
conserved element polypeptide comprises at least 7 conserved
elements from different regions of p24gag; and each of the
conserved elements has a conserved element sequence shown in Table
2.
[0021] Embodiment 9: The method of Embodiment 8, wherein one
conserved element polypeptide comprises conserved elements that
each have a sequence set forth in SEQ ID NOS:26, 27, 28, 29, 30,
31, and 32; and a second conserved element polypeptide comprises
conserved elements that each have a sequence set forth in SEQ ID
NOS: 33, 34, 35, 36, 37, 38, and 39.
[0022] Embodiment 10: The method of Embodiment 9, wherein one Gag
conserved element polypeptide comprises the amino acid sequence of
SEQ ID NO:40 and the second Gag conserved element polypeptide
comprises the amino acid sequence of SEQ ID NO:41.
[0023] Embodiment 11: The method of Embodiment 9 or 10, wherein the
conserved element polypeptides encoded by the first and second
nucleic acid Gag conserved element nucleic acid constructs are each
fused to a GM-CSF signal peptide.
[0024] Embodiment 12: The method of any one of Embodiments 1 to 6,
wherein protein of interest is an HIV-1 Env sequence.
[0025] Embodiment 13: The method of Embodiment 12, wherein one
conserved element polypeptide comprises at least 12 conserved
elements from different conserved regions of Env of HIV-1 Group M
and each of the conserved elements has a conserved element sequence
shown in Table 1.
[0026] Embodiment 14: The method of Embodiment 13, wherein one
conserved element polypeptide comprises conserved elements that
each have a sequence set forth in SEQ ID NOS:1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, and 22; and a second conserved element polypeptide
comprises conserved elements that each have a sequence set forth in
SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23.
[0027] Embodiment 15: The method of Embodiment 14, wherein one Env
conserved element polypeptide comprises the amino acid sequence of
SEQ NO:24 and the second Env conserved element polypeptide
comprises the amino acid sequence of SEQ ID NO:25.
[0028] Embodiment 16: The method of Embodiment 14 or 15, wherein
the conserved element polypeptides encoded by the first and second
nucleic acid Env conserved element nucleic acid constructs are each
fused to a GM-CSF signal peptide.
[0029] Embodiment 17: The method of any one of Embodiments 4 to 16,
wherein the first and second nucleic acid conserved element nucleic
acid constructs are contained in the same vector.
[0030] Embodiment 18: The method of any one of Embodiments 4 to 16,
wherein the first and second nucleic acid constructs are contained
in different vectors.
[0031] Embodiment 19: The method of any one of Embodiments 1 to 18,
wherein boosting is performed at least two weeks after the priming
step.
[0032] Embodiment 20: The method of any one of Embodiments 1 to 19,
wherein the priming step comprises two administrations of the one
or more conserved element polypeptides, each separated by at least
2 weeks; and the boosting step comprises two co-administrations,
each separated by at least 2 weeks.
[0033] Embodiment 21: A method of inducing an immune response to
HIV Gag in a patient, the method comprising: priming an immune
response to HIV Gag, wherein priming comprises administering to the
patient a CE-encoding expression vector that comprises a nucleic
acid sequence encoding SEQ ID N 40 fused at the N-terminus to a
human GM-CSF signal peptide and a nucleic acid sequence encoding
SEQ ID NO: 41 fused at the N-terminus to a human GM-CSF signal
peptide; and boosting an immune response to HIV Gag, wherein
boosting comprises co-delivering the CE-encoding expression vector
with an expression vector that encodes full-length HIV-1 p55Gag, or
substantially full-length Gag.
[0034] Embodiment 22: The method of Embodiment 21, wherein the
boosting step is performed about two months after the priming
step.
[0035] Embodiment 23: The method of Embodiment 21 or 22, wherein
the method comprises two priming steps, each separated by about two
months; and two boosting steps, each separated by about two months.
In some embodiments, the priming steps are separated by at least
one month and/or the boosting steps are separated by at least one
month. In some embodiments, the priming steps are separated by
about three months and/or the boosting steps are separated by about
three month.
[0036] Embodiment 24: A method of inducing an immune response to a
protein of interest, wherein the protein of interest is HIV Gag or
HIV Env, the method comprising: priming an immune response to the
protein of interest, wherein priming comprises administering at
least one nucleic acid construct encoding a conserved element
polypeptide, wherein the conserved elements are from the protein of
interest and the polypeptide comprises at least three conserved
elements, each from 8 to 50 amino acids in length where the
conserved elements are joined by non-naturally occurring linkers,
with the proviso that a nucleic acid construct encoding the
full-length protein, or a substantially full-length region thereof,
is not administered in the priming step; boosting the immune
response to the protein of interest, wherein hosting comprises
co-administering (i) a nucleic acid construct encoding the
full-length protein, or substantially full-length protein and (ii)
the nucleic acid construct encoding the conserved element
polypeptide.
[0037] Embodiment 25: The method of Embodiment 24, wherein the
boosting step comprises administering the nucleic acid of (i) and
the nucleic acid of (ii) at the same time.
[0038] Embodiment 26: The method of Embodiment 24 or 25, wherein
the nucleic acid constructs are administered intramuscularly by in
vivo electroporation.
[0039] Embodiment 27: The method of any one of Embodiments 24 to
26, wherein the priming and boosting steps further comprise
administering a second conserved element nucleic acid construct
that encodes a second conserved element polypeptide that comprises
at least one variant of a conserved element contained in the first
conserved element polypeptide, wherein the variant in the second
polypeptide differs from the variant in the first polypeptide by 1,
2, 3, 4, or 5 amino acids.
[0040] Embodiment 28: The method of Embodiment 27, the first and
second conserved element nucleic acid constructs are administered
sequentially.
[0041] Embodiment 29: The method of Embodiment 27, the first and
second conserved element nucleic acid constructs are administered
concurrently.
[0042] Embodiment 30: The method of any one of Embodiments 24 to
29, wherein the first and second nucleic acid conserved element
nucleic acid constructs are contained in the same vector.
[0043] Embodiment 31: The method of any one of Embodiments 24 to
29, wherein the first and second nucleic acid constructs are
contained in different vectors.
[0044] Embodiment 32: The method of any one of Embodiments 24 to
31, wherein boosting is performed at least two weeks after the
priming step.
[0045] Embodiment 33: The method of any one of Embodiments 24 to
32, wherein the priming step comprises two administrations of the
one or more conserved element polypeptides, each separated by at
least 2 weeks; and the boosting step comprises two
co-administrations, each separated by at least 2 weeks.
[0046] Embodiment 34: A method of inducing an immune response to
HIV Gag in a patient, the method comprising: priming an immune
response to HIV Gag, wherein priming comprises administering to the
patient a CE-encoding expression vector that comprises a nucleic
acid sequence encoding p24GagCE1 as set forth in Table 2 fused at
the N-terminus to a human GM-CSF signal peptide and a nucleic acid
sequence encoding p24GagCE2 as set forth in Table 2 fused at the
N-terminus to a human GM-CSF signal peptide; and boosting an immune
response to HIV Gag, wherein boosting comprises co-delivering the
CE-encoding expression vector with an expression vector that
encodes full-length HIV-1 p55gag, or substantially full-length
HIV-1 p55gag.
[0047] Embodiment 35: The method of Embodiment 34 wherein the
boosting step is performed about two months after the priming
step.
[0048] Embodiment 36: The method of Embodiment 34 or 35, wherein
the method comprising two priming steps, each separated by about
two months; and two boostep steps, each separated by about two
months. In some embodiments, the priming steps are separated by at
least one month and/or the boosting steps are separated by at least
one month. In some embodiments, the priming steps are separated by
about three months and/or the boosting steps are separated by about
three month.
[0049] Embodiment 37: A nucleic acid encoding an immunogenic HIV
Env conserved element polypeptide, wherein the HIV Env conserved
element polypeptide comprises at least five conserved elements
selected from SEQ ID NO:1, SEQ II) NO:2, SEQ ID NO:4, SEQ NO:6, SEQ
ID NO:8. SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,
SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
[0050] Embodiment 38: The nucleic acid of Embodiment 37, wherein
the HIV Env conserved element polypeptide comprises at least six,
seven, eight, nine, ten, or eleven conserved elements selected from
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8.
SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID
NO:18, SEQ ID NO:20, and SEQ ID NO:22
[0051] Embodiment 39: The nucleic acid of Embodiment 37, wherein
the HIV Env conserved element polypeptide comprises the conserved
elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ
ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,
SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0052] Embodiment 40: The nucleic acid of Embodiment 37, 38, or 39,
wherein the HIV Env conserved element polypeptide further comprises
a V1V2 variable region sequence.
[0053] Embodiment 41: The nucleic acid of Embodiment 37, wherein
the HIV Env conserved element polypeptide comprises SEQ ID
NO:24.
[0054] Embodiment 42: A nucleic acid encoding an immunogenic HIV
Env conserved element polypeptide, wherein the HIV Env conserved
element polypeptide comprises at least five conserved elements
selected from SEQ II) NO:1, SEQ ID NO:3, SEQ IL) NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23; and the
conserved elements are separated by linkers of 1 to 5 amino acids
in length.
[0055] Embodiment 43: The nucleic acid of Embodiment 42, wherein
the HIV Env conserved element polypeptide comprises at least six,
seven, eight, nine, ten, or eleven conserved elements selected from
SEQ ID NO:1, SEQ NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ
ID NO:11, SEQ NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ
ID NO:21, and SEQ ID NO:23.
[0056] Embodiment 44: The nucleic acid of Embodiment 42, wherein
the HIV Env conserved element polypeptide comprises the conserved
elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ
ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0057] Embodiment 45: The nucleic acid of Embodiment 42, 43, or 44,
further comprising a V1V2 variable region sequence.
[0058] Embodiment 44: The nucleic acid of Embodiment 42, wherein
the HIV E conserved element polypeptide comprises SEQ ID NO:25.
[0059] Embodiment 45: The nucleic acid of any one of Embodiments 42
to 44, wherein the HIV Env conserved element polypeptide comprises
a signal peptide.
[0060] Embodiment 46: The nucleic acid of Embodiment 45, wherein
the signal peptide is the signal peptide of GM-CSF.
[0061] Embodiment 47: An immunogenic HIV Env conserved element
polypeptide comprising at least five conserved elements selected
from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ
ID NO:18, SEQ ID NO:20, and SEQ ID NO:22, wherein the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
[0062] Embodiment 48: The immunogenic HIV Env conserved element
polypeptide of Embodiment 47, wherein the polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22
[0063] Embodiment 49: The immunogenic HIV Env conserved element
polypeptide of Embodiment 47, wherein the polypeptide comprises the
conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0064] Embodiment 50: The immunogenic HIV Env conserved element
polypeptide of Embodiment 47, 48, or 49, wherein the polypeptide
further comprises a V1V2 variable region sequence.
[0065] Embodiment 51: The immunogenic HIV Env conserved element
polypeptide of Embodiment 47, wherein the polypeptide comprises SEQ
ID NO:24.
[0066] Embodiment 52: An immunogenic HIV Env conserved element
polypeptide comprising at least five conserved elements selected
from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ Ill NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, and SEQ ID NO:23; wherein the conserved
elements are separated by linkers of 1 to 5 amino acids in
length.
[0067] Embodiment 53: The immunogenic HIV Env conserved element
polypeptide of Embodiment 52, wherein the polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0068] Embodiment 54: The immunogenic HIV Env conserved element
polypeptide of Embodiment 52, wherein the polypeptide comprises the
conserved elements of SEQ NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID
NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0069] Embodiment 55: The immunogenic HIV Env conserved element
polypeptide of Embodiment 52, 53, or 54, wherein the polypeptide
further comprises a V1V2 variable region sequence.
[0070] Embodiment 56: The immunogenic HIV Env conserved element
polypeptide of Embodiment 52, wherein the polypeptide comprises SEQ
ID NO:25.
[0071] Embodiment 57: A composition comprising: a first nucleic
acid encoding a first immunogenic HIV Env conserved element
polypeptide that comprises at least five conserved elements
selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length; and a second nucleic acid encoding a second immunogenic
HIV Env conserved element polypeptide comprises at least five
conserved elements selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23,
wherein the conserved elements are separated by linkers of 1 to 5
amino acids in length.
[0072] Embodiment 58: The composition of Embodiment 57, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
at least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ NO:20, and SEQ NO:22; and the second
immunogenic HIV Env conserved element polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17,
SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0073] Embodiment 59: The composition of Embodiment 57, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0074] Embodiment 60: The composition of Embodiment 57, wherein the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ NO:3, SEQ ID NO:5, SEQ
ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ
ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0075] Embodiment 61: The composition of Embodiment 57, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO 6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ NO:21, and SEQ ID NO:23.
[0076] Embodiment 62: The compositions of any one of Embodiments 57
to 61, wherein the first immunogenic HIV Env conserved element
polypeptide or the second immunogenic HIV Env conserved element
polypeptide; or both the first immunogenic HIV Env conserved
element polypeptide and the second immunogenic HIV Env conserved
element polypeptide further comprise a V1V2 variable region
sequence.
[0077] Embodiment 63: The composition of Embodiment 57, wherein the
first immunogenic HIV Env conserved element comprises the amino
acid sequence of SEQ ID NO:24 and the second immunogenic HIV Env
conserved element polypeptide comprises the amino acid sequence of
SEQ ID NO:25.
[0078] Embodiment 64: The composition of any one of Embodiments 57
to 63, wherein the first nucleic acid and second nucleic acid are
contained in separate expression vectors.
[0079] Embodiment 65: The composition of any one of Embodiments 57
to 63, wherein the first nucleic acid and second nucleic acid are
contained in the same expression vector.
[0080] Embodiment 66: A composition comprising: a first immunogenic
HIV Env conserved element polypeptide comprising at least five
conserved elements selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22,
wherein the conserved elements are separated by linkers of 1 to 5
amino acids in length; and a second immunogenic HIV Env conserved
element polypeptide comprises at least five conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length.
[0081] Embodiment 67: The composition of Embodiment 66, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
at least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second
immunogenic HIV Env conserved element polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0082] Embodiment 68: The composition of Embodiment 67, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0083] Embodiment 69: The composition of Embodiment 67, wherein the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0084] Embodiment 70: The composition of Embodiment 67, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID
NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID
NO:23.
[0085] Embodiment 71: The composition of any one of Embodiments 67
to 70, wherein the first immunogenic HIV Env conserved element
polypeptide or the second immunogenic HIV Env conserved element
polypeptide, or both the first immunogenic HIV Env conserved
element polypeptide and the second immunogenic HIV Env conserved
element polypeptide further comprise a V1V2 variable region
sequence.
[0086] Embodiment 72: The composition of Embodiment 67, wherein the
first immunogenic HIV Env conserved element comprises the amino
acid sequence of SEQ ID NO:24 and the second immunogenic HIV Env
conserved element polypeptide comprises the amino acid sequence of
SEQ NO:25.
[0087] Embodiment 73: A method of inducing an immune response to
HIV Env, the method comprising administering a nucleic acid of any
one of Embodiments 37 to 46 to a subject or an HIV Env conserved
element polypeptide of any one of Embodiment 47 to 56 to the
subject; or administering a compositions of any one of Embodiments
57 to 72.
[0088] Embodiment 74: A method of inducing an immune response to
HIV Env, the method comprising administering: a first nucleic acid
encoding a first immunogenic HIV Env conserved element polypeptide
that comprises at least five conserved elements selected from SEQ
ID NO:1, SEQ NO:2, SEQ NO:4, SEQ NO:6, SEQ ID NO:8, SEQ ID NO:10,
SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID
NO:20, and SEQ ID NO:22, wherein the conserved elements are
separated by linkers of 1 to 5 amino acids in length; and a second
nucleic acid encoding a second immunogenic HIV Env conserved
element polypeptide comprises at least five conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length.
[0089] Embodiment 75: The method of Embodiment 74, wherein: the
first immunogenic HIV Env conserved element polypeptide comprises
at least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ HIV NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second
immunogenic HIV Env conserved element polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID
NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ
ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0090] Embodiment 76: The method of Embodiment 74, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ NO:2, SEQ ID NO:4, SEQ
NO:6, SEQ ID NO:8, SEQ NO:10, SEQ NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0091] Embodiment 77 The method of Embodiment 74, wherein the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0092] Embodiment 78: The method of Embodiment 74, wherein: the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ NO:23.
[0093] Embodiment 79. The method of any one of Embodiments 74 to
78, wherein the first immunogenic HIV Env conserved element
polypeptide, or the second immunogenic HIV Env conserved element
polypeptide; or both the first immunogenic HIV Env conserved
element polypeptide and the second immunogenic HIV Env conserved
element polypeptide further comprise a V1V2 variable region
sequence.
[0094] Embodiment 80: The method of Embodiment 74, wherein the
first immunogenic HIV Env conserved element comprises the amino
acid sequence of SEQ NO:24 and the second immunogenic HIV Env
conserved element polypeptide comprises the amino acid sequence of
SEQ ID NO:25.
[0095] Embodiment 81: The method of any one of Embodiments 74 to
80, wherein the first nucleic acid and second nucleic acid are
contained in separate expression vectors.
[0096] Embodiment 82: The method of any one of Embodiments 74 to
80, wherein the first nucleic acid and second nucleic acid are
contained in the same expression vector.
[0097] Embodiment Embodiment 83: The method of any one of
Embodiments 74 to 80, wherein the first and the second nucleic
acids are administered sequentially.
[0098] Embodiment 84: The method of any one of Embodiments 74 to
80, wherein the first and second nucleic acids are administered
concurrently.
[0099] Embodiment 85: The method of any one of Embodiments 74 to
84, wherein a nucleic acid encoding a full-length Env polypeptide,
or substantially full-length Env polypeptide, is administered after
the first and the second nucleic acid encoding the first and the
second HIV conserved element polypeptides; or a full-length Env
polypeptide, or substantially full-length Env polypeptide, is
administered after the first and the second nucleic acid encoding
the first and the second HIV conserved element polypeptides,
[0100] Embodiment 86: The method of any one of Embodiments 74 to
85, wherein the nucleic acid constructs are administered
intramuscularly by in vivo electroporation.
[0101] Embodiment 87: A method of inducing an immune response to
HIV Env, the method comprising administering: a first immunogenic
HIV Env conserved element polypeptide that comprises at least five
conserved elements selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID
NO.-4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22,
wherein the conserved elements are separated by linkers of 1 to 5
amino acids in length; and a second immunogenic HIV Env conserved
element polypeptide comprises at least five conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23, wherein the
conserved elements are separated by linkers of 1 to 5 amino acids
in length.
[0102] Embodiment 88: The method of Embodiment 87, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
at least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the second
immunogenic HIV Env conserved element polypeptide comprises at
least six, seven, eight, nine, ten, or eleven conserved elements
selected from SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7,
SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID
NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0103] Embodiment 89: The method of Embodiment 87, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ NO:4, SEQ
ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ NO:14, SEQ ID
NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.
[0104] Embodiment 90: The method of Embodiment 87, wherein the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO. 9, SEQ ID NO: 11, SEQ ID NO:13, SEQ NO:15,
SEQ NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ NO:23.
[0105] Embodiment 91: The method of Embodiment 87, wherein the
first immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4,
SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14,
SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22; and the
second immunogenic HIV Env conserved element polypeptide comprises
the conserved elements of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,
SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15,
SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, and SEQ ID NO:23.
[0106] Embodiment 92: The method of any one of Embodiments 87 to
91, wherein the first immunogenic HIV Env conserved element
polypeptide or the second immunogenic. HIV Env conserved element
polypeptide; or both the first immunogenic HIV Env conserved
element polypeptide and the second immunogenic HIV Env conserved
element polypeptide further comprise a V1V2 variable region
sequence.
[0107] Embodiment 93: The method of Embodiment 87, wherein the
first immunogenic HIV Env conserved element comprises the amino
acid sequence of SEQ ID NO:24 and the second immunogenic HIV Env
conserved element polypeptide comprises the amino acid sequence of
SEQ ID NO:25.
[0108] Embodiment 94: The method of Embodiments 87 to 93, wherein
the first and the second immunogenic HIV Env conserved element
polypeptides are administered sequentially.
[0109] Embodiment 95: The method of Embodiments 87 to 93, wherein
the first and the second immunogenic HIV Env conserved element
polypeptides are administered concurrently.
[0110] Embodiment 96: The method of any one of Embodiments 87 to
95, wherein a full-length Env polypeptide, or substantially
fill-length Env polypeptide, is administered after the first and
the second second HIV conserved element polypeptides; or a
full-length Env polypeptide, or substantially full-length Env
polypeptide, is administered after the first and the second nucleic
acid encoding the first and the second HIV conserved element
polypeptides.
[0111] Embodiment 97: An HIV Gag CE polypeptide comprising: (a) SEQ
ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ NO:35,
and/or SEQ NO:37; or (b) SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,
SEQ ID NO:34, SEQ ID NO:36, and/or SEQ ID NO:38.
[0112] Embodiment 98: An HIV Gag CE polypeptide comprising:
P24CE8-1, p24CE9-1, p24CE3-1, p24CE10-1, p24CE5-1, p24CE11-1,
p24CE6-1, p24CE12-1, p24CE13-1 as shown in FIG. 23; or P24CE8-1,
p24CE9-2, p24CE3-2, p24CE10-21, p24CE5-2, p24CE11-2, p24CE6-2,
p24CE12-2, p24CE13-2 as shown in FIG. 23.
[0113] Embodiment 99: The method of any one of Embodiments 87 to
96, further comprising administering an immunogenic composition to
induce an immune response to HIV Gag, wherein the immunogenic
composition is an HIV Gag CE polypeptide comprising p24 CE1, CE2,
CE3, CE4, CE5, CE6, and CE7 as shown in FIG. 23; or an HIV Gag CE
polypeptide of claim 109 or 110.
[0114] Embodiment 100: The method of any one of Embodiments 87 to
96, further comprising administering an immunogenic composition to
induce an immune response to HIV Gag, wherein the method comprises
administering an HIV Gag CE polypeptide comprising P24CE8-1,
p24CE9-1, p24CE3-1, p24CE10-1, p24CE5-1, p24CE11-1, p24CE6-1,
p24CE12-1, p24CE13-1 as shown in FIGS. 23 and P24CE8-1, p24CE9-2,
p24CE3-2, p24CE10-21, p24CE5-2, p24CE11-2, p24CE6-2, p24CE12-2,
p24CE13-2 as shown in FIG. 23.
[0115] Embodiment 101: A nucleic acid construct encoding an HIV Gag
CE polypeptide of Embodiment 97 or 98.
[0116] Embodiment 102: The method of Embodiment 99 or 100, wherein
the HIV Gag CE polypeptide is administered as a nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0117] FIG. 1: Derivation of SIV p27 CE and conservation relative
to HIV-1 and SIV strains from multiple species. All sequences were
compared to HIV-1 p24CE1, with a "." indicating homology. Toggle
positions that distinguish SIV p27CE1 and p27CE2 are shown with red
type. Amino acid differences that distinguished the SIV and HIV-1
CE but were conserved in other SIV strains are shown in light blue
type. A protocol of including only one toggle site per CE was
adhered to except for CE4, in which 2 additional amino acids were
substituted since those amino acid variants were always found
together in the database. No toggled amino acid was included for
SIV p27 CE1, CE6 or CE7 due to the extreme conservation observed in
those segments among available SIV sequences. The sequences were
aligned to the consensus of those obtained from the Los Alamos HIV
database. Blanks indicate that sequences corresponding to CE region
were available. Two representative sequences are shown: SIVmac
(species of origin: macaque), N=495; SIVsmm (sooty mangabey),
N=272. Additional sequences were considered for the definition of
CE including SIVver (vervet), N=3; SIVIst (I'Hoest), N=4; SIVmnd
(mandrill), N=3; SIVgsn (greater spot-nosed), N=2; SIVdrl (drill),
N=2; SIVden (Dent's Mona); N=1; SIVmus (mustached). N=1; SIVmon
(mona) N=1; SIN/deb (De Brazza's), N=2; SIVsyk (Sykes), N=1; SIVtal
(talapoin), N=2; SIVsun (sun-tailed), N=1.
[0118] FIG. 2: CE-specific T cell responses in p27CE pDNA
vaccinated macaques. Animals (N=14) were vaccinated with a mixture
of p27CE1 and p27CE2 pDNA. The CE-specific T cell responses were
measured by intracellular flow cytometry in blood at 2 weeks after
the 2nd vaccination. PBMC were stimulated with a pool of peptides
spanning the CE (15-mer overlapping by 11 AA and 10-mer overlapping
by 9 AA). Macaques T129 through T152 were also vaccinated with HIV
Env CE pDNA vaccine at the same time when the gag CE pDNA vaccine
was administered. No difference in the induced cellular responses
was found compared to animals (L986 through R684) which only
received the gag CE pDNA vaccine, demonstrating lack of
interference of gag and env CE induced immune responses.
[0119] FIG. 3: Evaluation of two Prime-boost Regimens in Macaques.
All animals received priming vaccination with conserved element
(CE) DNA vaccine. The animals were boosted by DNA expressing the
full-length gag DNA or by co-delivery of CE and gag DNA.
[0120] FIG. 4. CE DNA primed T cell Responses Are Boosted by a
Single p57gag DNA Vaccination. After receiving the priming
vaccination with p27CE pDNA, the animals received a heterologous
booster vaccination with the full-length p57gag pDNA. The
CE-specific T cell responses were measured by intracellular flow
cytometry in blood at 2 weeks after the priming, 2 months later
(day of booster vaccination) and 2 weeks later. PBMC were
stimulated with a pool of peptides spanning the CE (15-mer
overlapping by 11 AA and 10-mer overlapping by 9 AA). The increase
of total CE-specific IFN-.gamma.+T cells upon boost is shown.
[0121] FIG. 5: Increase of CE-primed T cell Responses by
Co-delivery of CE&gag DNA as Booster Vaccination. The of
CE-specific IFN-.gamma.+T cells was measured by flow cytometry
after the 1st and 2nd co-delivery booster vaccination. CE-specific
responses were measured by intracellular cytokine staining after
stimulation of PBMC with a CE-specific peptide pool covering all 7
CE (mixture of 15-mer overlapping by 11 AA and 10-mer overlapping
by 9 AA). The statistical analysis using paired t test is
shown.
[0122] FIG. 6: CE DNA Prime and Co-delivery of CE&gag DNA as
Boost Induces Broadest Responses. The total CE-specific responses
was de-convoluted using peptide pools specific sub-pools (mixture
of 15- and 10-mer peptides) to each individual CE using PBMC from
animals vaccinated with CE DNA (N=14), animals that received a gag
DNA boost (N=6) and animals that received co-delivery of CE and gag
DNA as boost.
[0123] FIG. 7: CE&gag DNA Co-delivery as Boost Increases the
Levels of Cytotoxic CE-specific T cells. The cells (CD4 and CD8
subsets) were analyzed for their granzyme B (GzmB) content and
their ability to degranulate (CD107a) by flow cytometry.
Statistical analysis of GzmB+CD107a+IFN-.gamma.+T cells of the 2
vaccine regimens are shown.
[0124] FIG. 8: A. Schematic of p24GagCE plasmid. B. Schematic of
p55gag plasmid.
[0125] FIG. 9 provides a schematic of the localization of conserved
regions within Env and the distribution of conserved elements of
the invention within the conserved regions.
[0126] FIG. 10 depicts two Env CE Env immunogenic polypeptides.
Env-CE1 and Env-CE2 in accordance with the invention. Env-CE1 and
Env CE2 span 220 amino acids. They are highly related, differing by
24 amino acids. The Env-CE1 and Env-CE2 sequence are each 282 amino
acids in length, including linkers that are each three amino acids
in length and a 29-amino acid signal peptide.
[0127] FIG. 11 provides an illustrative protocol for the
vaccination of macaques with HIV Env-CE DNA. CE-specific immune
responses in blood were evaluated at 2 weeks after V2 (second
administration of vaccine) and V3 (third administration of vaccine)
and after a rest period of 3 months.
[0128] FIG. 12 provides illustrative data showing T cell responses
of DNA-vaccinated macaques. Peripheral blood mononuclear cells
(PBMCs) were incubated with a peptide pool spanning all 12
conserved elements, The percent of CE-specific CD4 and CD8 cells
producing IFN-.gamma. upon peptide stimulation was determined. The
results show the induction of both CD4 and CD8 responses, with a
skewing to CD8 responses.
[0129] FIG. 13 provides data illustrative that vaccination with
Env-CE vaccines induces memory T cell responses. PBMC were
incubated with a peptide pool spanning all 12 conserved elements
and the % CE-specific IFN-.gamma.-producing CD4 and CD8 central
memory (CM;CD28.sup.+CD95.sup.+) and effector memory
(EM;CD28.sup.+CD95.sup.30 ) T cells was determined following
peptide stimulation.
[0130] FIG. 14 provides data illustrating that Env-CE DNA
vaccination induces CE-specific responses with a significant
fraction of multifunctional CDB.sup.+ T cells, PBMC were incubated
with a peptide pool spanning all 12 CE and the % of
IFN-.gamma.-producing CE-specific CD4 and CD8 T cells harboring
Granzyme B and expressing CD107a was determined following peptide
stimulation.
[0131] FIG. 15 shows the results of mapping of CE-specific
responses. This demonstrated recognition of six of the twelve CEs
in four animals. PBMC were incubated with individual peptide pools
spanning the 12 CE's and the % of CE-specific CD4 and CD8 cells
producing IFN-.gamma. was determined following peptide stimulation.
Each animal recognized 2-4 CE, CE6, CE1, CE10, CE12, CE13, and CE15
did not show cellular responses in these animals in this
experiment.
[0132] FIG. 16 provides data illustrating that vaccination of 9
macaques with DNA-expressing full-length Env induces no, or poor,
CE-specific responses. Env-specific responses were observed in all
animals (using a peptide pool of HIV Env clade B strain BaL
spanning gp120) and CE-specific responses are found in about 55% of
vaccinees. The CE-specific responses exhibited narrow breadth (0-2
CE/animal) compared to the HIV Env-CE vaccine (2-4CE/animal: 100%
response rate).
[0133] FIG. 17 illustrates a prime boost vaccination protocol using
HIV env-CE DNA as prime and env DNA as boost. The boost was a
mixture of env DNA composed of clade B Bal and 6101 and clade C
1086 env DNA covering all of the 24 CE sequences present in HIV Env
CE1 and CE2 proteins. The intact Env molecule produced (gp145)
lacks the immunodominant loop in the extracellular portion of gp41.
Macaque IL-12 DNA was included in the vaccine mixture. DNA was
delivered by intramuscular injection followed by in vivo
electroporation.
[0134] FIG. 18 provides data illustrating that env-CE DNA (CE1 and
CE2) primed responses are boosted upon vaccination with intact Env,
The results show that CE-specific responses were significantly
increased upon a first boost with gp145 env DNA and were further
augmented with a 2.sup.nd boost. In contrast, env DNA vaccination
only induced poor, or no, CE-specific responses. Priming with
env-CE DNA followed by boosting with DNA encoding intact Env is an
effective regimen to increase the breadth and magnitude of
Env-specific responses, including memory and cyotoxic T cell
responses.
[0135] FIG. 19 shows a comparison of responses to individual CE
before and after boosting with DNA plasmids expressing intact Env.
The analysis shows that upon boost, 7 of the 12 segments (58%) of
the HIV Env-CE are immunogenic.
[0136] FIG. 20 shows data illustrating that an env-CE DNA vaccine
induced antibodies that can recognize gp120 Env by ELISA (HIV-1
IIIB). In this experiment, serial dilutions of plasma from
vaccinated animals were tested using an HIV-1 IIIB ELISA assay. The
results show that one of the animals (P574) developed an antibody
binding response to gp120 after CE prime vaccination (22 weeks
after 3rd vaccination V3wk2). The responses were boosted by each of
the fill-length env DNA vaccination 4th and 5th vaccination (4th
and 5th vaccination V4wk2, V5wk2). Animals L985, R288, and RO67
developed ELISA antibody responses only after booster vaccination
(4.sup.th and 5.sup.th vaccination).
[0137] FIG. 13 shows the results of a western immunoblot analysis
of humoral immune responses. Proteins from cells transfected with
HIV env-CE1 DNA or HIV env-CE2 DNA were separated on denaturing
gels and transferred onto membranes. Individual strips of membranes
were incubated with plasma (1:100 dilution) from the vaccinated
macaques and visualized using standard western blot methodology.
The results show that all four macaques immunized with HIV env-CE
DNA developed antibody responses to the immunogens. Env-CE2 was
better recognized, likely because higher levels of this protein
were effectively loaded because the protein is slightly more stable
than Env-CE1. The antibodies recognizing the CE immunogen are
boosted in animals L985, R288 and to a lesser extent, in P574 and
RO67.
[0138] FIG. 22 provides data illustrating that antibodies induced
by the HIV Env-CE vaccine recognized intact HIV Env proteins from
both clade B and clade C. gp145 protein from transfected cells was
separated on denaturing gels and transferred onto membranes. The
membranes were incubated with plasma from macaques primed using
env-CE DNA and boosted with intact env DNA. Proteins from
mock-transfected cells served as a negative control.
[0139] FIG. 23 shows HIV Gag p24CE sequences. The term "initial" CE
in FIG. 15 refers to HIV Gagp24 CE sequences described in U.S.
Patent Application Publication No. 20150056237. "Alternate" CE
sequences are described herein.
[0140] FIG. 24 shows additional HIV Env CE sequences.
[0141] FIG. 25 shows and illustrative HIV Env CE polypeptide
(Env-CE1_v1V2 BaL) comprising a V1V2 sequence.
DETAILED DESCRIPTION OF THE INVENTION
Terminology
[0142] A "conserved region" as used herein refers to a protein
sequence that is conserved across a protein that has high sequence
diversity in nature, e.g, a viral protein such as HIV Env or HIV
Gag. A "conserved region" need not have 100% sequence identity
across the diversity of naturally occurring sequence of the
protein, but the amino acid sequence variability in the naturally
occurring conserved region sequences is low, typically 10% or less.
A "conserved element" in the context of the present invention is a
segment of a conserved region that is at least 8 amino acids, or
greater, in length. In some embodiments, a "conserved element" is
greater than 8 amino acids in length, e.g.. 9. 10, 11, 12, 13, 14,
15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 or more
amino acids in length. Typically, a conserved element is less than
50 amino acids in length, A "conserved element" need not be 100%
conserved across the diversity of HIV sequences, e.g., HIV Gag
sequences or HIV Env sequences. The sequence variability in the
naturally occurring conserved element sequence is low, however,
typically 10% or less.
[0143] In the context of this invention, a "conserved element pair"
as it relates to a conserved element immunogenic composition,
conserved elements of HIV Gag or HIV Env, refers to two versions of
a conserved element sequence that have amino acid changes relative
to one another such that the two sequence together cover at least
90% of naturally occurring sequences. For example, an HIV Env
"conserved element pair" refers to two versions of a conserved
element sequence that have amino acid changes relative to one
another such that the two sequence together cover at least 90% of
naturally occurring HIV Env variants belonging to the HIV-1 M
group.
[0144] A "variable region" element in the context of HIV Env is a
sequence from a variable region of HIV Env, e.g., the V1V2 region,
that may also be included in an immunogenic HIV Env CE polypeptide
of the invention if recognition of this region is associated with
lower viral loads in published studies or if amino acid
substitutions in that region are known to decrease viral
replication fitness, or if recognition of the region has been
associated with vaccine efficacy, or if amino acid substitutions in
that region are associated with changes in predicted protein
stability.
[0145] A "nucleic acid vaccine" as used herein includes both naked
DNA vaccines, e.g., plasmid vaccine, and viral vector-based nucleic
acid vaccines that are comprised by a viral vector and/or delivered
as viral particles.
[0146] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences, as well as the sequence explicitly
indicated. Degenerate codon substitutions can be achieved by
generating sequences in which the third position of one or more
selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081
(1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985);
Rossolini et al., Mol. Cell, Probes 8:91-98 (1994)). The term
"nucleic acid" is used interchangeably with gene, cDNA,
oligonucleotide, and polynucleotide. A "nucleic acid" encompasses
RNA as well as DNA.
[0147] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (e.g., about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or higher, identity over a specified region (e.g., a
polypeptide sequence comprising collinear conserved elements), when
compared and aligned for maximum correspondence over a comparison
window or designated region) as measured using a BLAST or BLAST 2.0
sequence comparison algorithms with default parameters (see, e.g.,
NCBI web site or the like), or by manual alignment and visual
inspection. In the present invention, in the context of comparison
of a particular conserved element sequence with a variant of that
sequence, identity is defined over the length of the conserved
element reference sequence. In some embodiments, percent identity
of one conserved element to a corresponding variant conserved
element is determined by visual inspection.
[0148] The term "operably linked" refers to a functional linkage
between a first nucleic acid sequence and a second nucleic acid
sequence, such that the first and second nucleic acid sequences are
transcribed into a single nucleic acid sequence. Operably linked
nucleic acid sequences need not be physically adjacent to each
other. The tem "operably linked" also refers to a functional
linkage between a nucleic acid expression control sequence (such as
a promoter, or array of transcription factor binding sites) and a
transcribable nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the transcribable sequence.
[0149] Amino acids can be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, can be referred to by their commonly
accepted single-letter codes.
[0150] The terms "mammal" or "mammalian" refer to any animal within
the taxonomic classification mammalia. A "mammal" can refer to a
human or a non-human primate. A "mammal" can also refer to a
domestic animal, including for example, canine, feline, rodentia,
including lagomorpha, murine, rattus, Cricetinae (hamsters), etc. A
mammal can refer to an agricultural animal, including for example,
bovine, ovine, porcine, equine, etc.
[0151] The terms "treating" and "treatment" refer to delaying the
onset of, retarding or reversing the progress of, or alleviating or
preventing either the disease or condition to which the term
applies, or one or more symptoms of such disease or condition.
[0152] An "immunogen" refers to a molecule, typically a protein
molecule in the current invention, containing one or more epitopes
(either linear, conformational or both) that will stimulate a
host's immune system to make a humoral and/or cellular
antigen-specific response. Normally, an epitope will comprise
between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino
acids, The term "immunogen" includes isolated immunogens as well as
inactivated organisms, such as viruses.
[0153] In the present description, any concentration range,
percentage range, ratio range, or integer range is to be understood
to include the value of any integer within the recited range and,
when appropriate, fractions thereof (such as one tenth and one
hundredth of an integer), unless otherwise indicated, As used
herein, "about" means +10% of the indicated range, value, sequence,
or structure, unless otherwise indicated. It should be understood
that the terms "a" and "an" as used herein refer to "one or more"
of the enumerated components unless otherwise indicated or dictated
by its context. The use of the alternative (e.g., "or") should be
understood to mean either one, both, or any combination thereof of
the alternatives unless otherwise indicated.
Aspects of the Disclosure
[0154] The invention is based, in part, on the discovery that
administration of one or more polypeptides comprising conserved
elements, separated by non-naturally occurring linkers and
collinearly arranged, from an immunogen of interest, e.g., a viral
antigen such as HIV Gag or HIV Env, can provide an enhanced immune
response when one or more conserved element nucleic acid constructs
is administered to a subject as a prime followed by
co-administration to the subject of a nucleic acid construct
encoding a full-length antigen, or substantially a full-length
antigen, with the conserved element construct(s) as a boost. In
typical embodiments, the prime and/or boost components of an
immunization protocol in accordance with the invention are
administered as nucleic acids that encode the polypeptides. In some
embodiments, prime and/or boost immunization components are
administered as polypeptides.
[0155] The immunogen can be any protein for which it is desired to
induce an immune response, but is often a viral protein that
exhibits sequence diversity in naturally occurring variants. In
some embodiments, the viral protein is a retrovirus protein, such
as a lentiviral protein. In some embodiments, the viral protein is
a retroviral Gag or Env protein, such as an HIV Gag or HIV Env
protein.
[0156] The invention is additionally based, in part, on the
discovery that administration of one or more polypeptides
comprising conserved elements, separated by linkers and collinearly
arranged, of HIV Env conserved element proteins as described herein
can provide a robust immune response compared to administration of
a full-length Env protein to a subject. In some aspects, the
disclosure thus provides HIV Env conserved element polypeptides,
nucleic acids encoding HIV Env conserved element polypeptides, and
methods of using such polypeptide and nucleic acids to induce an
immune response.
HIV Env Conserved Element Immunogenic Compositions.
[0157] In one aspect, the disclosure provides HIV Env conserved
element compositions. In some embodiments, an HIV Env conserved
element composition is administered in the form of a nucleic acid
construct that encodes an HIV Env conserved element immunogenic
polypeptide. Alternatively, the HIV Env conserved element
immunogenic composition may may be administered in polypeptide
from. Accordingly, the invention provide immunogenic HIV Env
conserved element polypeptides and nucleic acid constructs that
encode these polypeptides.
[0158] A nucleic acid construct encoding an HIV Env conserved
element polypeptide in accordance with the invention typically
encodes a polypeptide that comprises at least six of the conserved
elements set forth in SEQ ID NOS:1-23 and 70-85. In some
embodiments, the nucleic acid construct encodes a polypeptide that
comprises at least 8, typically, at least 9, 10, 11, 12, 14, 15,
16, 17, 18, 19, 20, 25, 30, 35, or more consecutive amino acids
from the conserved elements of SEQ ID NOS:1-23 and 70-85.
[0159] In some embodiments, a nucleic acid construct encoding an
HIV Env conserved element polypeptide encodes a polypeptide that
comprises at least one, two, three, four, five, six, seven, eight,
nine, ten, or eleven, or all twelve of the conserved elements set
forth in SEQ ID NOs. 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22.
In some embodiments, a nucleic acid construct encoding an HIV Env
conserved element polypeptide encodes a polypeptide that comprises
at least one, two, three, four, five, six, seven, eight, nine, ten,
or eleven, or all twelve of the conserved elements set forth in SEQ
ID NOs. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23. In some
embodiments, a conserved element sequence has at least 8,
typically, at least 9, 10, or 11 consecutive amino acids from CE6
(SEQ ID NO:1). In some embodiments, a conserved element sequence
has at least 10, typically at least 11, 12, 13, or 14 consecutive
amino acids from CE1(SEQ ID NO:2 or SEQ ID NO:3). In some
embodiments, a conserved element sequence has at least 15,
typically at least 16, 17, 18, 19, 20, or 21 consecutive amino
acids from CE7 (SEQ NO:4 or SEQ ID NO:5). In some embodiments, a
conserved element sequence has at least 10, typically at least 11,
12, 13, 14, or 15 consecutive amino acids from CE8 (SEQ ID NO:6 or
SEQ ID NO:7). In some embodiments, a conserved element sequence has
at least 15, typically at least 16, 17, 18, 19, 20, 21, 22, or 23
consecutive amino acids from CE9 (SEQ ID NO:8 or SEQ ID NO:9). In
some embodiments, a conserved element sequence has at least 15,
typically at least 16, 17, 18, 19, 20, or 21 consecutive amino
acids from CE10 (SEQ ID NO:10 or SEQ ID NO:11). In some
embodiments, a conserved element sequence has at least 8,
typically, at least 9, 10, 11, 12, or 13 consecutive amino acids
from CE11 (SEQ ID NO:12 or SEQ ID NO:13). In some embodiments, a
conserved element sequence has at least 8, typically, at least 9,
10, 11, or 12 consecutive amino acids from CE12 (SEQ ID NO:14 or
SEQ NO:15). In some embodiments, a conserved element sequence has
at least 9, typically, at least 10, 11, 12, 13, or 14 consecutive
amino acids from CE 13 (SEQ NO:16 or SEQ ID NO:17). In some
embodiments, a conserved element sequence has at least 20, and
typically at least 21, 22, 23, 24, 25, 26, 26, 27, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 consecutive amino
acids from CE4 (SEQ ID NO:18 or SEQ ID NO:19). In some embodiments,
a conserved element sequence has at least 15, typically at least
16, 17, 18, 19, or 20 consecutive amino acids from CE15 (SEQ ID
NO:20 or SEQ ID NO:21), In some embodiments, a conserved element
sequence has at least 8, typically, at least 9, 10, 11, 12, or 13
consecutive amino acids from CE16 (SEQ ID NO:22 or SEQ ID
NO:23).
[0160] In some embodiments, an HIV Env CE polypeptide may comprise
a CE set forth in any one of SEQ ID NOS:70-85. In some embodiments,
a conserved element sequence has at least 10, typically, at least
11, 12, 13, 14, 15, or 16 consecutive amino acids of EnvCE17 (SEQ
ID NO:70 or SEQ ID NO:71). In some embodiments, a conserved element
sequence has at least 20, 21, 22, 23, 24, 25, or 26 consecutive
amino acids of CE18 (SEQ ID NO:72 or SEQ ID NO:73). In some
embodiments, a conserved element sequence has at least 15,
typically at least 16, 17, 18, or 19 consecutive amino acids from
CE19 (SEQ ID NO:74 or SEQ ID NO:75). In some embodiments, a
conserved element sequence has at least 8, 9, or 10 consecutive
amino acids of CE20 (SEQ ID NO:76 or SEQ ID NO:77). In some
embodiments, a conserved element sequence has at least 14,
typically at least 15, 16, or 17 consecutive amino acids of CE21
(SEQ ID NO:78 or SEQ ID NO:79). In some embodiments, a conserved
element sequence has at least 25, typically at least 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or
45 consecutive amino acids of CE22 (SEQ NO:80 or SEQ ID NO:81. In
some embodiments, a conserved element sequence has at least 12,
typically at least 13, 14, or 15 consecutive amino acids of CE23
(SEQ ID NO:82 or SEQ ID NO:83). In some embodiments, a conserved
element sequence has at least 15, typically at least 16, 17, 19, or
19 consecutive amino acids of CE24 (SEQ ID NO:84 or SEQ ID
NO:85).
[0161] In some embodiments, an HIV Env CE polypeptide comprises the
twelve HIV Env conserved element sequences CE6, CE1, CE7, CE8, CE9,
CE 10, CE11, CE12, CD13, CE14, CE15 and CE16, in this order or in
any other order. In some embodiments, an HIV Env CE polypeptide may
further comprise HIV Env conserved element sequence CE20 and/or
CE23. In some embodiments, an HIV Env CE polypeptide conserved
element polypeptide comprises CE17, CE18, CE19, CE21, CE22, and/or
CE24 in replace of CE8, CE9, CE10, CE11, CE14, and/or CE15,
respectively.
[0162] In some embodiments, an HIV Env CE polypeptide of the
invention comprises Env conserved elements CE6, CE1, CE7, CE17,
CE18, CE19, CE20, CE21, CE12, CE13, CE22, CE23, CE24, and CE16.
[0163] In some embodiments, an HIV Ens' CE polypeptide in
accordance with the invention has at least 90%, or at least 91%,
92%, 93%, 94%. 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:24
or SEQ ID NO:25. In some embodiments, the HIV Env CE polypeptide
has at least 90%, or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity to the Env-CE1-V1V2 Bal polypeptide sequence
shown in FIG. 25 (SEQ ID NO:69).
[0164] In the present disclosure, the HIV Env conserved elements
contained within conserved element polypeptides of the invention
are not contiguous in the native Env proteir sequence. Further, the
conserved elements present in an HIV Env conserved element
polypeptide are separated by polypeptide sequences that don't
naturally occur in native protein sequences. The individual
conserved elements are typically joined to one another in the
nucleic acid construct by a peptide linker, such as an
alanine-containing linker.peptide linker sequences contain Ala and
may also include other amino acids such as Gly, Val, Glu, Asp, Lys,
or Phe. The linker sequence may range in length, e.g., from 1 to 5
amino acids, or even longer, in length, but is typically no longer
than 6, 7, or 8 amino acids in length. In some embodiments, the
linker sequence is 3 amino acids in length. In some embodiments,
the linker sequence is AAV, AAE, GAK, AAD, AAK, GAV, VAV, or
AAF.
[0165] The conserved elements may be present in any order in the
construct, they need not occur in the order of the naturally
occurring sequence. For example, a conserved element that occurs
toward the N-terminus of a protein may be encoded at the C-terminal
end of the construct.
[0166] In some embodiments, a nucleic acid encoding an HIV Env
conserved element polypeptide in accordance with the invention
encodes a polypeptide that comprises at least five or more, or at
least six, seven, eight, nine, ten, or eleven, of the conserved
elements set forth in SEQ ID NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, or 22. In sonic embodiments, a nucleic acid encoding an HIV Env
conserved element polypeptide in accordance with the invention
encodes a polypeptide that comprises the twelve conserved elements
set forth in SEQ IL) NOS:1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and
22. In some embodiments, such a nucleic acid construct encodes a
polypeptide comprising the amino acid sequence of SEQ ID NO:24. In
sonic embodiments, a nucleic acid encoding an HIV Env conserved
element polypeptide in accordance with the invention encodes a
polypeptide comprising at least five or more, or at least six,
seven, eight, nine, ten, or eleven, of the conserved elements set
forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23.
In some embodiments, a nucleic acid encoding an HIV Env conserved
element polypeptide in accordance with the invention encodes a
polypeptide that comprises the twelve conserved elements set forth
in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23. In
some embodiments, such a nucleic acid construct encodes a
polypeptide that comprises the amino acid sequence of SEQ ID NO:25.
In some embodiments, a nucleic acid construct encoding a
polypeptide comprising SEQ ID NO:24 is administered in conjunction
with a nucleic acid construct encoding a polypeptide comprising SEQ
ID NO:25.
[0167] In some embodiments, an HIV Env CE polypeptide comprising
Env conserved elements SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ
ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78,
SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:80, SEQ ID NO:82, SEQ ID
NO:84, and SEQ ID NO:22 is administered in conjunction with an HIV
Env CE polypeptide comprising SEQ NO:1, SEQ NO:3, SEQ NO:5, SEQ ID
NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ
ID NO:15, SEQ ID NO:17, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85,
and SEQ ID NO:23.
[0168] In some embodiments, a nucleic acid encoding an HIV Env
conserved element polypeptide in accordance with the invention
encodes a polypeptide that differs from SEQ ID NO:24 by no more
than 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 amino acids. in some embodiments, a
nucleic acid encoding an HIV Env conserved element polypeptide in
accordance with the invention encodes a polypeptide that differs
from SEQ ID NO:25 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino
acids.
[0169] In some embodiments, an HIV Env conserved element
polypeptide further comprises one or more sequences from an HIV Env
variable region, e.g., the V1V2 region. The variable region
sequences are well known in the art and are hounded by cysteine
residues.
[0170] In the present disclosure, an HIV Env conserved element
nucleic acid construct as described herein is typically employed in
a vaccination regimen that also employs a nucleic acid encoding
full-length Env protein, or substantially full-length Env protein,
from which the conserved elements are obtained. In the context of
the present invention, "substantially full-length" refers to the
region of the Env protein that includes all of the conserved
elements, i.e., a sufficient length of a naturally occurring Env
protein is provided that includes all of the conserved elements
that are used in the conserved element construct.
Administrative Regimens
[0171] In a further aspect, the disclosure provides an enhanced
administration regimen for conserved element immunogenic
compositions that provides a robust immune response. Thus, the
invention additionally provides a method of inducing an immune
response, where the method comprises administering one or more
nucleic acid constructs encoding a conserved element polypeptide,
followed by administering a nucleic acid construct encoding a
full-length or substantially full-length polypeptide, where the
nucleic acid construct encoding a full-length or substantially
full-length polypeptide is administered in conjunction with the
nucleic acid construct encoding the conserved element
polypeptide.
[0172] In some embodiments, the immunogenic compositions employed
in the enhanced administration regimens as described in this
section relate to a viral protein, e.g., a retrovirus protein such
as Gag. Illustrative conserved elements of Gag are described
herein. See, also e.g., U.S. Patent Application Publication No.
20110269937; Rolland et al., PLoS Pathog 3: e157, 2007; Mothe et
al., PLoS One 7: e29717, 2012; and US20150056237.
[0173] Each conserved element included in a CE polypeptide in
accordance with the enhanced methods of generating an immune
response of the disclosure is generally 50 amino acids or fewer in
length, but is at least 8 amino acids in length. In some
embodiments, the conserved element is at least 8 amino acids in
length and 45, 40, 35, 30, 25, 20, or 15 amino acids, or fewer, in
length. In some embodiments, the conserved element is 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or
45 amino acids in length.
[0174] In preferred embodiments, more than one nucleic acid
construct encoding the conserved elements of interest are used
where one construct encodes a first set of conserved elements,
e.g., from HIV Gag or HIV Env, and the second construct encodes a
second set of conserved elements where one or more elements, often
most or all of the conserved elements, of the second set of
conserved elements differ from the first set by 5 or fewer amino
acids. That is, one conserved element present in the two sets
contains a limited number of substitutions (e.g., 1, 2, 3, 4, or 5)
relative to the corresponding conserved element. The residues where
the sequences differ, however, are at sites of naturally occurring
variation in the naturally occurring protein sequences, so that
each of the conserved elements in the first arid second sets
corresponds to a naturally occurring protein sequence. In some
embodiments, each element of the second set is at least 80% or at
least 90% identical to the corresponding element in the first set
of conserved sequences. The nucleic acid construct encoding the
first set of conserved elements and the nucleic acid construct
encoding the second set of conserved elements may be present in the
same vector or different vectors.
[0175] As explained above regarding HIV Env conserved element
constructs, in the enhanced methods of generating an immune
response as described herein, the conserved elements contained
within conserved element polypeptides are not contiguous in the
naturally occurring protein sequence. Further, the conserved
elements present in a conserved element polypeptide generated in
accordance with the invention are separated by polypeptide
sequences that do not naturally occur in the protein sequence. The
individual conserved elements are typically joined to one another
in the nucleic acid construct by a peptide linker, such as an
alanine-containing linker. Linker sequences are well known in the
art. Typical peptide linker sequences contain Ala and may also
include other amino acids such as Gly, Val, Glu, Asp, Lys, or Phe.
The linker sequence may range in length, e.g., from 1 to 5 amino
acids, or even longer, in length, but is typically no longer than
6, 7, or 8 amino acids in length. In some embodiments, the linker
sequence is 3, 4, or 5 amino acids in length. In sonic embodiments,
the linker sequence is AAV, AAE, GAK, AAD, AAK, GAV, VAV, or AAF.
In some embodiments, the linker is AA, AAAE, AAAA, AAK, AG, AA,
LAK, AAK, AAAAL, and the like.
[0176] The conserved elements may be present in any order in the
construct, they need not occur in the order of the naturally
occurring sequence. For example, a conserved element that occurs
toward the N-terminus of a protein may be encoded at the C-terminal
end of the construct.
[0177] In some embodiments, the protein of interest is HIV Gag. A
nucleic acid construct encoding a conserved element polypeptide for
use in the invention encodes a polypeptide that comprises at least
one, two, three, four, five, six, or seven conserved elements set
forth in Table 2. In some embodiments, the nucleic acid construct
encodes a polypeptide that comprises at least 8, typically, at
least 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20, or more
consecutive amino acids from a conserved element set forth in Table
2. See also, e.g. US20150056237, incorporated by reference, which
teaches HIV Gag conserved elements. In some embodiments, a Gag CE
polypeptide used in accordance with the methods of the invention
comprises a p24Gag CE polypeptide having the sequence of the
p24GagCE1 polypeptide of Table 2:
TABLE-US-00001 (SEQ ID NO: 40)
VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEP
RGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRF
YKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKV.
[0178] In some embodiments, a Gag CE polypeptide used in accordance
with the methods of the invention comprises a p24Gag CE polypeptide
having the sequence of the p24GagCE2 polypeptide of Table 2:
TABLE-US-00002 (SEQ ID NO: 41)
VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEP
RGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRF
FKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV.
[0179] In some embodiments, a nucleic acid encoding an HIV Gag
conserved element polypeptide for use in the invention encodes a
polypeptide that comprises the conserved elements set forth in
Table 2 SEQ ID NOS:26-32. In some embodiments, such a nucleic acid
construct encodes a polypeptide comprising the amino acid sequence
of p24 Gag CE1 as shown in Table 2. In some embodiments, a nucleic
acid encoding a conserved element encodes a polypeptide comprising
the conserved elements set forth in SEQ ID NOS:33-39. In some
embodiments, such a nucleic acid construct encodes a polypeptide
that comprises the p24 Gag CE2 amino acid sequence as shown in
Table 2. In some embodiments, a nucleic acid construct encoding a
polypeptide comprising the p24 Gag CEI sequences SEQ ID NOs 26-32
set forth in Table 2 is administered with a nucleic acid construct
encoding a polypeptide comprising the p24 Gag CE2 sequences SEQ ID
NOS:33-39 set forth in Table 2. In some embodiments, the nucleic
acid sequence encoding p24gag CE1 is present in the same vector as
the nucleic acid sequence encoding p24gag CE2.
[0180] In some embodiments, a nucleic acid encoding p24GagCE1 as
set forth in Table 2 is co-administered with a nucleic acid
encoding p24GagCE2 as set forth in Table 2 where each of the
p24GagCE1 and p24GagCE2 polypeptides is expressed as a fusion
protein having a human GM-CSF signal peptide at the N-terminus.
[0181] In some embodiments, a nucleic acid encoding a Gag conserved
element polypeptide encodes at least one conserved element set
forth in the alternative CE sequences shown in Table 2.
[0182] In some embodiments, an immunogen of interest is HIV Env. In
some embodiments, a nucleic acid construct encoding an HIV Env
conserved element polypeptide employed in accordance with the
invention typically encodes a polypeptide that comprises a
conserved element as decribed herein. Thus, in some embodiments,
the nucleic acid construct encodes a polypeptide that comprises at
least 8, typically, at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 25, 30, 35, or more consecutive amino acids from the Env
conserved elements shown in Table 1. In sonic embodiments, an Env
CE polypeptide used in accordance with the methods of the invention
comprises an Env CE1 polypeptide comprising 12 conserved element:
CE6, CE1-1, CE7-1, CE8-1, CE9-1, CE10-1, CE11-1, CE12-1, CE13-1,
CE14-1, CE15-1, and CE16-1 as shown in Table 1. In some
embodiments, the Env CE1 polypeptide comprises the Env CE1
polypeptide sequence of Table 1 (linkers are underlined):
TABLE-US-00003 WVTVYYGVPVWAAVHNVWATHACVPTDPAAEISLWDQSLKPCVKLTPLC
VTLGAKFEPIPIHYCTPAGFAGAKVQCTHGIRPVVSTQLLLNGSLAEAA
DSGGDPEIVMHSFNCGGEFFYCGAKDNWRSELYKYKVVAAKARRRVVQR
EKRAGAVGFLGTAGSTMGAASVAVLTVQARLLLSGIVQQQNNLLRAIEA
QQHLLQLTVWGIKQLQARAADWLWYIKIFIMIVGGLVGLRIAAFRVRKG YSPLSLQT.
[0183] In some embodiments, an Env CE polypeptide used in
accordance with the methods of the invention comprises an Env CE2
polypeptide comprising 12 conserved element: CE6, CE1-2, CE7-2,
CE8-2, CE9-2, CE10-2, CE11-2, CE12-2, CE13-2, CE14-2, CE15-2, and
CE16-2 as shown in Table 1. In some embodiments, the Env CE2
polypeptide comprises the sequence of the Env CE2 polypeptide of
Table 1 (linkers are underlined):
TABLE-US-00004 WVTVYYGVPVWAAVHNIWATHACVPTDPAAEISLWDESLKPCVKLTPLC
VTLGAKFDPIPIHYCAPAGYAGAKVQCTHGIKPVVSTQLLLNGSLAEAA
DAGGDLEITTHSFNCRGEFFYCGAKNNWRSELYKYKVVAAKAKRRVVER
EKRAGAVGFLGAAGSTMGAASVAVLTVQARQLLSGIVQQQSNLLKAIEA
QQHMLQLTVWGIKQLQTRAADWLWYIRIFIMIVGGLIGLRIAAFRVRQG YSPLSFQT.
[0184] In some embodiments, a nucleic acid encoding an HIV Env
conserved element polypeptide for use in accordance with the
disclosure encodes a polypeptide that comprises at the twelve
conserved elements set forth in SEQ ID NOS:1, 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, and 22. In some embodiments, a nucleic acid
encoding an HIV Env conserved element polypeptide in accordance
with the disclosure encodes a polypeptide comprising the twelve
conserved elements set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, and 23.
[0185] In some embodiments, an HIV Env CE polypeptide comprising
Env conserved elements SEQ ID NO:1, SEQ ID NO:2, SEQ NO:4, SEQ
NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ
ID NO:14, SEQ ID NO:16, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,
and SEQ ID NO:22 is administered in conjunction with an HIV Env CE
polypeptide comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ
ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48,
SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:50, SEQ ID NO:52, SEQ ID
NO:54, and SEQ ID NO:23.
[0186] In some embodiments, a nucleic acid encoding an HIV Env
conserved element polypeptide in accordance with the invention
encodes a polypeptide that differs from SEQ ID NO:24 by no more
than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, or 25 amino acids. In some embodiments, a
nucleic acid encoding an HIV Env conserved element polypeptide in
accordance with the invention encodes a polypeptide that differs
from SEQ ID NO:25 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino
acids.
[0187] An HIV Env conserved element construct can be administered
in conjunction with immunogenic constructs to induce immune
response to additional regions of HIV, e.g., Gag or a
non-structural protein such as Nef. In typical embodiments, the
additional immunogenic compositions encode conserved element
polypeptides to the HIV protein of interest, e.g., Gag.
[0188] In some aspects, the disclosure further provides Gag
conserved elements in addition to those described above for use in
a CE immunogenic compositions. In some embodiments, an HIV Gag CE
polypeptide comprises a CE as set forth below:
TABLE-US-00005 p24CE8-1 (SEQ ID NO: 57) QPISPRTLNAWVKV p24CE8-2
(SEQ ID NO: 58) QALSPRTLNAWVKV p24CE9-1 (SEQ ID NO: 59)
EEKAFSPEVIPMFSALSEGATPQDLNTMLN p24CE9-2 (SEQ ID NO: 60)
EEKGFNPEVIPMFTALSEGATPQDLNMMLN p24CE10-1 (SEQ ID NO: 61)
PRGSDIAGTTSTLQEQIGWMT p24CE10-2 (SEQ ID NO: 62)
PRGSDIAGTTSTLQEQIAWMT p24CE11-1 (SEQ ID NO: 63)
SILDIRQGPKEPFRDYVDRF p24CE11-2 (SEQ ID NO: 64) SILDIKQGPKEPFRDYVDRF
p24CE12-1 (SEQ ID NO: 65) QNSNPDCKTILKALG p24CE12-2 (SEQ ID NO: 66)
QNANPDCKTILKALG p24CE13-1 (SEQ ID NO: 67) LEEMMTACQGVGGPGHKARILAEAM
p24CE13-2 (SEQ ID NO: 68) LEEMMTACQGVGGPSHKARVLAEAM.
[0189] Thus, in one aspect the disclosure provides an HIV Gag CE
polypeptide that comprises one or more, or two, or three, or four,
or five, or all of, the CEs of SEQ ID NOs:57, 59, 61, 63, 65, or
67; or one or more, or two, or three, or four, or five, or all of,
the CEs of SEQ ID NOs:58, 60, 62, 64, 66, or 68.
[0190] These alternative CE elements are typically employed with
Gag CE elements from U.S. Patent Application Publication No.
20150056237, the sequences of which are shown in FIG. 23.
[0191] In some embodiments, the invention provides Gag CE
polypeptides that includethe following CE: [0192] P24CE8-1,
p24CE9-1, p24CE3-1, p24CE10-1, p24CE5-1, p24CE11-1, p24CE6-1,
p24CE12-1, p24CE13-1; or a Gag CE polypeptide that comprises
P24CE8-2, p24CE9-2, p24CE3-2, p24CE10-2, p24CE5-2, p24CE11-2,
p24CE6-2, p24CE12-2, p24CE13-2. The two polypeptides are typically
used in conjunction with one another to cover almost all of the
diversity observed in those conserved elements in naturally
occurring HIV Gag sequences. Each of the two Gag CE polypeptides
include CE elements that correspond to one another, but that
different at a small number of amino acid positions see, FIG. 23).
In some embodiments, the CE of a Gag CE polypeptide may be
presented in an order that differs from the order shown for the
illustrative Gag CE polypeptides above.
[0193] In some embodiments, an HIV Gag CE polypeptide may comprise
p24CE8 instead of p24CE1; p24CE9 instead of p24CE2; and/or p24CE13
instead of p24CE7 as shown in FIG. 23. In some embodiments, an HIV
Gag CE polypeptide may additionally comprise CE12, and CE6. In some
embodiments, an HIV Gag CE polypeptide may additionally comprise
CE11.
[0194] In the present disclosure, a conserved element nucleic acid
construct is employed in an immunization regimen that also employs
a nucleic acid encoding full-length protein, or substantially
fill-length protein, from which the conserved elements are
obtained. In the context of the present disclosure, "substantially
full-length" refers to the region of the protein that includes all
of the conserved elements, i.e., a sufficient length of a naturally
occurring protein is provided that includes all of the conserved
elements that are used in the conserved element construct. In the
present disclosure, administration of a nucleic acid construct
encoding a full-length protein excludes administration of a viral
genome, e.g., an HIV genome, that comprises a nucleic acid sequence
that encodes the protein. In some embodiments, a nucleic acid
construct used in accordance with a treatment protocol of the
present disclosure that encodes a full-length Gag or Env protein
may also encode additional viral proteins, but does not encode a
full complement of viral proteins. For example, such a nucleic acid
construct may exclude sequences that encode one or more regulatory
polypeptides such as Tat.
[0195] A nucleic acid construct encoding a full-length protein, or
substantially full-length protein, is administered following
administration of the one or more constructs encoding the CE
polypeptide(s), such that the CE polypeptide(s) acts as a prime and
the full-length polypeptide, or substantially full-length
polypeptide, is a boost. In the present disclosure, the boost
preferably comprises co-administering the one or more nucleic acid
constructs encoding CE polypeptide(s) with the nucleic acid
construct encoding the full-length polypeptide, or substantially
full-length polypeptides. The boost is typically administered
anywhere from about one, about two, about three, or about four
months; or even about one year, or longer, following administration
of the initial priming vaccines. Multiple boost vaccinations may be
used, and different full-length proteins may be used in a sequence
of boosts. A priming vaccination can itself be one or multiple,
e.g., 2, 3, 4, or 5, administrations of the CE polypeptide(s). CE
polypeptides and a full-length polypeptide, or substantially
full-length polypeptide, are preferably administered to the host by
way of administration of expression constructs that encode the
polypeptides, although in some embodiments, the polypeptides are
administered in a protein form. Protein forms may be employed in
either the priming or boosting administrations.
[0196] In the present disclosure, nucleic acid constructs encoding
the CE polypeptides that are administered in the priming component
of the vaccine regimen are administered first, i.e., the subject
has not been administered a nucleic acid that encodes the
full-length, or substantially full-length, polypeptide either prior
to or concurrently with administration of the CE constructs. Thus,
for example, a method of the invention for inducing an immune
response in the subject can comprise administering one or more
nucleic acid constructs encoding a Gag CE polypeptide construct as
the priming immunization followed by administration of a
full-length, or substantialliy full-length Gag polypeptide as a
boosting step, where the boosting step also comprises
co-administration of the CE constructs again.
[0197] The initial priming administration of the CE constructs may
also comprise administering CE constructs for another polypeptide
for which it is desired to elicit an immune response. Thus, one or
more Gag CE constructs may be administered sequentially or
concurrently with one or more Env CE constructs in the initial
priming phase of the vaccination regimen. As noted above, the
priming administration occurs before administration of either
full-length, or substantially full-length, form of either
antigen.
[0198] Nucleic acids encoding multiple CE polypeptides, typically
two CE polypeptides, i.e., a conserved element polypeptide pair,
are administered in combination. In the context of the current
invention, nucleic acid constructs encoding CE polypeptides
"administered in combination" also referred to herein as
"co-administration", may be administered together or separately,
For example, a nucleic acid construct encoding a second CE
polypeptide may be administered after (e.g., anywhere from 1
minutes to 60 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, or up to 2 weeks) administration of a first
nucleic acid construct encoding a CE polypeptide, but is typically
administered at the same time as the first nucleic acid construct.
In some embodiments, the nucleic acid construct encoding the second
CE polypeptide is administered within 24 hours of administration of
the nucleic acid construct encoding the first CE polypeptide.
[0199] Similarly, for co-administration of one or more CE
polypeptides with the full-length, or substantially full-length
polypeptide, administered as the boost, the co-administration may
be formed by administering the constructs together, or they may be
administered separately. For example, one or more nucleic acid
constructs encoding a CE polypeptide(s) may he administered shortly
before (e.g., anywhere from 1 minutes to 60 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, usually within 24 hours) or
after, a nucleic acid construct encoding the full-length
polypeptide, or the substantially full-length polypeptide.
[0200] In some mbodiments, in a boosting administration, the one or
more CE nucleic acid constructs are co-delivered with a nucleic
acid construct encoding a full-length, or substantially
full-length, protein. In the context of this disclosure,
"co-delivery" refers to administering the nucleic acid
constructions together at the same site, e.g., administering them
in the same mixture.
[0201] In some embodiments, a nucleic acid immunization regimen in
accordance with the disclosure comprises performing at least two
priming administrations with one or more CE nucleic acids
constructs, which encode a conserved element pair, either on
separate vectors or the same vector, followed by performing at
least two boosting administrationsof the CE nucleic acid
construct(s) co-delivered with the construct encoding the
full-length polypeptide or substantially full-length polypeptide.
In some embodiments, priming vaccinations can be performed at least
about two weeks apart. In some embodiments, priming vaccinations
are performed at least about one month apart or separated by
several months. Boost vaccinations are typically administered at
least about one month, often at least about 2, 3, 4, 5, 6, 7, 8, 9,
10, or 11 months; or 1 or more years after the priming
vaccinations.
[0202] In the methods of the disclosure, a nucleic acid construct
encoding a CE polypeptide is directly introduced into the cells of
the individual receiving the immunogenic composition, i.e., the CE
polypeptide is administered to the host via expression in the host
cells of the nucleic acid construct encoding the polypeptide. This
approach is described, for instance, in Wolff et. at., Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; and WO 98/04720.
Examples of DNA-based delivery technologies include, "naked DNA",
facilitated (hupivicaine, polymers, peptide-mediated) delivery, and
cationic lipid complexes or liposomes. In some embodiments, nucleic
acids are administered using ballistic delivery as described, for
instance, in U.S. Pat. No. 5,204,253 or pressure (see, e.g., U.S.
Pat. No. 5,922,687).
[0203] In some mbodiments, the immunogenic compositions of the
disclosure are administered by injection or electroporation, or a
combination of injection and electroporation. For example, the
nucleic acid can be administered using intramuscular or intradermal
injection and may include in vivo electroporation to enhance DNA
uptake.
[0204] In some embodiments, e.g., where a nucleic acid construct of
the invention is encoded by a viral vector, the nucleic acid
construct can be delivered by infecting the cells with the virus
containing the vector. This can be performed using well-known
delivery.
[0205] For embodiments that employ administration of a polypeptide
as a protein form, rather than expressing the protein in the host
cell using a nucleic acid construct, the proteins are typically
produced in an in vitro cell expression system and isolated for
administration to the subject. Such cellular expression systems are
well known in the art.
[0206] Nucleic acid constructs may be employed as plasmid
expression vectors or may be administered as a virus. In some
embodiments, the nucleic acid constructs encoding the conserved
elements and/or full-length HIV polypeptides are one or more
purified nucleic acid molecules, for example, one or more DNA
plasmid-based vectors ("naked" DNA).
[0207] In some embodiments, a nucleic acid construct encoding a CE
immunogenic polypeptide is contained within a viral vector and
administered as a virus. Viral delivery systems include adenovirus
vectors, adeno-associated viral vectors, herpes simplex viral
vectors, retroviral vectors, pox viral vectors, lentiviral vectors,
alphavirus vectors, poliovirus vectors, and other positive and
negative stranded RNA viruses, viroids, and virusoids, or portions
thereof. Methods of constructing and using such vectors are well
known in the art.
[0208] For example, recombinant viruses in the pox family of
viruses can be used for delivering the nucleic acid molecules.
These include vaccinia viruses and avian poxviruses, such as the
fowlpox and canarypox viruses. Methods for producing recombinant
pox viruses are known in the art and employ genetic recombination.
See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545. A detailed
review of this technology is found in U.S. Pat. No. 5,863,542.
Representative examples of recombinant pox viruses include ALVAC,
TROVAC, and NYVAC.
[0209] A number of adenovirus vectors, including Ad2, Ad5, and Ad7
have also been described that can be used to deliver one or more of
the nucleic acid constructs as described herein (Haj-Ahmad and
Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993)
67:5911-5921; Mittereder et al., Human Gene Therapy (1994)
5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al.,
Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)
6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).
Additionally, various adeno-associated virus (AAV) vector systems
have been developed for gene delivery. AAV vectors can he readily
constructed using techniques well known in the art. See, e.g., U.S.
Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos.
WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4
Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988)
8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor
Laboratory Press); Carter, B. J. Current Opinion in Biotechnology
(1992) 3:533-539; Muzvczka, N. Current Topics in Microhiol. and
Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994)
5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and
Zhou et al., J. Exp. Med. (1994) 179:1867-1875.
[0210] Retroviruses also provide a platform for gene delivery
systems. A number of retroviral systems have been described (U.S.
Pat. No. 5,219,740; Miller and Rosman. BioTechniques (1989)
7:980-990; Miller, A. D., Human Gene Therapy (1990) 1:5-14; Scarpa
et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl.
Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrie and Temin,
Cur, Opin. Genet. Develop. (1993) 3:102-109. Additional gene
delivery systems include lentiviral vectors that employ lentiviral
vector backbones.
[0211] Members of the Alphavirus genus, such as, but not limited
to, vectors derived from the Sindhis, Semliki Forest, and
Venezuelan Equine Encephalitis viruses, can also be used as viral
vectors to deliver one or more nucleic acid constructs of the
disclosure. For a description of Sindbis-virus derived vectors
useful for the practice of the instant methods, see, Dubensky et
al, J. Virol. (1996) 70:508-519; and International Publication Nos.
WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T. W., et
al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky,
Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4, 1998).
Expression Constructs Encoding Fusion Polypeptides Comprising a
Degradation Signal or Signal Peptide Sequence
[0212] In some embodiments, a nucleic acid encoding a conserved
element polypeptide encodes a form in which the conserved element
is fused to a sequence to enhance the immune response, such as a
signal peptide sequence or a sequence that targets the protein for
lysosomal degradation. Such embodiments typically results in
enhanced immune responses in comparison to embodiments where the
conserved element vaccine is not fused to a signal peptide or
degradation signal.
Lysosomal Targeting Sequence
[0213] In some embodiments, signals that target proteins to the
lysosome may be employed. For example, the lysosome associated
membrane proteins1 and 2 (LAMP-1 and LAMP-2) include a region that
targets proteins to the lysosome. Examples of lysosome targeting
sequences are provided, e.g., in U.S. Pat. Nos. 5,633,234;
6,248,565; and 6,294,378.
[0214] Destabilizing sequences present in particular proteins are
well known in the art. Exemplary destabilization sequences include
c-myc aa 2-120; cyclin A aa 13-91; Cyclin B aa 13-91; IkB.alpha. aa
20-45; .beta.-Catenin aa 19-44; .beta.-Catenin aa 18-47, c-Jun
aa1-67; and c-Mos aa1-35; and fragments and variants, of those
segments that mediate destabilization. Such fragments can be
identified using any method. For example, polypeptide half-life can
be determined by a pulse-chase assay that detects the amount of
polypeptide that is present over a time course using an antibody to
the polypeptide, or to a tag linked to the polypeptide. Exemplary
assays are described, e.g., in WO02/36806, which is incorporated by
reference.
[0215] Variants of such sequences, e.g., that have at least 90%
identity, usually at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or greater, identity to the sequences noted above, e.g., a
LAMP degradation sequence, can be employed in this invention.
[0216] Additional degradation signals that can be used to modify
retroviral antigens, e.g., HIV antigens in accordance with the
invention include the F-box degradation signal, such as the F- BOX
signal 47aa (182-228) from protein beta-TrCP (Liu, et al., Biochem
Biophys Res Comm. 313:1023-1029, 2004). Accordingly, in sonic
embodiments, an expression vector for use in the invention may
encode a fusion protein where an F-box degradation signal is
attached to an HIV polypeptide, e.g., an HIV Env CE polypeptide as
described herein.
Targeting to the Proteasome and Other Degradation Signals
[0217] Many polypeptide sequences that target a protein for
degradation are known in the art. One example of destabilizing
sequences are so-called PEST sequences, which are abundant in the
amino acids Pro, Asp, Glu, Ser, Thr (they need not be in a
particular order), and can occur in internal positions in a protein
sequence. A number of proteins reported to have PEST sequence
elements are rapidly targeted to the 26S proteasome. A PEST
sequence typically correlates with a) predicted surface exposed
loops or turns and b) serine phosphorylation sites, e.g., the motif
S/TP is the target site for cyclin dependent kinases.
[0218] Additional destabilization sequences relate to sequences
present in the N-terminal region. In particular the rate of
ubiquitination, which targets proteins for degradation by the 26S
proteasome can be influenced by the identity of the N-terminal
residue of the protein. Thus, destabilization sequences can also
comprise such N-terminal residues, "N-end rule" targeting (see,
e.g., Tobery et at., J Exp. Med. 185:909-920.).
[0219] Other targeting signals include the destruction box sequence
that is present, e.g., in cyclins. Such a destruction box has a
motif of 9 amino acids, R1(A/T)2(A)3L4(G)5X6(1/V)7(G/T)8(N)9, in
which the only invariable residues are R and L in positions 1 and
4, respectively. The residues shown in parenthesis occur in most
destruction sequences. (see, e.g., Hershko Ciechanover, Annu. Rev.
Biochem. 67:425-79, 1998). In other instances, destabilization
sequences lead to phosphorylation of a protein at a serine residue
(e.g., 1.kappa.b.alpha.).
[0220] In some embodiments, a conserved element polypeptide of the
invention is fused to a LAMP degradation sequence. For example, the
methods of the invention may employ a polypeptide in which SEQ ID
NO:24 or SEQ ID NO:25, or SEQ ID NO:40 or 41, is fused to a LAMP
degradation sequence.
Expression Constructs that Encode Secreted Fusion Proteins
[0221] A secretory polypeptide in the context of this invention is
a polypeptide signal sequence that results in secretion of the
protein to which it is attached. In some embodiments, the secretory
polypeptide is a chemokine, cytokine, or lymphokine, or a fragment
of the chemokine, cytokine, or lymphokine that retains
immunostimulatory activity. Examples of chemokine secretory
polypeptides include MCP-3 and IP-10. In other embodiments, the
secretory polypeptide is a polypeptide signal sequence from a
secreted protein such as tissue plasminogen activator (tPA)
protein, growth hormone, GM-CSF, a cytokine, or an immunoglobulin
protein. Constructs encoding secretory fusion proteins are
disclosed, e.g., in WO02/36806.
[0222] In some embodiments, the signal peptide is a GM-CSF
sequence, e.g., a mammalian GM-CSF sequence such as a human GM-CSF
signal peptide sequence.
[0223] In some embodiments, a secretory signal for use in the
invention is MCP-3 amino acids 33-109, e.g., linked to an IP-10
secretory peptide.
[0224] In some embodiments, a conserved element polypeptide may SEQ
ID NO:24 fused to a signal peptide; or may comprise SEQ ID NO:25
fused to a signal peptide. The signal peptide may be a native HIV
Env signal peptide or a heterologous signal peptide. In some
embodiments, a conserved element polypeptide may SEQ ID NO:40 fused
to a signal peptide; or may comprise SEQ ID NO:41 fused to a signal
peptide. The signal peptide may be a native HIV signal peptide or a
heterologous signal peptide.
[0225] Similarly, an expression construct encoding a full-length
polypeptide, or substantially fill-length polypeptide, may also be
modified with a secretory sequence, e.g., a heterologous signal
peptide or degradation peptide sequence. Moreover, more than one
construct encoding the full-length polypeptide, or substantially
full-length polypeptide,s may be administered. For example, a
construct in which Gag (or Env) is fused to a signal polypeptide
may be used in conjunction with a construct in which Gag (or ENv)
is fused to a degradation sequence.
Additional Properties of Expression Constructs
[0226] Within each expression cassette, sequences encoding an
antigen for use in the nucleic acid vaccines of the invention will
be operably linked to expression regulating sequences. "Operably
linked" sequences include both expression control sequences that
are contiguous with the nucleic acid of interest and expression
control sequences that act in trans or at a distance to control the
gene of interest. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that promote RNA export (e.g., a constitutive transport
element (CIE), a RNA transport element (RTE), or combinations
thereof; sequences that enhance translation efficiency (e.g., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion.
[0227] Any of the conventional vectors used for expression in
eukaryotic cells may be used for directly introducing nucleic acids
into tissue. Expression vectors containing regulatory elements from
eukaryotic viruses are often used in eukaryotic expression vectors.
Such regulatory elements include, e.g., human CMV, simian CMV,
viral LTRs, and the like. Typical vectors may comprise, e.g., those
with a human CMV promoter, bovine growth hormone polyA site and an
antibiotic resistance gene for selective growth in bacteria.
[0228] Other expression vector components are well known in the
art, including, but not limited to, the following: transcription
enhancer elements, transcription termination signals,
polyadenylation sequences, splice sites, sequences for optimization
of initiation of translation, and translation termination
sequences.
[0229] In some embodiments, the nucleic acid component may comprise
one or more RNA molecules, such as viral RNA molecules or mRNA
molecules that encode the antigen of interest.
[0230] In typical embodiments, the nucleic acid constructs are
codon-optimized for expression in a human.
[0231] As noted here, in the present disclosure, a "nucleic acid"
molecule can include cDNA and genomic DNA sequences, RNA, and
synthetic nucleic acid sequences. Thus, "nucleic acid" also
encompasses embodiments in which analogs of DNA and RNA are
employed.
[0232] An immunogenic composition of the invention can be
administered as one or more constructs. For example, where two sets
of conserved elements are employed, e.g., HIV Env conserved element
polypeptides of SEQ ID NOS:24 and 25, or HIV Gag conserved elment
polypeptides of SEQ ID NOS: 40 and 41, nucleic acid construct can
encode both sets, or each set may be encoded by a separate
expression vector. Thus, the expression constructs administered in
accordance with the invention may be administered as multiple
expression vectors, or as one or more expression vectors encoding
multiple expression units, e.g., a discistronic, or otherwise
multicistronic, expression vectors. For example, an expression
vector may be employed that encodes both SEQ NO:24 and SEQ ID NO:25
or multiple expression vectors may be employed where SEQ ID NO:24
is encoded by one vector and SEQ ID NO:25 is encoded by another
vector. Similarly, an expression vector may be employed that
encodes both SEQ ID NO:40 and SEQ ID NO:41 or multiple expression
vectors may be employed where SEQ ID NO:40 is encoded by one vector
and SEQ ID NO:41 is encoded by another vector.
[0233] In some embodiments, multiple nucleic acid constructs that
encode an HIV Gag or Env CE polypeptide may be employed. For
example, a nucleic acid construct that encodes an HIV-Env CE
polypeptide fused to a signal peptide may be used in combination
with a nucleic acid construct that encodes the HIV-Env CE
polypeptide fused to a degradation signal; and/or a nucleic acid
construct that encodes an HIV-Gag CE polypeptide fused to a signal
peptide may be used in combination with a nucleic acid construct
that encodes the HIV-Gag CE polypeptide fused to a degradation
signal. Thus, multiple constructs that encode a desired HIV CE
polypeptide may be used in a vaccine.
Administration of Immunogenic Compositions as Peptides
[0234] In some embodiments, CE polypeptides, e.g., HIV Gag or Env
CE polypeptides as of the present discosure, are administered to a
subject in the form of a protein. Thus, in some embodiments, a
subject is administered multiple, typically two, HIV CE
polypeptides. As explained in the context of the administration of
a nucleic acid construct, CE polypeptides "administered in
combination" may be administered together or separately. For
example, a first CE polypeptide may be administered prior to (e.g.,
anywhere from 1 minutes to 60 minutes, 1 hour, 2 hours, 4 hours, 6
hours, 12 hours, 24 hours. 48 hours, 72 hours, 96 hours, or 1 week,
or more) administration of a second CE polypeptide.
[0235] A full-length protein or substantially full-length protein
may also he administered in the form of a protein. In one
embodiment, a full-length polypeptide or substantially full-length
polypeptide is administered following administration of a CE
polypeptide, such that the CE polypeptide acts as a prime and the
full-length polypeptide, or substantially full-length polypeptide
is a boost. In preferred embodiments, the full-length or
substantially full-length polypeptide is administered in a boosting
step that also comprises administration of the CE polypeptide(s).
The boost is typically administered anywhere from two weeks to one,
two, three, or four months, or longer, following administration of
the initial priming vaccines. A priming vaccination can itself be
one or multiple administrations of the CE polypeptide. In some
embodiments, a boost of a full-length, or substantially
full-length, polypeptide is administered in combination with a
further administration of the CE polypeptide (s), such that the CE
polypeptide is not only administered as a priming step, but as part
of the boosting step that comprises administering full-length, or
substantially full-length polyeptide.
[0236] In some embodiments, both nucleic acids CE polypeptides in
accordance with the disclosure and protein forms of the CE
polypeptides may be administered to a subject at various times in a
vaccination regimen. Similarly, nucleic acid constructs encoding a
full-length protein, or substantially full-length protein, may be
used in vaccine regimens that also employ protein forms of the
full-length protein, or substantially full-length protein. In some
embodiments, a full-length protein may be administered in the form
of a viral particle.
Assessment of Immunogenic Response
[0237] To assess a patient's immune system during and after
treatment and to further evaluate the treatment regimen, various
parameters can be measured. Measurements to evaluate immunogenic
responses include: antibody measurements in the plasma, serum, or
other body fluids; analysis of in vitro cell proliferation in
response to a specific antigen, indicating the function of CD4+
cells, and analysis of CD8+ responses. Such assays are well known
in the art. For example, for measuring CD4+ T cells, many
laboratories measure absolute CD4+ T-cell levels in whole blood by
a multi-platform, three-stage process. Systems for measuring CD4+
cells are commercially available. For example commercially
available FAC sytems are available that automatically measure
absolutes CD4+, CD8+, and CD3+ T lymphocytes.
[0238] Other measurements of immune response include assessing CD8+
responses. These techniques are well known. CD8+ T-cell responses
can be measured, for example, by using tetramer staining of fresh
or cultured PBMC (see, e.g., Altman, et al., Proc. Natl. Acad. Sci.
USA 90:10330, 1993; Altman, et al., Science 274:94, 1996), or
.gamma.-interferon release assays such as ELISPOT assays (see,
e.g., Lalvani, et al., J. Exp. Med. 186:859, 1997; Dunbar, et al.,
Curr. Biol. 8:413, 1998; Murali-Krishna, et al. Immunity 8:177,
1998), or by using functional cytotoxicity assays.
Viral Titer
[0239] Viremia is measured by assessing viral titer in a patient.
There are a variety of methods of perform this. For example, plasma
HIV RNA concentrations can be quantified by either target
amplification methods (e.g., quantitative RT polymerase chain
reaction [RT-PCR], Amplicor HIV Monitor assay, Roche Molecular
Systems; or nucleic acid sequence-based amplification,
[NASBA.RTM.], NucliSens.TM. HIV-1 QT assay, Organon Teknika) or
signal amplification methods (e.g., branched DNA [bDNA],
Quantiplex.TM. HIV RNA bDNA assay, Chiron Diagnostics). The bDNA
signal amplification method amplifies the signal obtained from a
captured HIV RNA target by using sequential oligonucleotide
hybridization steps, whereas the RT-PCR and NASBA.RTM. assays use
enzymatic methods to amplify the target HIV RNA into measurable
amounts of nucleic acid product. Target HIV RNA sequences are
quantitated by comparison with internal or external reference
standards, depending upon the assay used.
Administration of Nucleic Acid Constructs
[0240] Nucleic acids for administration to a subject are formulated
for pharmaceutical administration. While any suitable carrier known
to those of ordinary skill in the art may be employed in the
pharmaceutical compositions of this invention, the type of carrier
will vary depending on the mode of administration. For parenteral
administration, including intranasal, intradermal, subcutaneous or
intramuscular injection or electroporation, the carrier preferably
comprises water, saline, and optionally an alcohol, a frit, a
polymer, a wax, one or more stabilizing amino acids or a buffer.
General formulation technologies are known to those of skill in the
art (see, for example, Remington: The Science and Practice of
Pharmacy (22.sup.nd edition), Allan, ed; 2012, Lippincott Williams
& Wilkins; Injectable Dispersed Systems: Formulation,
Processing And Performance, Burgess, ed., 2005, CRC Press; and
Pharmaceutical Formulation Development of Peptides and Proteins,
2000, Taylor & Francis).
[0241] DNA immunogenic compositions can be administered once or
multiple times. DNA vaccination is performed more than once, for
example, 2, 3, 4, 5, 6, 7, 8, 10, 15, 20 or more times as needed to
induce the desired response (e.g., specific antigenic response or
proliferation of immune cells). Multiple administrations can be
administered, for example, hi-weekly, weekly, bi-monthly, monthly,
or more or less often, as needed, for a time period sufficient to
achieve the desired response.
[0242] The nucleic acid constructs in accordance with the invention
are administered to a mammalian host. The mammalian host usually is
a human or a primate. In some embodiments, the mammalian host can
be a domestic animal, for example, canine, feline, lagomorpha,
rodentia, rattus, hamster, murine. In other embodiment, the
mammalian host is an agricultural animal, for example, bovine,
ovine, porcine, equine, etc.
[0243] Immunogenic compositions containing the DNA expression
constructs can be formulated in accordance with standard techniques
well known to those skilled in the pharmaceutical art. Such
compositions can be administered in dosages and by techniques well
known to those skilled in the medical arts taking into
consideration such factors as the age, sex, weight, and condition
of the particular patient, and the route of administration.
[0244] In therapeutic applications, the vaccines are administered
to a patient in an amount sufficient to elicit a therapeutic
effect, e.g, a CD8.sup.30, CD4.sup.+, and/or antibody response to
the HIV-1 antigens encoded by the vaccines that at least partially
arrests or slows symptoms, or and/or complications of HIV
infection. An amount adequate to accomplish this is defined as
"therapeutically effective dose." Amounts effective for this use
will depend on, e.g., the particular composition of the vaccine
regimen administered, the manner of administration, the stage and
severity of the disease, the general state of health of the
patient, and the judgment of the prescribing physician.
[0245] Nucleic acid vaccines are administered by methods well known
in the art as described in Donnelly et al. (Ann. Rev. Immunal.
15:617-648 (1997)); Feigner et al. (U.S. Pat. No. 5,580,859, issued
Dec. 3, 1996); Feigner (U.S. Pat. No. 5,703,055, issued Dec. 30,
1997); and Carson et al. (U.S. Pat. No. 5,679,647, issued Oct. 21,
1997), each of which is incorporated herein by reference. One
skilled in the art would know that the choice of a pharmaceutically
acceptable carrier, including a physiologically acceptable
compound, depends, for example, on the route of administration of
the expression vector.
[0246] As noted above, immunogenic DNA compositions can be
delivered via a variety of routes. Typical delivery routes include
parenteral administration, e.g., intradermal, intramuscular or
subcutaneous routes. Administration of expression vectors of the
invention to muscle and by electroporation can be a particularly
effective method of administration, including intradermal and
subcutaneous injections and transdermal administration. Transdermal
administration, such as by iontophoresis, is also an effective
method to deliver expression vectors of the invention to muscle.
Epidermal administration of expression vectors of the invention can
also be employed. Epidermal administration involves mechanically or
chemically irritating the outermost layer of epidermis to stimulate
an immune response to the irritant (Carson et al., U.S. Pat. No.
5,679,647).
[0247] Nucleic acids can be administered in solution (e.g., a
phosphate-buffered saline solution) by injection, usually by an
intravenous, subcutaneous or intramuscular route. In embodiments
that employ a naked nucleic acid composition, the dose of a naked
nucleic acid composition is from about 1.0 ng to about 10 mg for a
typical 70 kilogram patient. Subcutaneous or intramuscular doses
for naked nucleic acid (typically DNA) may range from 0.1 ug to 100
ug for a 70 kg patient in generally good health. For example, an
HIV DNA vaccine, e.g., naked DNA or polynucleotide in an aqueous
carrier, can be injected into tissue, e.g., intramuscularly or
intradermally, in amounts of from 10 .mu.l per site to about 1 ml
per site. The concentration of polynucleotide in the formulation is
usually from about 0.1 .mu.g/ml to about 4 mg/ml. In some
embodiments, the DNA may be administered in ng amounts, for example
at a level of 1 to 100 ng.
[0248] In embodiments in which one or more of the components of a
vaccine regimen is administered in the form of a protein, the
protein is typically administered at a concentration that is
determined by known techniques taking into account medical
considerations regarding the subject. Dosages for administration of
a polypeptide composition included milligram or microgram amounts
per kilogram. Illustrative doses are about 0.1 ug/kg to about 500
mg/kg. For example, doses may range from about 1 ug/kg to about 100
ug/kg or from about 0.1 ug/kg to about 50 ug/kg. I some
embodiments, the dosage is about 0.5 ug/kg or more, 1 ug/kg or
more, 2 ug/kg or more, 5 ug/kg or more, 10 ug/kg or more 25 ug/kg
or more or 50 ug/kg or more.
[0249] As understood by one in the art, dosages may vary depending
on the route of aministration.
[0250] The protein is formulated using well-known techniques for
administration employing various routes, e.g., intravenous,
subcutaneous, intramuscular, intranasal, transdermal, or
intraperitoneal administration.
[0251] The vaccine may be delivered in a physiologically compatible
solution such as sterile PBS. The vaccines may also be lyophilized
prior to delivery. As well known to those in the art, the dose may
he proportional to weight.
[0252] The compositions included in the regimen descried herein for
inducing an immune response can be administered alone, or can be
co-administered or sequentially administered with other
immunological, antigenic, vaccine, or therapeutic compositions.
[0253] Compositions that may also be administered with the vaccines
include other agents to potentiate or broaden the immune response,
e.g., IL-15, IL-12, IL-2 or CD40 ligand, which can be administered
at specified intervals of time, or continuously administered.
[0254] The vaccines can additionally be complexed with other
components such as peptides, polypeptides and carbohydrates for
delivery. For example, expression vectors, i.e., nucleic acid
vectors that are not contained within a viral particle, can be
complexed to particles or beads that can he administered to an
individual, for example, using a vaccine gun.
[0255] The immunogenic compositions can also be formulated for
administration via the nasal passages. Formulations suitable for
nasal administration, wherein the carrier is a solid, include a
coarse powder having a particle size, for example, in the range of
about 10 to about 500 microns which is administered in the manner
in which snuff is taken, i.e., by rapid inhalation through the
nasal passage from a container of the powder held close up to the
nose. Suitable formulations wherein the carrier is a liquid for
administration as, for example, nasal spray, nasal drops, or by
aerosol administration by nebulizer, include aqueous or oily
solutions of the active ingredient. For further discussions of
nasal administration of AIDS-related vaccines, references are made
to the following patents, U.S. Pat. Nos. 5,846,978, 5,663,169,
5,578,597, 5,502,060, 5,476,874, 5,413,999, 5,308,854, 5,192,668,
and 5,187,074.
[0256] The vaccines can be incorporated, if desired, into
liposomes, microspheres or other polymer matrices (see, e.g.,
Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome
Technology, Vols. I to III (2nd ed. 1993). Liposomes, for example,
which consist of phospholipids or other lipids, are nontoxic,
physiologically acceptable and metabolizable carriers that are
relatively simple to make and administer. Liposomes include
emulsions, foams, micelles, insoluble monolayers, liquid crystals,
phospholipid dispersions, lamellar layers and the like.
EXAMPLES ILLUSTRATING THE INVENTION
Example 1
Illustration of Enhanced Immune Responses Obtained with CE
Construct Prime Followed by CE and Full-Length Polypeptides as
Boost
[0257] This example evaluated prime-boost combination vaccines to
increase breadth of immunity. In addition to the CE prime-full
length molecule boost, it was found that using a boost involving
co-delivery of CE and full-length molecule resulted in
significantly further improved breadth of the responses. This boost
further elicited higher levels of cytotoxic T cells focusing the
immune responses to the highly conserved epitopes.
[0258] Method: this study was performed using a simian
immunodeficiency virus (SIV) derived conserved element pDNA vaccine
that was designed from the SIV p27.sup.gag capsid protein by
analogy to an HIV CE vaccine. The SIV p27.sup.gag is the
proteolytic processing product derived from the full length SIV
p57.sup.gag protein. Seven highly conserved elements were
identified within the p27.sup.gag capsid protein by (i) analogy to
HIV CE sequences and (ii) sequence conservation among available SW
sequences (FIG. 1). Two sets of sequences, p27CE1 and p27CE2,
differing in six `toggle` AA, were generated. DNA vectors
expressing the SW p27CE1 and p27CE2 proteins were tested in
macaques.
[0259] Macaques (N=14) received vaccinations with a mixture of SW
p27CE1 pDNA and p27CE2 pDNA, referred to SIV p27 pDNA (FIG. 2). The
CE-specific cellular immune responses were compared in blood. PBMC
were stimulated with a SIV CE-specific peptide pool and showed the
induction of robust CE-specific responses ranging from 0.03-0.8% of
total T lymphocytes. The CE-specific T cells showed both central
(CD28.sup.+, CD95.sup.+) and effector memory phenotype (EM,
CD28.sup.+, CD95.sup.+) with a significant fraction of cytotoxic T
cells (IFN-y.sup.+, granzyme B.sup.+, CD107a.sup.+). In contrast,
only .about.60% of the macaques (18 of 31 vaccinated animals)
vaccinated with a plasmid expressing the full length SIV Gag
developed responses recognizing any CE, albeit they developed
robust cellular immune responses to variable regions of gag. These
data are similar to results found in HIV gag DNA vaccinated animals
(Kulkami et al PLoS One;9:e86254, 2014).
[0260] To further expand the potency of booster vaccination, two
different regimens were compared using the SIV CE pDNA primed
macaques (FIG. 3): (i) As prime, the animals (N=6) received p27CE
pDNA vaccinations, and as boost (1.times.) gag DNA as described
above (by analogy to HIV boosting regimen). (ii) Another group of
animals (N=6) received a single p27CE pDNA vaccine as prime, and as
boost co-delivery (2.times.) of CE and gag DNA. The CE-specific
immune responses were measured in blood and the responses were
mapped using peptide pools specific for each of the individual SIV
CE.
[0261] The SIV p27CE pDNA primed animals (N=6) received a booster
vaccination with a plasmid expressing the full length SW
p57.sup.gag pDNA (FIG. 4). This resulted in a significant increase
in CE-specific responses, corroborating the observation from the
HIV p24CE prime-gag DNA boost studies. Thus, both the SW p27CE pDNA
and the HIV p24CE pDNA vaccine share the unique feature of
effectively inducing immune responses to subdominant Gag epitopes,
which is only inefficiently achieved by DNA vaccination expressing
full length Gag. It was also demonstrated that for both CE
vaccines, a gag pDNA boost significantly increased the magnitude of
these responses.
[0262] The other group of CE primed macaques (N=6) received a
booster vaccination by co-delivery of the CE DNA and a plasmid
expressing the full length SW p57.sup.gag pDNA (FIG. 5). Comparison
of the magnitude of the immune responses obtained after the
1.sup.st and 2.sup.nd booster vaccination showed a significant
increase (p=0.0022) of the magnitude of CE-specific T cell
responses upon the 2.sup.nd boost (FIG. 5) reaching responses up to
.about.2% of total circulating T cells. These data show that the
inclusion of two booster vaccinations is advantageous to increase
the magnitude of the CE-specific responses.
[0263] While both booster regimens led to marked increase in
magnitude of the CE responses, reaching similar levels, a
fundamental difference in the breadth of the responses was found
(FIG. 6). Mapping of responses to individual CE in the CE-primed
macaques (N-14) showed a response rate of 1-4 CE per animals (FIG.
6). Mapping the responses elicited by the gag DNA only boost
revealed recognition of 1-3 CE per animal (FIG. 6). Thus, no change
in breadth was found upon inclusion of additional gag DNA booster
vaccination (both SW and HIV model systems). In contrast, boosting
of CE primed animals by co-delivery of CE and gag DNA resulted in
increased breadth of the responses recognizing of 2-6 CE per animal
with all 7 CE being immunogenic (FIG. 6). Comparison of the
response rate (number of positive CE per animal) showed a
significant (p=0.0195, Mann Whitney test) increase in breadth using
co-delivery of CE and gag pDNA as boost.
[0264] The induced CE-specific immune responses were further
examined for cytotoxicity markers (FIG. 7). Analysis of the CD4 and
CD8 T cell subsets showed significant increase of CE-specific cells
harboring granzyme B and being able to degranulate (CD107a.sup.+)
in the animals that received the CE and gag DNA co-delivery boost.
The cytotoxic CE-specific responses within the total T cell
population are plotted. Thus, SW gag&CE DNA co-delivery as
boosts not only broadened the Gag T cell responses, it also induced
more robust cytotoxic CE-specific T cell responses.
[0265] The data obtained with the SIV p27CE DNA vaccine therefore
demonstrate that a booster vaccination including the co-delivery of
CE and gag pDNA provided a significant increase in breadth of the
CE-specific responses compared to a regimen using only the gag pDNA
boost. The co-delivery of CE pDNA and pDNA expressing the full
length protein as boost is proposed to maximize the induction of T
cell responses to subdominant HIV Gag epitopes and this strategy is
currently tested.
[0266] Together, these data corroborate the original observation
demonstrating that priming with CE as vaccine (DNA, viral vector,
protein) is important to allow development of immune responses to
subdominant epitopes. Fine-tuning the booster vaccination by
including the co-delivery CE and the intact full-length molecule
offers an additional advantage to optimize breadth and quality of
the immune responses. This regimen is more effective than the CE
DNA prime-gag DNA boost even when additional gag DNA boosts were
given.
[0267] In humans, immunogenic responses will be evaluated in a
trial that includes priming with the HIV p24CE DNA (0, month 2),
followed by 2 booster vaccinations using codelivery of p24CE DNA
and p55gag DNA (Month 4 and 6). In macaques, there were no
significant differences identified between 2 and 3 CE DNA
vaccinations, thus, two priming vaccinations are planned to
strengthen the priming step. Two booster vaccinations will be given
because in the macaque model, 2 co-delivery boosts provided
improved results. This trial employs ae dual-promoter p24CE1/2 pDNA
(plasmid code 306H, FIG. 8A) that spans 5577 base pairs (bp) and
encodes the two expression-optimized p24CE genes from a single
eukaryotic expression vector pDP.CMVkan, a derivative of pVR1012
(Hartikka et al., 1996). The p24CE1 gene is expressed from the
human cytomegalovirus (hCMV) promoter and is terminated by the
bovine growth hormone (BGH) polyadenylation (polyA) signal. The
p24CE2 gene is expressed from the simian CMV (sCMV) promoter and is
terminated by the simian vacuolating virus 40 (SV40) polyA signal.
P24CE1 and p24CE2 proteins contain the 17-aminon acid GM-CSF signal
peptide (GenBank: AAA98768.1), which favorably affects localization
and stability of p24CE1 and p24CE2 proteins as well as the nature
of elicited immune responses (Kulkarni et al., 2013, supra). The
sequences of both genes are optimized to enhance expression in
human cells.
[0268] A plasmid encoding p55gag is used in the boost along with
the p24CE plasmid. The p55gag pDNA (plasmid code 114H, FIG. 8B)
spans 5518 bp and encodes the fill-length p55gag protein of HIV-1
(clade B; GenBank NP_057850.1). The p55gag protein is 500 amino
acids in length and contains the proteolytic processing product,
p24gag, located between AA 133-363. The expression-optimized p55gag
gene was chemically synthesized and inserted into the eukaryotic
expression plasmid pCMVkan under the control of the hCMV immediate
early enhancer/promoter and terminated by the BGH polyA signal. The
pCMVkan plasmid backbone is derived from pVR1012 (Hartikka et al.,
1996). pCMVkan shares the backbone with pDP.CMVkan, but lacks the
simian CMV promoter and the SV40 polyadenylation signal.
[0269] Application of this method is not limited to HIV but can
modulated to address the induction of immune responses to any
subdominant epitopes (cellular and or humoral) to increase breadth,
magnitude and quality of the immune responses.
Example 2
Env DNA Vaccination Induces Cross-Clade Specific Cellular Immune
Responses in Mice
Result:
Conserved Element DNA Vaccines
[0270] A set of conserved elements (CE) was identified in the HIV
Env protein (FIG. 9). This example describes the identification of
12 conserved elements in HIV Env of 11, 14, 21, 15, 23, 21, 13, 12,
14, 43, 20, and 13 AA in length (FIG. 10). Each CE segment is
separated by linkers 3 amino acids in length, composed of alanine
and some of which also contain a valine, glutamic acid, lysine,
aspartic acid, phenylalanine or glycine, designed to facilitate
processing of the protein. Expression-optimized synthetic Env-CE1
and Env-CE2 genes were inserted into an eukaryotic expression
vector pCMVkan between the human CMV promoter and bovine growth
hormone poly A signal. pCMVkan is optimized for optimal growth in
bacteria (kanR) and expression of the insert in mammalian
cells.
[0271] Regions for inclusion/exclusion from the vaccine were
selected based on whether immune responses to such regions were
associated with virologic control or lack of control. We designed
and synthesized two versions of these synthetic proteins (Env-CE1
and Env-CE2), each of which is composed of 12 conserved elements
that differ by 0-5 amino acid per conserved element (CE) to
maximize the inclusion of commonly detected variants (See Table 1).
These CE were collinearly arranged and separated by short amino
acid linkers (e.g., 3 amino acids) designed to facilitate
processing of the protein and avoidance of neo-antigens. The coding
sequences were RNA/codon-optimized according to Pavlakis and
Felber's RNA optimization method (U.S. Pat. Nos. 5,972,596;
5,965,726; 6,174,666; 6,791,664, 6,414,132; 6,794,498) and designed
to use alternative codons to maximize expression in human cells.
Expression-optimized sequences were placed into a eukaryotic DNA
plasmid vector.
[0272] In an illustrative embodiment, a combination of the two
plasmids was used to vaccinate 4 macaques (FIG. 11). This study
showed induction of robust cellular CE-specific immune responses
(0.1-0.8% of T cells), mediated primarily by CD8+ T cells. (FIG.
12). Fine mapping showed that 6 of the 12 CE were immunogenic (FIG.
15). The vaccine induced both CD8 and CD4 memory T cell responses
of the central and effector type (FIG. 13). This vaccine also
induced CE-specific responses with a significant fraction of CDS+ T
cells with cytotoxic phenotype (Granzyme B+ and CD107a+) (FIG. 14).
Thus, the Env-CE DNA vaccine induced cellular immune responses with
the desired features for an effective T cell vaccine. In addition
to the cellular immunity, the Env-CE DNA vaccine also induced
Immoral immune responses. The antibodies could detect the immunogen
as well as clade B and clade C Env by Western blotting (FIG. 22).
The antibody could also detect HIV gp120 by ELISA (FIG. 20).
[0273] In contrast, vaccination of macaques (N=9) with complete Env
DNA failed to develop CE-specific linear peptide responses in 50%
of the macaques or induced poor responses to only 1 or 2 CE (FIG.
16) and could not detect the immunogen on Western blots. This
indicates that the 12 Env CE are not immunogenic when present
within with the complete Env protein, likely due to immunological
interference with other epitopes and thus, unable to induce de novo
responses.
[0274] Implementation of a prime-boost regimen using the Env-CE DNA
prime followed by the intact Env boost (FIGS. 17, 18, and 19),
demonstrated great increase of both cellular (including cytotoxic)
responses as well as humoral responses. In particular, we found a
great augmentation of linear peptide response to the CE, which is
not found in Env DNA vaccinated macaques. Although inefficient in
generating de novo responses to the CE, boosting with the intact
Env DNA potently augmented pre-existing immunity. This vaccine
regimen thus achieved an effective alteration in the immunological
hierarchy.
[0275] These data thus indicate that HIV Env-CE1 and Env-CE2 are
effective immunogens in inducing cellular and humuoral responses
and fulfill the basic criteria of inducing responses to highly
conserved region of Env that are not seen by the immune system as
part of the intact Env (altered immunohierachy) and the responses
are cross-clade reactive (improved cross-clade breadth).
[0276] As shown in the illustrative data provided herein, the use
of full-length Env fails or only poorly induces responses to the
conserved regions of the protein. This led to the hypothesis that
CE epitopes within the Env protein are not immunogenic due to
either suboptimal processing and presentation or immunodominance
hierarchy focusing the responses to variable epitopes. To address
this experimentally, Env-CE DNA primed macaques received an Env DNA
booster vaccine with the expectation of either no boost of CE
responses (if no processing and presentation of CE-containing
peptides from Env) or a boost of the CE responses (if
immunodominance hierarchy can be altered by CE priming). Indeed,
robust increase in immunity was found in CE-primed macaques given a
boost with DNA expressing intact Env.
[0277] Therefore, priming with Env-CE DNA and boosting with plasmid
expressing intact Env may solve a major obstacle in HIV vaccine
development, demonstrating alteration of the hierarchy of epitope
recognition and development of immune responses to potentially
protective subdominant highly conserved epitopes. Boosting with Env
DNA vaccine increases magnitude, breadth and polyfunctionality of
the cellular responses as well as the breadth of humoral immunity
against Env, targeting epitopes within the highly conserved
elements as well as outside Env-CE.
Identification of Conserved Element Regions
[0278] The success of a conserved elements vaccine approach (1)
depends on the ability to identify important features of viral
proteins. Recent studies have shown that mutations at highly
conserved sites showed varying degrees of fitness impact, from
deleterious to negligible (2-5) to increasing (6).
[0279] Furthermore, mutations at sites that remain conserved
through time have been shown to have greater fitness cost than
mutations of amino acids that became dominant (and hence calculated
to be conserved) later in the pandemic (2). Indeed, HIV strains are
continuously being imprinted by the human HLA types encountered in
different human populations (7,8). Thus, sequence conservation may
change as the virus continues to evolve and adapt to host immunity,
and contemporary sequence conservation may not be sufficient for
pinpointing sites with crucial functional or structural roles. We
therefore use sequence conservation as well as other features to
identify the conserved elements to be used in our vaccines. The
degree of conservation required for inclusion as an Env CE was at
least 90% across the entire HIV-1 M group, and usually at least
98%. However, this requirement could be relaxed if data was
available in the literature that associated a CTL or antibody
epitope, within the CE, with virologic control (as evidenced by low
viral load). If an epitope was associated with lack of virologic
control (high viral load), its sequence was excluded from inclusion
in a CE. Criteria for assigning virologic control or lack of
control van in the literature. For example, Mothe (9) and Matthews
(10) used viral load <2,000 HIV RNA molecules per milliliter of
blood plasma to indicate control.
[0280] Toggle sites were used to create two versions of most CE. A
toggle site represents an amino acid site at which conservation may
be low but at which two amino acids combined account for the vast
majority of sequences of all HIV-1 M group sequences known, usually
98-100%. HIV-1 M group subtypes B, primarily found in the Americas
and Western Europe, and subtype C primarily found in South Africa
and India, represent most of the available sequence data, and
together they represent >60% of all HIV-1 infections. Toggle
sites often represent AA that are highly conserved in either
subtype B or C, and the CE1 and CE2 sequences correspond to the AA
most associated with subtypes B and C, respectively, if the
consensus or second most common variant residues differed. Another
means of coordinating toggle sites is to associate covarying amino
acids (11, 12).
[0281] Other features that resulted in a relaxation of the 90%
requirement was an association with known function or CTL escape
resulting in a loss in viral fitness, or to substantially extend
the length of a CE. For example, the CD4 binding loop region of the
HIV Env protein corresponds to a region that binds to broadly
neutralizing antibodies and this region is included as CE10 and
CE19. To include this region, seven toggle sites in were employed
and one residue was included in CE10 without a toggle that had a
conservation level of only 79%. In another example, a very long CE
(CE14; 43 AA) was included by allowing five toggle sites and one
residue was conserved only at a level of 84% across the HIV-1 M
group. However, this site was conserved at 97% and 99% in HIV-1 M
group subtypes B and C and no obvious toggle site was evident.
Alternate CE were generated in some cases by relaxing the criteria
further to extend a first generation CE in an attempt to increase
its immunogenicity.
[0282] Next, we tested the ability of structure-based computational
models to assign sites of mutations that would destabilize the
protein structure as a means of identifying vulnerable targets for
inclusion in CE vaccines. This approach was first applied to the
HIV capsid protein for which more comprehensive three-dimensional
structural data is available. Predicted destabilizing mutations
were rarely found in a database of 5811 HIV-1 capsid protein coding
sequences, with none being present at a frequency greater than 2%.
However, 90% of variants with high instability scores from a set of
184 capsid variants whose replication fitness or infectivity has
been studied in vitro had aberrant capsid structures and reduced
viral infectivity. Based on these instability scores, we identified
45 sites in the capsid protein prone to destabilizing mutations.
Applying this method to capsid protein sequences, 9 additional
sites were included in a second generation of CE. More than half of
these sites are targets of one or more known inhibitors of capsid
function (Table 3). The capsid regions enriched with these sites
also overlap with peptides shown to induce cellular immune
responses associated with lower viral loads in infected
individuals.
[0283] A joint scoring metric was also developed that takes into
account both sequence conservation and protein structure stability.
This performed better at identifying deleterious mutations than
sequence conservation or structure stability information alone
(Table 2). The computational sequence-structure stability approach
thus provides an additional method of identifying conserved
elements suitable for use in an immunogenic composition employing
conserved elements as described herein for HIV polypeptides.
Twenty-one sites in Env were also identified as prone to
destabilizing mutations, 11 of which are included in HIV CE.
[0284] A joint scoring metric was also developed that takes into
account both sequence conservation and protein structure stability.
This performed better at identifying deleterious mutations than
sequence conservation or structure stability information alone
(Table 2). The computational sequence-structure stability approach
provides an additional method of identifying conserved elements
suitable for use in an immunogenic composition employing conserved
elements as described herein for HIV polypeptides.
Detailed Methods for Structural Stability Prediction.
[0285] Starting 3-dimensional structures. The capsid hexamer, PDBID
3H4E (13), and the dimer form of the carboxy-terminal domain of the
capsid, PDB ID 1A43 (14), were used as template structures for
mutations in the amino-terminal domain (NTD; residue 1 to 147) and
the carboxy-terminal domain (CID residue 148 to 219, including the
linker region), respectively. While the known capsid hexamer
structure also includes CTD, it does not contain the CTD
dimerization interface, shown to be crucial for mature capsid
structure and function (15-17). Hence, the CTD dimer (14) was also
used. The last 13 residues were missing from the C-terminus of the
template structures and, hence, excluded from analysis.
[0286] A high-resolution crystal structure of a trimeric pre-fusion
Env with the antibodies removed www website
rcsb.org/pdb/explore/explore.do?structureId=4TVP was used as
template. As no high-resolution structure of the HIV-I Env protein
with all of the variable loop sequences identified has yet been
elucidated, these predictions may not cover all mutations that
affect protein function.
[0287] In silico mutagenesis. All 19 possible amino acid changes
were introduced in silico at each position in the HIV-1 capsid and
Env proteins, one at a time. Reference structures in which the
starting amino acid was re-introduced into the structure were also
generated and used as a control set.
[0288] Two in silico mutation modeling approaches were applied:
Fixed-backbone models explore the best-fit side-chain conformation
while the main chain atoms of the mutated residue are kept
unchanged from the original position in the template structure. The
side-chain atoms of the mutated residue were replaced with those of
the new amino acid. The best-fit side chain conformation was
selected using the SCWRL program version 4.0 (18). The model was
then run through 200 steps of energy minimization to remove atomic
clashes or internal constraints generated by side-chain replacement
using the CHARMM force field, as implemented in the program NAMD
version 2.8 (19). Flexible-backbone models allow the main chain
atoms to move along with the side chains and the best-fit
combination is selected. These were generated in the FOLDX program
suite (20) using the BUILDMODEL function with default parameters.
This method allows the neighboring side-chains to be moved in order
to explore alternative backbone conformations.
[0289] Proteins stability scores. Two types of protein-scoring
functions were used to assess mutant models: The atomic
distance-dependent statistical based scoring function referred to
as Discrete Optimized Protein Energy (DOPE) (21) and the empirical
based force field energy function called FOLD-X energy function
(FOLDEF) (22). DOPE is part of the protein-modeling package
MODELLER (http://salilab.org/modeller/) (23). FOLDEF is part of the
FOLDX program suite (http://foldx.crg.es/) (20). Both programs were
run using default parameters.
[0290] Sequence dataset and amino acid database frequencies.
Full-length HIV-1 subtype B Gag and Env coding sequences were
downloaded from the HIV database (HIVDB, http://www.hiv/lanl.gov/).
Any sequences with hypermutations (24) early stop codons,
frame-shift mutations or ambiguous amino acids were excluded. A
multiple sequence alignment was prepared using MUSCLE (25) and then
manually edited using Mesquite (26). The database frequency of each
amino acid at each site in the final alignment was then calculated
using a perl script
(http://indra.mullins.microbiol.washington.edu/perlscript/docs/CountAAFre-
q.html).
[0291] To identify candidate regions for CTL vaccine immunogens
design, we searched the Env and capsid sequences in sliding windows
of 15 amino acids, one amino acid at a time, and counted the number
of sites prone to destabilizing mutations within each window. The
window of size 15 was selected to cover the length of known CD4 and
CD8 epitopes (27). If less than 70% of the possible AA
substitutions were predicted to result in a stable structure, then
the site was considered to be structurally vulnerable. As possible,
given our other criteria, structurally vulnerable sites were
included in CE.
[0292] Composite sequence-structure stability score. A composite
score was derived from the mutation database frequency and FOLDEF
score of the mutant flexible backbone model. First, all mutations
were ranked by database frequency in ascending order. Mutations
with the lowest database frequency, i.e., 0%, were given the lowest
frequency rank. Next, all mutations were ranked by FOLDEF score in
descending order, i.e., mutant models with the highest stability
score were given the lowest stability rank. For each mutation, the
two ranks were added to get a composite score.
[0293] The combined sequence-structure approach described here has
the potential to serve as a target-screening tool for HIV drug and
vaccine development. One limitation of this work is that only
single amino acid changes were studied, whereas compensatory
mutations can arise during viral infection that can restore protein
stability and function (Chang, 2011; Gong, 2013; Liu, 2014). Also,
all sequence, structure and experimental data used in our analyses
were obtained using HIV-1 subtype B viruses. Studies of the effect
of more complicated mutational patterns on protein stability in
multiple genetic background will provide further insight for
identifying desirable targets for HIV vaccines and therapies.
Lastly, amino acid conservation can also be evaluated by weighting
specific amino acid substitutions by the similarity in
physico-chemical properties to the initial amino acid.
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TABLE-US-00006 TABLE 1 Illustrative HIV Env Conserved Element
Sequences The amino acid residues that differ between the two
alternative conserved element sequences in each set are underlined.
EnvCE6 WVTVYYGVPVW (SEQ ID NO: 1) EnvCE1-1 HNVWATHACVPTDP (SEQ ID
NO: 2) EnvCE1-2 HNIWATHACVPTDP (SEQ ID NO: 3) EnvCE7-1
ISLWDQSLKPCVKLTPLCVTL (SEQ ID NO: 4) EnvCE7-2 ISLWDESLKPCVKLTPLCVTL
(SEQ ID NO: 5) EnvCE8-1 FEPIPIHYCTPAGFA (SEQ ID NO: 6) EnvCE8-2
FDPIPIHYCAPAGYA (SEQ ID NO: 7) EnvCE9-1 VQCTHGIRPVVSTQLLLNGSLAE
(SEQ ID NO: 8) EnvCE9-2 VQCTHGIKPVVSTQLLLNGSLAE (SEQ ID NO: 9)
EnvCE10-1 SGGDPEIVMHSFNCGGEFFYC (SEQ ID NO: 10) EnvCE10-2
AGGDLEITTHSFNCRGEFFYC (SEQ ID NO: 11) EnvCE11-1 DNWRSELYKYKVV (SEQ
ID NO: 12) EnvCE11-2 NNWRSELYKYKVV (SEQ ID NO: 13) EnvCE12-1
ARRRVVQREKRA (SEQ ID NO: 14) EnvCE12-2 AKRRVVEREKRA (SEQ ID NO: 15)
EnvCE13-1 GFLGTAGSTMGAAS (SEQ ID NO: 16) EnvCE13-2 GFLGAAGSTMGAAS
(SEQ ID NO: 17) EnvCE14-1
LTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQAR (SEQ ID NO: 18)
EnvCE14-2 LTVQARQLLSGIVQQQSNLLKAIEAQQHMLQLTVWGIKQLQTR (SEQ ID NO:
19) EnvCE15-1 WLWYIKIFIMIVGGLVGLRI (SEQ ID NO: 20) EnvCE15-2
WLWYIRIFIMIVGGLIGLRI (SEQ ID NO: 21) EnvCE16-1 RVRKGYSPLSLQT (SEQ
ID NO: 22) EnvCE16-2 RVRQGYSPLSFQT (SEQ ID NO: 23) Alternative
EnvCE: EnvCE17-1 PIPIHYCAPAGFAILKC (SEQ ID NO: 70) EnvCE17-2
PIPIHYCTPAGYAILKC (SEQ ID NO: 71) EnvCE18-1
NVSTVQCTHGIRPVVSTQLLLNGSLAE (SEQ ID NO: 72) envCE18-2
NVSSVQCTHGIKPVVSTQLLLNGSLAE (SEQ ID NO: 73) EnvCE19-1
GGDPEIVMHTFNCGGEFFYC (SEQ ID NO: 74) envCE19-2 GGDLEITTHSFNCRGEFFYC
(SEQ ID NO: 75) EnvCE20-1 CRIKQIINMWQ (SEQ ID NO: 76) EnvCE20-2
CKIRQIVNRWQ (SEQ ID NO: 77) EnvCE21-1 GGDMRDNWRSELYKYKVV (SEQ ID
NO: 78) EnvCE21-2 GGNMKDNWRSELYKYKVV (SEQ ID NO: 79) envCE22-1
LTVQARLLLSGIVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARVLA (SEQ ID NO: 80)
envCE22-2 LTVQARQLLSGIVQQQSNLLKAIEAQQHMLQLTVWGIKQLQTRVLA (SEQ ID
NO: 81) EnvCE23-1 DQQLLGIWGCSGKLIC (SEQ ID NO: 82) EnvCE23-2
DQQLLGLWGCSGKLIC (SEQ ID NO: 83) EnvCE24-1 WLWYIKIFIMIVGGLVGLRI
(SEQ ID NO: 84) EnvCE24-2 WLWYIRIFIMIVGGLIGLRI (SEQ ID NO: 85) SEQ
ID NO: 24. Env conserved element 1 immunogenic polypeptide sequence
comprising 12 conserved element: CE6, CE1-1, CE7-1, CE8-1, CE9-1,
CE10-1, CE11-1, CE12-1, CE13-1, CE14-1, CE15-1, and CE16-1. The
linker amino acids between the individual conserved elements are
underlined.
WVTVYYGVPVWAAVHNVWATHACVPTDPAAEISLWDQSLKPCVKLTPLCVTLGAKFEPIPIH
YCTPAGFAGAKVQCTHGIRPVVSTQLLLNGSLAEAADSGGDPEIVMHSFMCGGEFFYCGAKD
NWRSELYKYKVVAAKARRRVVQREKRAGAVGFLGTAGSTMGAASVAVLTVQARLLLSGIVQQ
QNNLLRAIEAQQHLLQLTVWGIKQLQARAADWLWYIKIFIMIVGGLVGLRIAAFRVRKGYSP
LSLQT SEQ ID NO: 25. Env conserved element 2 immunogenic
polypeptide sequence comprising 12 conserved element: CE6, CE1-2,
CE7-2, CE8-2, CE9-2, CE10-2, CE11-2, CE12-2, CE13-2, CE14-2,
CE15-2, and CE16-2. The linker amino acids between the individual
conserved elements are underlined.
WVTVYYGVPVWAAVHNIWATHACVPTDFAAEISLWDESLKPCVKLTPLCVTLGAKFDPIPIH
YCAPAGYAGAKVQCTHGIKPVVSTQLLLNGSLAEAADAGGDLEITTHSFNCRGEFFYCGAKN
NWRSELYKYKVVAAKAKRRVVEREKRAGAVGFLGAAGSTMGAASVAVLTVQARQLLSGIVQQ
QSNLLKAIEAQQHMLQLTVWGIKQLQTRAADWLWYIRIFIMIVGGLIGLRIAAFRVRQGYSP
LSFQT SEQ ID NO: 69 FIG. 25 Env-CE1_V1V2-containing CE construct
amino acid sequence
MRVTEIRKSYQHWWRWGIMLLGILMICNAEEKLWVTVYYGVPVWKEATTTLFCAS
DRKAYDTEVHNVWATHACVPTDPNPQEVELKNVTENFNMWKNNMVEQMHEDIISL
WDQSLKPCVKLTPLCVTLNCTDLRNATNGNDTNTTSSSRGMVGGGEMKNCSFNIT
TNIRGKVQKEYALFYKLDIAPIDNNSNNRYRLISCGAKFEPIPIHYCTPAGFAGA
KVQCTHGIRPVVSTQLLLNGSLAEAADSGGDPEIVMHSFNCGGEFFYCGAKDNWR
SELYKYKVVAAKARRRVVQREKRAGAVGFLGTAGSTMGAASVAVLTVQARLLLSG
IVQQQNNLLRAIEAQQHLLQLTVWGIKQLQARAADWLWYIKIFIMIVGGLVGLRI
AAFRVRKGYSPLSLQT Alternative EnvCE arrangements (Env CE order):
EnvCE6, EnvCE1-1, EnvCE7-1, EnvCE17-1, EnvCE18-1, EnvCE19-1,
EnvCE20-1, EnvCE21-1, EnvCE12-1, EnvCE13-1, EnvCE22-1, EnvCE23-1,
EnvCE24-1, EnvCE16-1 and EnvCE6, EnvCE1-2, EnvCE7-2, EnvCE17-2,
EnvCE18-2, EnvCE19-2, EnvCE20-2, EnvCE21-2, EnvCE12-2, EnvCE13-2,
EnvCE22-2, EnvCE23-2, EnvCE24-2, EnvCE16-2
TABLE-US-00007 TABLE 2 Illustrative HIV Gag conserved element
sequences: p24 Gag conserved elements for p24CE1 vaccine ("also
referred to as "p24 CE1"): SEQ ID NO: 26 conserved element 1 (CE1)
ISPRTLNAWVKV SEQ ID NO: 27 conserved element 2 (CE2)
VIPMFSALSEGATPQDLN SEQ ID NO: 28 conserved element 3 (CE3)
VGGHQAAMQMLKDTINEEAAEWDR SEQ ID NO: 29 conserved element 4 (CE4)
PRGSDIAGTTSTLQEQIGW SEQ ID NO: 30 conserved element 5 (CE5)
KRWIILGLNKIVRMYSPTSI SEQ ID NO: 31 conserved element 6 (CE6)
YVDRFYKTLRAEQA SEQ ID NO: 32 conserved element 7 (CE7)
LEEMMTACQGVGGPGHK p24 Gag conserved elements for p24CE2 vaccine
("also referred to as p24 CE2"): SEQ ID NO: 33 conserved element 1
(CE1) LSPRTLNAWVKV SEQ ID NO: 34 conserved element 2 (CE2)
VIPMFTALSEGATPQDLN SEQ ID NO: 35 conserved element 3 (CE3)
VGGHQAAMQMLKETINEEAAEWDR SEQ ID NO: 36 conserved element 4 (CE4)
PRGSDIAGTTSTLQEQIAW SEQ ID NO: 37 conserved element 5 (CE5)
KRWIILGLNKIVRMYSPVSI SEQ ID NO: 38 conserved element 6 (CE6)
YVDRFFKTLRAEQA SEQ ID NO: 39 conserved element 7 (CE7)
LEEMMTACQGVGGPSHK SEQ ID NO: 40 amino acid sequence of p24 Gag CE1
polypeptide:
VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAG
TTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEE
MMTACQGVGGPGHKAAISPRTLNAWVKV SEQ ID NO: 41 amino acid sequence of
p24 Gag CE2 polypeptide:
VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAG
TTSTLQEQTAWAAAKRWITLGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEE
MMTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 42 polypeptide sequence of
p24CE1 including a GM-CSF signal peptide
MWLQSLLLLGTVACSISVIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEA
AEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYV
DRFYKTLRAEQAAGLEEMMTACQGVGGPGHKAAISPRTLNAWVKV SEQ ID NO: 43
polypeptide sequence of p24CE2 including a GM-CSF signal peptide
MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEA
AEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYV
DRFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 44
LAMP-p24CE1 (p24CE1 underlined)
MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDT
KSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV
QLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTL
HDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNG
TCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVL
LFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEH
VRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFSALSEGATPQ
DLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAA
KRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEMMTACQGVGGPGHK
AAISPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI SEQ ID NO:
45 LAMP-p24CE2 fusion (p24CE2 underlined)
MAPRSARRPLLLLLLLLLLGLMHCASAAMFMVKNGNGTACIMANFSAAFSVNYDT
KSGPKNMTLDLPSDATVVLNRSSCGKENTSDPSLVIAFGRGHTLTLNFTRNATRYSV
QLMSFVYNLSDTHLFPNASSKEIKTVESITDIRADIDKKYRCVSGTQVHMNNVTVTL
HDATIQAYLSNSSFSRGETRCEQDRPSPTTAPPAPPSPSPSPVPKSPSVDKYNVSGTNG
TCLLASMGLQLNLTYERKDNTTVTRLLNINPNKTSASGSCGAHLVTLELHSEGTTVL
LFQFGMNASSSRFFLQGIQLNTILPDARDPAFKAANGSLRALQATVGNSYKCNAEEH
VRVTKAFSVNIFKVWVQAFKVEGGQFGSVEECLLDENSLEDIVIPMFTALSEGATPQ
DLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLGQEQIAWAAA
KRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEMMTACQGVGGPSHK
AALSPRTLNAWVKVGSEFTLIPIAVGGALAGLVLIVLIAYLVGRKRSHAGYQTI SEQ ID NO:
46 SP-p24CE2 (p24CE1 underlined)
MWLQSLLLLGTVACSISVIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEA
AEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVD
RFFKTLRAEQAAGLEEMMTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 47
MCP3-p24CE1 (p24CE1 underlined)
MWKPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQR
LESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
VIPMFSALSEGATPQDLNAAVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAG
TTSTLQEQIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQAAGLEEM
MTACQGVGGPGHKAAISPRTLNAWVKV SEQ ID NO: 48 MCP3-p24CE2 (p24CE2 is
underlined)
MWKPMPSPSNMKASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQR
LESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL
VIPMFTALSEGATPQDLNAAVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAG
TTSTLQEQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQAAGLEEM
MTACQGVGGPSHKAALSPRTLNAWVKV SEQ ID NO: 49 SP-p24CE1c (p24CE1c
underlined) alternative CE polypeptide. Includes GM-CSF signal
peptide, CE1 and CE2 replaced by CE8 and CE9, respectively
(relative to p24 CE "Core1"); lacks CE7; arranged in the
configuration of conserved elements: CE 8-9-3-4-5-6
MWLQSLLLLGTVACSISQGQMVHQAISPRTLNAWVKVLAKEEKAFSPEVIPMFSALS
EGATPQDLNAAKVGGHQAAMQMLKETINEEAAEWDRAAAEPRGSDIAGTTSTLQE
QIGWAAAKRWIILGLNKIVRMYSPTSIAAKYVDRFYKTLRAEQADYKDDDDKL SEQ ID NO: 50
Gag conserved element 8 (CE8) QGQMVHQAISPRTLNAWVKV SEQ ID NO: 51
Gag conserved element 9 (CE9) EEKAFSPEVIPMFSALSEGATPQDLN SEQ ID NO:
52 SP-p24CE2c- alternative CE polypeptpide p24CE2c underlined
MWLQSLLLLGTVACSISQGQMVHQALSPRTLNAWVKVLAKEEKGFNPEVIPMFTAL
SEGATPQDLNAAKVGGHQAAMQMLKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQ
EQIAWAAAKRWIILGLNKIVRMYSPVSIAAKYVDRFFKTLRAEQA SEQ ID NO: 53
SP-p24CE2d alternative CE polypeptide construct; in order
CE9-3-4-5-6-8
MWLQSLLLLGTVACSISEEKGFNPEVIPMFTALSEGATPQDLNAAKVGGHQAAMQM
LKDTINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIAWAAAKRWIILGLNKIVRMYSP
VSIAAKYVDRFFKTLRAEQAALQGQMVHQALSPRTLNAWVKV SEQ ID NO: 54 SP-24CE1d-
alternative CE polypeptide construction; in order CE9-3-4-5-6-8
MWLQSLLLLGTVACSISEEKAFSPEVIPMFSALSEGATPQDLNAAKVGGHQAAMQM
LKETINEEAAEWDRAAAEPRGSDIAGTTSTLQEQIGWAAAKRWIILGLNKIVRMYSP
TSIAAKYVDRFYKTLRAEQAALQGQMVHQAISPRTLNAWVKV p24CE1d has 6 CE (is
identical to p24CE1c except for the CE arrangement within the
protein) GM-CSF signal peptide CE1 and C2 replaced by CE8 and CE9
respectively, lacks CE7 and has the CE arranged in the
configuration CE9-3-4-5-6-8 SEQ ID NO: 55 Gag conserved element 8
(CE8- variant for CE2 constructs) QGQMVHQALSPRTLNAWVKV SEQ ID NO:
56 Gag conserved element 9 (CE9) EEKGENPEVIPMFTALSEGATPQDLN
Differences between CE for p24 CE polypeptides and variant p24CE
polypeptides is one amino acid per CE except CE9, which differs by
3 amino acids Alternative Gag p24 conserved elements. p24CE8-1
QPISPRTLNAWVKV (SEQ ID NO: 57) p24CE8-2 QALSPRTLNAWVKV (SEQ ID NO:
58) p24CE9-1 EEKAFSPEVIPMFSALSEGATPQDLNTMLN (SEQ ID NO: 59)
p24CE9-2 EEKGFNPEVIPMFTALSEGATPQDLNMMLN (SEQ ID NO: 60) p24CE10-1
PRGSDIAGTTSTLQEQIGWMT (SEQ ID NO: 61) p24CE10-2
PRGSDIAGTTSTLQEQIAWMT (SEQ ID NO: 62) p24CE11-1
SILDIRQGPKEPFRDYVDRF (SEQ ID NO: 63) p24CE11-2 SILDIKQGPKEPFRDYVDRF
(SEQ ID NO: 64) p24CE12-1 QNSNPDCKTILKALG (SEQ ID NO: 65) p24CE12-2
QNANPDCKTILKALG (SEQ ID NO: 66) p24CE13-1 LEEMMTACQGVGGPGHKARILAEAM
(SEQ ID NO: 67) p24CE13-2 LEEMMTACQGVGGPSHKARVLAEAM (SEQ ID NO: 68)
Alternative p24CE arrangements: P24CE8-1, p24CE9-1, p24CE3-1,
p24CE10-1, p24CE5-1, p24CE11-1, p24CE6-1, p24CE12-1, p24CE13-1
P24CE8-2, p24CE9-2, p24CE3-2, p24CE10-2, p24CE5-2, p24CE11-2,
p24CE6-2, p24CE12-2, p24CE13-2
TABLE-US-00008 TABLE 3 Accuracy of using mutation frequency or
change in structural stability to predict viral infectivity in
binary classification manner Predictor Sensitivity.sup.d
Specificity.sup.e Precision.sup.f Accuracy.sup.g Mutation
frequency.sup.a 59.5% 70.0% 67.10% 64.10% Stability of 72.34%
77.78% 77.30% 75.00% reference models.sup.b Composite Score.sup.c
80.00% 79.57% 78.16% 79.78% .sup.aMutations with a database
frequency of 0.2% or less are predicted to result in non-infectious
virus .sup.bMutants with structural stability higher than the
reference models are predicted to result in non-infectious virus
.sup.cMutants with a composite score (sum of ranks of frequency and
stability scores) higher than 175 are predicted to result in
non-infectious virus .sup.dSensitivity = (True positive)/(True
positive + False negative) .sup.eSpecificity = (True
negative)/(True negative + False positive) .sup.fPrecision = (True
positive)/(True positive + False positive) .sup.gAccuracy = (True
positive + True negative)/(True positive + True negative + False
positive + False negative)
TABLE-US-00009 TABLE 4 Amino acid sites within HIV-1 CA prone to
destabilizing mutations Solvent Site Consensus Structural element
and location in accessibility Binding site of CA (HXB2) AA
Frequency capsid hexamer and/or CTD dimer surface area (A.sup.2)
inhibitors 2 ILE 0.98 .beta.-hairpin 10.22 None 8 GLY 0.99
.beta.-hairpin 40.98 None 20 LEU 0.99 Helix 1; capsid
hexamerization 2.1 None (NTD-NTD) interface 23 TRP 0.99 Helix1 2.81
CAP-1, BD3, BM4 Inhibitor3 32 PHE 0.99 .beta. turn in loop region
between 5.97 CAP-1; BD3, helix 1 and 2 BM4; Inhibitor3
Benzodiazepine series 33 36 VAL 0.97 Helix 2 0.65 I-XW-053; BD3
Inhibitor3 37 ILE 0.97 Helix 2 17.74 Inhibitor3 40 PHE 0.99 Helix 2
0 Inhibitor3 43 LEU 0.98 Helix 2; capsid hexamerization 13.74 None
(NTD-NTD) interface 49 PRO 0.99 Helix 3 0 None 52 LEU 0.99 Helix 3
0.02 None 55 MET 0.99 Helix 3 0.69 None 56 LEU 0.99 Helix 3 12.75
Inhibitor3 {ref} 65 ALA 0.99 Helix 4 0 CAP-1; Inhibitor3;
Benzodiazepine series 33 66 MET 0.99 Helix 4 13.09 Inhibitor3; PF-
3450074 69 LEU 0.99 Helix 4 2.91 Inhibitor3 73 ILE 0.99 Helix 4
13.8 PF-3450074; 80 TRP 0.99 Helix 4 40.65 Inhibitor 4 99 PRO 0.99
Loop between helix 4 and 5 8.6 None 101 GLY 0.99 Helix 5 0.1 None
104 ILE 0.99 Helix 5 0 None 106 GLY 0.99 .beta. turn in loop region
between 18.65 None helix 5 and 6 109 SER 0.99 .beta. turn in loop
region between 13.19 None helix 5 and 6 111 LEU 0.99 Helix 6 53.08
None 117 TRP 0.99 Helix 6 0.24 Inhibitor4; 126 VAL 0.99 Helix 7 0
None 133 TRP 0.99 Helix 7 10.88 None 134 ILE 0.99 Helix 7 0
Inhibitor3 138 LEU 0.98 Helix 7 0.66 Inhibitor3 141 ILE 0.99 Helix
7 0.22 CAP-1; Inhibitor3 142 VAL 0.99 Helix 7 0.84 Inhibitor3 144
MET 0.99 Helix 7 20.6 None 150 ILE 0.99 Linker region between NTD
23.25 CAC1; CP4 peptide and CTD; CTD dimerization interface 153 ILE
0.99 Linker region between NTD 11.75 None and CTD; CTD dimerization
interface 161 PHE 0.99 Helix 8; MHR 6.88 None 165 VAL 0.99 Helix 8;
MHR; capsid 0 I-XW-053; CAI- hexamerization (NTD-CTD) compound
series; interface H8; NYAD peptides 168 PHE 0.99 Helix 8; MHR 0.12
I-XW-053; NYAD peptides 169 TYR 0.99 Helix 8; MHR; capsid 30.73
I-XW-053; CAI hexamerization (NTD-CTD) peptide; CAI- interface
compound series; NYAD peptides 172 LEU 0.99 Helix 8; MHR; CTD 5.4
I-XW-053; CAC1, dimerization interface H8; NYAD peptides 189 LEU
0.99 Helix 9; CTD dimerization 24.94 CAC1 interface 198 CYS 0.99
Helix 10 12.6 CAC1 202 LEU 0.99 Helix 10 7.67 CAC1 205 LEU 0.98
Helix 10 77.49 None 206 GLY 0.99 .beta. turn in loop region between
28.12 None helix 10 and 11 211 LEU 0.99 Helix 11; capsid
hexamerization 4.46 CAI-compound (NTD-CTD) interface series; CAC1;
NYAD peptides; CP4 peptide
Sequence CWU 1
1
89111PRTArtificial Sequencesynthetic peptide construct 1Trp Val Thr
Val Tyr Tyr Gly Val Pro Val Trp 1 5 10 214PRTArtificial
Sequencesynthetic peptide construct 2His Asn Val Trp Ala Thr His
Ala Cys Val Pro Thr Asp Pro 1 5 10 314PRTArtificial
Sequencesynthetic peptide construct 3His Asn Ile Trp Ala Thr His
Ala Cys Val Pro Thr Asp Pro 1 5 10 421PRTArtificial
Sequencesynthetic peptide construct 4Ile Ser Leu Trp Asp Gln Ser
Leu Lys Pro Cys Val Lys Leu Thr Pro 1 5 10 15 Leu Cys Val Thr Leu
20 521PRTArtificial Sequencesynthetic peptide construct 5Ile Ser
Leu Trp Asp Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro 1 5 10 15
Leu Cys Val Thr Leu 20 615PRTArtificial Sequencesynthetic peptide
construct 6Phe Glu Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Phe
Ala 1 5 10 15 715PRTArtificial Sequencesynthetic peptide construct
7Phe Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr Ala 1 5 10
15 823PRTArtificial Sequencesynthetic peptide construct 8Val Gln
Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu 1 5 10 15
Leu Asn Gly Ser Leu Ala Glu 20 923PRTArtificial Sequencesynthetic
peptide construct 9Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser
Thr Gln Leu Leu 1 5 10 15 Leu Asn Gly Ser Leu Ala Glu 20
1021PRTArtificial Sequencesynthetic peptide construct 10Ser Gly Gly
Asp Pro Glu Ile Val Met His Ser Phe Asn Cys Gly Gly 1 5 10 15 Glu
Phe Phe Tyr Cys 20 1121PRTArtificial Sequencesynthetic peptide
construct 11Ala Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys
Arg Gly 1 5 10 15 Glu Phe Phe Tyr Cys 20 1213PRTArtificial
Sequencesynthetic peptide construct 12Asp Asn Trp Arg Ser Glu Leu
Tyr Lys Tyr Lys Val Val 1 5 10 1313PRTArtificial Sequencesynthetic
peptide construct 13Asn Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val
Val 1 5 10 1412PRTArtificial Sequencesynthetic peptide construct
14Ala Arg Arg Arg Val Val Gln Arg Glu Lys Arg Ala 1 5 10
1512PRTArtificial Sequencesynthetic peptide construct 15Ala Lys Arg
Arg Val Val Glu Arg Glu Lys Arg Ala 1 5 10 1614PRTArtificial
Sequencesynthetic peptide construct 16Gly Phe Leu Gly Thr Ala Gly
Ser Thr Met Gly Ala Ala Ser 1 5 10 1714PRTArtificial
Sequencesynthetic peptide construct 17Gly Phe Leu Gly Ala Ala Gly
Ser Thr Met Gly Ala Ala Ser 1 5 10 1843PRTArtificial
Sequencesynthetic peptide construct 18Leu Thr Val Gln Ala Arg Leu
Leu Leu Ser Gly Ile Val Gln Gln Gln 1 5 10 15 Asn Asn Leu Leu Arg
Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu 20 25 30 Thr Val Trp
Gly Ile Lys Gln Leu Gln Ala Arg 35 40 1943PRTArtificial
Sequencesynthetic peptide construct 19Leu Thr Val Gln Ala Arg Gln
Leu Leu Ser Gly Ile Val Gln Gln Gln 1 5 10 15 Ser Asn Leu Leu Lys
Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu 20 25 30 Thr Val Trp
Gly Ile Lys Gln Leu Gln Thr Arg 35 40 2020PRTArtificial
Sequencesynthetic peptide construct 20Trp Leu Trp Tyr Ile Lys Ile
Phe Ile Met Ile Val Gly Gly Leu Val 1 5 10 15 Gly Leu Arg Ile 20
2120PRTArtificial Sequencesynthetic peptide construct 21Trp Leu Trp
Tyr Ile Arg Ile Phe Ile Met Ile Val Gly Gly Leu Ile 1 5 10 15 Gly
Leu Arg Ile 20 2213PRTArtificial Sequencesynthetic peptide
construct 22Arg Val Arg Lys Gly Tyr Ser Pro Leu Ser Leu Gln Thr 1 5
10 2313PRTArtificial Sequencesynthetic peptide construct 23Arg Val
Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr 1 5 10
24253PRTArtificial Sequencesynthetic peptide construct 24Trp Val
Thr Val Tyr Tyr Gly Val Pro Val Trp Ala Ala Val His Asn 1 5 10 15
Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Ala Ala Glu Ile 20
25 30 Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro
Leu 35 40 45 Cys Val Thr Leu Gly Ala Lys Phe Glu Pro Ile Pro Ile
His Tyr Cys 50 55 60 Thr Pro Ala Gly Phe Ala Gly Ala Lys Val Gln
Cys Thr His Gly Ile 65 70 75 80 Arg Pro Val Val Ser Thr Gln Leu Leu
Leu Asn Gly Ser Leu Ala Glu 85 90 95 Ala Ala Asp Ser Gly Gly Asp
Pro Glu Ile Val Met His Ser Phe Asn 100 105 110 Cys Gly Gly Glu Phe
Phe Tyr Cys Gly Ala Lys Asp Asn Trp Arg Ser 115 120 125 Glu Leu Tyr
Lys Tyr Lys Val Val Ala Ala Lys Ala Arg Arg Arg Val 130 135 140 Val
Gln Arg Glu Lys Arg Ala Gly Ala Val Gly Phe Leu Gly Thr Ala 145 150
155 160 Gly Ser Thr Met Gly Ala Ala Ser Val Ala Val Leu Thr Val Gln
Ala 165 170 175 Arg Leu Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn
Leu Leu Arg 180 185 190 Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu
Thr Val Trp Gly Ile 195 200 205 Lys Gln Leu Gln Ala Arg Ala Ala Asp
Trp Leu Trp Tyr Ile Lys Ile 210 215 220 Phe Ile Met Ile Val Gly Gly
Leu Val Gly Leu Arg Ile Ala Ala Phe 225 230 235 240 Arg Val Arg Lys
Gly Tyr Ser Pro Leu Ser Leu Gln Thr 245 250 25253PRTArtificial
Sequencesynthetic peptide construct 25Trp Val Thr Val Tyr Tyr Gly
Val Pro Val Trp Ala Ala Val His Asn 1 5 10 15 Ile Trp Ala Thr His
Ala Cys Val Pro Thr Asp Pro Ala Ala Glu Ile 20 25 30 Ser Leu Trp
Asp Glu Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu 35 40 45 Cys
Val Thr Leu Gly Ala Lys Phe Asp Pro Ile Pro Ile His Tyr Cys 50 55
60 Ala Pro Ala Gly Tyr Ala Gly Ala Lys Val Gln Cys Thr His Gly Ile
65 70 75 80 Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu
Ala Glu 85 90 95 Ala Ala Asp Ala Gly Gly Asp Leu Glu Ile Thr Thr
His Ser Phe Asn 100 105 110 Cys Arg Gly Glu Phe Phe Tyr Cys Gly Ala
Lys Asn Asn Trp Arg Ser 115 120 125 Glu Leu Tyr Lys Tyr Lys Val Val
Ala Ala Lys Ala Lys Arg Arg Val 130 135 140 Val Glu Arg Glu Lys Arg
Ala Gly Ala Val Gly Phe Leu Gly Ala Ala 145 150 155 160 Gly Ser Thr
Met Gly Ala Ala Ser Val Ala Val Leu Thr Val Gln Ala 165 170 175 Arg
Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Lys 180 185
190 Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile
195 200 205 Lys Gln Leu Gln Thr Arg Ala Ala Asp Trp Leu Trp Tyr Ile
Arg Ile 210 215 220 Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg
Ile Ala Ala Phe 225 230 235 240 Arg Val Arg Gln Gly Tyr Ser Pro Leu
Ser Phe Gln Thr 245 250 2612PRTArtificial Sequencesynthetic peptide
construct 26Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val 1 5 10
2718PRTArtificial Sequencesynthetic peptide construct 27Val Ile Pro
Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 1 5 10 15 Leu
Asn 2824PRTArtificial Sequencesynthetic peptide construct 28Val Gly
Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr Ile Asn 1 5 10 15
Glu Glu Ala Ala Glu Trp Asp Arg 20 2919PRTArtificial
Sequencesynthetic peptide construct 29Pro Arg Gly Ser Asp Ile Ala
Gly Thr Thr Ser Thr Leu Gln Glu Gln 1 5 10 15 Ile Gly Trp
3020PRTArtificial Sequencesynthetic peptide construct 30Lys Arg Trp
Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser 1 5 10 15 Pro
Thr Ser Ile 20 3114PRTArtificial Sequencesynthetic peptide
construct 31Tyr Val Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala
1 5 10 3217PRTArtificial Sequencesynthetic peptide construct 32Leu
Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His 1 5 10
15 Lys 3312PRTArtificial Sequencesynthetic peptide construct 33Leu
Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val 1 5 10
3418PRTArtificial Sequencesynthetic peptide construct 34Val Ile Pro
Met Phe Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 1 5 10 15 Leu
Asn 3524PRTArtificial Sequencesynthetic peptide construct 35Val Gly
Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn 1 5 10 15
Glu Glu Ala Ala Glu Trp Asp Arg 20 3619PRTArtificial
Sequencesynthetic peptide construct 36Pro Arg Gly Ser Asp Ile Ala
Gly Thr Thr Ser Thr Leu Gln Glu Gln 1 5 10 15 Ile Ala Trp
3720PRTArtificial Sequencesynthetic peptide construct 37Lys Arg Trp
Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr Ser 1 5 10 15 Pro
Val Ser Ile 20 3814PRTArtificial Sequencesynthetic peptide
construct 38Tyr Val Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln Ala
1 5 10 3917PRTArtificial Sequencesynthetic peptide construct 39Leu
Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Ser His 1 5 10
15 Lys 40140PRTArtificial Sequencesynthetic peptide construct 40Val
Ile Pro Met Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 1 5 10
15 Leu Asn Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys
20 25 30 Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala
Ala Glu 35 40 45 Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr
Leu Gln Glu Gln 50 55 60 Ile Gly Trp Ala Ala Ala Lys Arg Trp Ile
Ile Leu Gly Leu Asn Lys 65 70 75 80 Ile Val Arg Met Tyr Ser Pro Thr
Ser Ile Ala Ala Lys Tyr Val Asp 85 90 95 Arg Phe Tyr Lys Thr Leu
Arg Ala Glu Gln Ala Ala Gly Leu Glu Glu 100 105 110 Met Met Thr Ala
Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala Ala 115 120 125 Ile Ser
Pro Arg Thr Leu Asn Ala Trp Val Lys Val 130 135 140
41140PRTArtificial Sequencesynthetic peptide construct 41Val Ile
Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 1 5 10 15
Leu Asn Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys 20
25 30 Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala
Glu 35 40 45 Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu
Gln Glu Gln 50 55 60 Ile Ala Trp Ala Ala Ala Lys Arg Trp Ile Ile
Leu Gly Leu Asn Lys 65 70 75 80 Ile Val Arg Met Tyr Ser Pro Val Ser
Ile Ala Ala Lys Tyr Val Asp 85 90 95 Arg Phe Phe Lys Thr Leu Arg
Ala Glu Gln Ala Ala Gly Leu Glu Glu 100 105 110 Met Met Thr Ala Cys
Gln Gly Val Gly Gly Pro Ser His Lys Ala Ala 115 120 125 Leu Ser Pro
Arg Thr Leu Asn Ala Trp Val Lys Val 130 135 140 42157PRTArtificial
Sequencesynthetic peptide construct 42Met Trp Leu Gln Ser Leu Leu
Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Val Ile Pro Met
Phe Ser Ala Leu Ser Glu Gly Ala Thr Pro Gln 20 25 30 Asp Leu Asn
Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met Leu 35 40 45 Lys
Asp Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala 50 55
60 Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu
65 70 75 80 Gln Ile Gly Trp Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly
Leu Asn 85 90 95 Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Ala
Ala Lys Tyr Val 100 105 110 Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu
Gln Ala Ala Gly Leu Glu 115 120 125 Glu Met Met Thr Ala Cys Gln Gly
Val Gly Gly Pro Gly His Lys Ala 130 135 140 Ala Ile Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Val 145 150 155 43157PRTArtificial
Sequencesynthetic peptide construct 43Met Trp Leu Gln Ser Leu Leu
Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Val Ile Pro Met
Phe Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln 20 25 30 Asp Leu Asn
Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met Leu 35 40 45 Lys
Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala 50 55
60 Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu
65 70 75 80 Gln Ile Ala Trp Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly
Leu Asn 85 90 95 Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Ala
Ala Lys Tyr Val 100 105 110 Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu
Gln Ala Ala Gly Leu Glu 115 120 125 Glu Met Met Thr Ala Cys Gln Gly
Val Gly Gly Pro Ser His Lys Ala 130 135 140 Ala Leu Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Val 145 150 155 44564PRTArtificial
Sequencesynthetic peptide construct 44Met Ala Pro Arg Ser Ala Arg
Arg Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Gly Leu
Met His Cys Ala Ser Ala Ala Met Phe Met Val 20 25 30 Lys Asn Gly
Asn Gly Thr Ala Cys Ile Met Ala Asn Phe Ser Ala Ala 35 40 45 Phe
Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro Lys Asn Met Thr Leu 50 55
60 Asp Leu Pro Ser Asp Ala Thr Val Val Leu Asn Arg Ser Ser Cys Gly
65 70 75 80 Lys Glu Asn Thr Ser Asp Pro Ser Leu Val Ile Ala Phe Gly
Arg Gly 85 90 95 His Thr Leu Thr Leu Asn Phe Thr Arg Asn Ala Thr
Arg Tyr Ser Val 100 105 110 Gln Leu Met Ser Phe Val Tyr Asn Leu Ser
Asp Thr His Leu Phe Pro 115 120 125 Asn Ala Ser Ser Lys Glu Ile Lys
Thr Val Glu Ser Ile Thr Asp Ile 130 135 140 Arg Ala Asp Ile Asp Lys
Lys Tyr Arg Cys Val Ser Gly Thr Gln Val 145 150 155 160 His Met Asn
Asn Val Thr Val Thr Leu His Asp Ala Thr Ile Gln Ala 165 170 175 Tyr
Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu Thr Arg Cys Glu Gln 180 185
190 Asp Arg Pro Ser Pro Thr Thr Ala Pro Pro Ala Pro Pro Ser Pro Ser
195 200
205 Pro Ser Pro Val Pro Lys Ser Pro Ser Val Asp Lys Tyr Asn Val Ser
210 215 220 Gly Thr Asn Gly Thr Cys Leu Leu Ala Ser Met Gly Leu Gln
Leu Asn 225 230 235 240 Leu Thr Tyr Glu Arg Lys Asp Asn Thr Thr Val
Thr Arg Leu Leu Asn 245 250 255 Ile Asn Pro Asn Lys Thr Ser Ala Ser
Gly Ser Cys Gly Ala His Leu 260 265 270 Val Thr Leu Glu Leu His Ser
Glu Gly Thr Thr Val Leu Leu Phe Gln 275 280 285 Phe Gly Met Asn Ala
Ser Ser Ser Arg Phe Phe Leu Gln Gly Ile Gln 290 295 300 Leu Asn Thr
Ile Leu Pro Asp Ala Arg Asp Pro Ala Phe Lys Ala Ala 305 310 315 320
Asn Gly Ser Leu Arg Ala Leu Gln Ala Thr Val Gly Asn Ser Tyr Lys 325
330 335 Cys Asn Ala Glu Glu His Val Arg Val Thr Lys Ala Phe Ser Val
Asn 340 345 350 Ile Phe Lys Val Trp Val Gln Ala Phe Lys Val Glu Gly
Gly Gln Phe 355 360 365 Gly Ser Val Glu Glu Cys Leu Leu Asp Glu Asn
Ser Leu Glu Asp Ile 370 375 380 Val Ile Pro Met Phe Ser Ala Leu Ser
Glu Gly Ala Thr Pro Gln Asp 385 390 395 400 Leu Asn Ala Ala Val Gly
Gly His Gln Ala Ala Met Gln Met Leu Lys 405 410 415 Asp Thr Ile Asn
Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala Glu 420 425 430 Pro Arg
Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln 435 440 445
Ile Gly Trp Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys 450
455 460 Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Ala Ala Lys Tyr Val
Asp 465 470 475 480 Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln Ala Ala
Gly Leu Glu Glu 485 490 495 Met Met Thr Ala Cys Gln Gly Val Gly Gly
Pro Gly His Lys Ala Ala 500 505 510 Ile Ser Pro Arg Thr Leu Asn Ala
Trp Val Lys Val Gly Ser Glu Phe 515 520 525 Thr Leu Ile Pro Ile Ala
Val Gly Gly Ala Leu Ala Gly Leu Val Leu 530 535 540 Ile Val Leu Ile
Ala Tyr Leu Val Gly Arg Lys Arg Ser His Ala Gly 545 550 555 560 Tyr
Gln Thr Ile 45564PRTArtificial Sequencesynthetic peptide construct
45Met Ala Pro Arg Ser Ala Arg Arg Pro Leu Leu Leu Leu Leu Leu Leu 1
5 10 15 Leu Leu Leu Gly Leu Met His Cys Ala Ser Ala Ala Met Phe Met
Val 20 25 30 Lys Asn Gly Asn Gly Thr Ala Cys Ile Met Ala Asn Phe
Ser Ala Ala 35 40 45 Phe Ser Val Asn Tyr Asp Thr Lys Ser Gly Pro
Lys Asn Met Thr Leu 50 55 60 Asp Leu Pro Ser Asp Ala Thr Val Val
Leu Asn Arg Ser Ser Cys Gly 65 70 75 80 Lys Glu Asn Thr Ser Asp Pro
Ser Leu Val Ile Ala Phe Gly Arg Gly 85 90 95 His Thr Leu Thr Leu
Asn Phe Thr Arg Asn Ala Thr Arg Tyr Ser Val 100 105 110 Gln Leu Met
Ser Phe Val Tyr Asn Leu Ser Asp Thr His Leu Phe Pro 115 120 125 Asn
Ala Ser Ser Lys Glu Ile Lys Thr Val Glu Ser Ile Thr Asp Ile 130 135
140 Arg Ala Asp Ile Asp Lys Lys Tyr Arg Cys Val Ser Gly Thr Gln Val
145 150 155 160 His Met Asn Asn Val Thr Val Thr Leu His Asp Ala Thr
Ile Gln Ala 165 170 175 Tyr Leu Ser Asn Ser Ser Phe Ser Arg Gly Glu
Thr Arg Cys Glu Gln 180 185 190 Asp Arg Pro Ser Pro Thr Thr Ala Pro
Pro Ala Pro Pro Ser Pro Ser 195 200 205 Pro Ser Pro Val Pro Lys Ser
Pro Ser Val Asp Lys Tyr Asn Val Ser 210 215 220 Gly Thr Asn Gly Thr
Cys Leu Leu Ala Ser Met Gly Leu Gln Leu Asn 225 230 235 240 Leu Thr
Tyr Glu Arg Lys Asp Asn Thr Thr Val Thr Arg Leu Leu Asn 245 250 255
Ile Asn Pro Asn Lys Thr Ser Ala Ser Gly Ser Cys Gly Ala His Leu 260
265 270 Val Thr Leu Glu Leu His Ser Glu Gly Thr Thr Val Leu Leu Phe
Gln 275 280 285 Phe Gly Met Asn Ala Ser Ser Ser Arg Phe Phe Leu Gln
Gly Ile Gln 290 295 300 Leu Asn Thr Ile Leu Pro Asp Ala Arg Asp Pro
Ala Phe Lys Ala Ala 305 310 315 320 Asn Gly Ser Leu Arg Ala Leu Gln
Ala Thr Val Gly Asn Ser Tyr Lys 325 330 335 Cys Asn Ala Glu Glu His
Val Arg Val Thr Lys Ala Phe Ser Val Asn 340 345 350 Ile Phe Lys Val
Trp Val Gln Ala Phe Lys Val Glu Gly Gly Gln Phe 355 360 365 Gly Ser
Val Glu Glu Cys Leu Leu Asp Glu Asn Ser Leu Glu Asp Ile 370 375 380
Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 385
390 395 400 Leu Asn Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met
Leu Lys 405 410 415 Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg
Ala Ala Ala Glu 420 425 430 Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr
Ser Thr Leu Gln Glu Gln 435 440 445 Ile Ala Trp Ala Ala Ala Lys Arg
Trp Ile Ile Leu Gly Leu Asn Lys 450 455 460 Ile Val Arg Met Tyr Ser
Pro Val Ser Ile Ala Ala Lys Tyr Val Asp 465 470 475 480 Arg Phe Phe
Lys Thr Leu Arg Ala Glu Gln Ala Ala Gly Leu Glu Glu 485 490 495 Met
Met Thr Ala Cys Gln Gly Val Gly Gly Pro Ser His Lys Ala Ala 500 505
510 Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Gly Ser Glu Phe
515 520 525 Thr Leu Ile Pro Ile Ala Val Gly Gly Ala Leu Ala Gly Leu
Val Leu 530 535 540 Ile Val Leu Ile Ala Tyr Leu Val Gly Arg Lys Arg
Ser His Ala Gly 545 550 555 560 Tyr Gln Thr Ile 46157PRTArtificial
Sequencesynthetic peptide construct 46Met Trp Leu Gln Ser Leu Leu
Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Val Ile Pro Met
Phe Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln 20 25 30 Asp Leu Asn
Ala Ala Val Gly Gly His Gln Ala Ala Met Gln Met Leu 35 40 45 Lys
Glu Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala 50 55
60 Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu
65 70 75 80 Gln Ile Ala Trp Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly
Leu Asn 85 90 95 Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Ala
Ala Lys Tyr Val 100 105 110 Asp Arg Phe Phe Lys Thr Leu Arg Ala Glu
Gln Ala Ala Gly Leu Glu 115 120 125 Glu Met Met Thr Ala Cys Gln Gly
Val Gly Gly Pro Ser His Lys Ala 130 135 140 Ala Leu Ser Pro Arg Thr
Leu Asn Ala Trp Val Lys Val 145 150 155 47249PRTArtificial
Sequencesynthetic peptide construct 47Met Trp Lys Pro Met Pro Ser
Pro Ser Asn Met Lys Ala Ser Ala Ala 1 5 10 15 Leu Leu Cys Leu Leu
Leu Thr Ala Ala Ala Phe Ser Pro Gln Gly Leu 20 25 30 Ala Gln Pro
Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr Arg Phe 35 40 45 Ile
Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser Tyr Arg Arg Thr 50 55
60 Thr Ser Ser His Cys Pro Arg Glu Ala Val Ile Phe Lys Thr Lys Leu
65 70 75 80 Asp Lys Glu Ile Cys Ala Asp Pro Thr Gln Lys Trp Val Gln
Asp Phe 85 90 95 Met Lys His Leu Asp Lys Lys Thr Gln Thr Pro Lys
Leu Val Ile Pro 100 105 110 Met Phe Ser Ala Leu Ser Glu Gly Ala Thr
Pro Gln Asp Leu Asn Ala 115 120 125 Ala Val Gly Gly His Gln Ala Ala
Met Gln Met Leu Lys Asp Thr Ile 130 135 140 Asn Glu Glu Ala Ala Glu
Trp Asp Arg Ala Ala Ala Glu Pro Arg Gly 145 150 155 160 Ser Asp Ile
Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile Gly Trp 165 170 175 Ala
Ala Ala Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg 180 185
190 Met Tyr Ser Pro Thr Ser Ile Ala Ala Lys Tyr Val Asp Arg Phe Tyr
195 200 205 Lys Thr Leu Arg Ala Glu Gln Ala Ala Gly Leu Glu Glu Met
Met Thr 210 215 220 Ala Cys Gln Gly Val Gly Gly Pro Gly His Lys Ala
Ala Ile Ser Pro 225 230 235 240 Arg Thr Leu Asn Ala Trp Val Lys Val
245 48249PRTArtificial Sequencesynthetic peptide construct 48Met
Trp Lys Pro Met Pro Ser Pro Ser Asn Met Lys Ala Ser Ala Ala 1 5 10
15 Leu Leu Cys Leu Leu Leu Thr Ala Ala Ala Phe Ser Pro Gln Gly Leu
20 25 30 Ala Gln Pro Val Gly Ile Asn Thr Ser Thr Thr Cys Cys Tyr
Arg Phe 35 40 45 Ile Asn Lys Lys Ile Pro Lys Gln Arg Leu Glu Ser
Tyr Arg Arg Thr 50 55 60 Thr Ser Ser His Cys Pro Arg Glu Ala Val
Ile Phe Lys Thr Lys Leu 65 70 75 80 Asp Lys Glu Ile Cys Ala Asp Pro
Thr Gln Lys Trp Val Gln Asp Phe 85 90 95 Met Lys His Leu Asp Lys
Lys Thr Gln Thr Pro Lys Leu Val Ile Pro 100 105 110 Met Phe Thr Ala
Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Ala 115 120 125 Ala Val
Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile 130 135 140
Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala Glu Pro Arg Gly 145
150 155 160 Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln Ile
Ala Trp 165 170 175 Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly Leu Asn
Lys Ile Val Arg 180 185 190 Met Tyr Ser Pro Val Ser Ile Ala Ala Lys
Tyr Val Asp Arg Phe Phe 195 200 205 Lys Thr Leu Arg Ala Glu Gln Ala
Ala Gly Leu Glu Glu Met Met Thr 210 215 220 Ala Cys Gln Gly Val Gly
Gly Pro Ser His Lys Ala Ala Leu Ser Pro 225 230 235 240 Arg Thr Leu
Asn Ala Trp Val Lys Val 245 49165PRTArtificial Sequencesynthetic
peptide construct 49Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val
Ala Cys Ser Ile 1 5 10 15 Ser Gln Gly Gln Met Val His Gln Ala Ile
Ser Pro Arg Thr Leu Asn 20 25 30 Ala Trp Val Lys Val Leu Ala Lys
Glu Glu Lys Ala Phe Ser Pro Glu 35 40 45 Val Ile Pro Met Phe Ser
Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp 50 55 60 Leu Asn Ala Ala
Lys Val Gly Gly His Gln Ala Ala Met Gln Met Leu 65 70 75 80 Lys Glu
Thr Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Ala Ala Ala 85 90 95
Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu 100
105 110 Gln Ile Gly Trp Ala Ala Ala Lys Arg Trp Ile Ile Leu Gly Leu
Asn 115 120 125 Lys Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Ala Ala
Lys Tyr Val 130 135 140 Asp Arg Phe Tyr Lys Thr Leu Arg Ala Glu Gln
Ala Asp Tyr Lys Asp 145 150 155 160 Asp Asp Asp Lys Leu 165
5020PRTArtificial Sequencesynthetic peptide construct 50Gln Gly Gln
Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp
Val Lys Val 20 5126PRTArtificial Sequencesynthetic peptide
construct 51Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser
Ala Leu 1 5 10 15 Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn 20 25
52156PRTArtificial Sequencesynthetic peptide construct 52Met Trp
Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15
Ser Gln Gly Gln Met Val His Gln Ala Leu Ser Pro Arg Thr Leu Asn 20
25 30 Ala Trp Val Lys Val Leu Ala Lys Glu Glu Lys Gly Phe Asn Pro
Glu 35 40 45 Val Ile Pro Met Phe Thr Ala Leu Ser Glu Gly Ala Thr
Pro Gln Asp 50 55 60 Leu Asn Ala Ala Lys Val Gly Gly His Gln Ala
Ala Met Gln Met Leu 65 70 75 80 Lys Asp Thr Ile Asn Glu Glu Ala Ala
Glu Trp Asp Arg Ala Ala Ala 85 90 95 Glu Pro Arg Gly Ser Asp Ile
Ala Gly Thr Thr Ser Thr Leu Gln Glu 100 105 110 Gln Ile Ala Trp Ala
Ala Ala Lys Arg Trp Ile Ile Leu Gly Leu Asn 115 120 125 Lys Ile Val
Arg Met Tyr Ser Pro Val Ser Ile Ala Ala Lys Tyr Val 130 135 140 Asp
Arg Phe Phe Lys Thr Leu Arg Ala Glu Gln Ala 145 150 155
53155PRTArtificial Sequencesynthetic peptide construct 53Met Trp
Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15
Ser Glu Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Thr Ala 20
25 30 Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Ala Ala Lys Val
Gly 35 40 45 Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr Ile
Asn Glu Glu 50 55 60 Ala Ala Glu Trp Asp Arg Ala Ala Ala Glu Pro
Arg Gly Ser Asp Ile 65 70 75 80 Ala Gly Thr Thr Ser Thr Leu Gln Glu
Gln Ile Ala Trp Ala Ala Ala 85 90 95 Lys Arg Trp Ile Ile Leu Gly
Leu Asn Lys Ile Val Arg Met Tyr Ser 100 105 110 Pro Val Ser Ile Ala
Ala Lys Tyr Val Asp Arg Phe Phe Lys Thr Leu 115 120 125 Arg Ala Glu
Gln Ala Ala Leu Gln Gly Gln Met Val His Gln Ala Leu 130 135 140 Ser
Pro Arg Thr Leu Asn Ala Trp Val Lys Val 145 150 155
54155PRTArtificial Sequencesynthetic peptide construct 54Met Trp
Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15
Ser Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala 20
25 30 Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Ala Ala Lys Val
Gly 35 40 45 Gly His Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile
Asn Glu Glu 50 55 60 Ala Ala Glu Trp Asp Arg Ala Ala Ala Glu Pro
Arg Gly Ser Asp Ile 65 70 75 80 Ala Gly Thr Thr Ser Thr Leu Gln Glu
Gln Ile Gly Trp Ala Ala Ala 85 90 95 Lys Arg Trp Ile Ile Leu Gly
Leu Asn Lys Ile Val Arg Met Tyr Ser 100 105 110 Pro Thr Ser Ile Ala
Ala Lys Tyr Val Asp Arg Phe Tyr Lys Thr Leu 115 120 125 Arg
Ala Glu Gln Ala Ala Leu Gln Gly Gln Met Val His Gln Ala Ile 130 135
140 Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val 145 150 155
5520PRTArtificial Sequencesynthetic peptide construct 55Gln Gly Gln
Met Val His Gln Ala Leu Ser Pro Arg Thr Leu Asn Ala 1 5 10 15 Trp
Val Lys Val 20 5626PRTArtificial Sequencesynthetic peptide
construct 56Glu Glu Lys Gly Phe Asn Pro Glu Val Ile Pro Met Phe Thr
Ala Leu 1 5 10 15 Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn 20 25
5714PRTArtificial Sequencesynthetic peptide construct 57Gln Pro Ile
Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val 1 5 10
5814PRTArtificial Sequencesynthetic peptide construct 58Gln Ala Leu
Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val 1 5 10
5930PRTArtificial Sequencesynthetic peptide construct 59Glu Glu Lys
Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu 1 5 10 15 Ser
Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn 20 25 30
6030PRTArtificial Sequencesynthetic peptide construct 60Glu Glu Lys
Gly Phe Asn Pro Glu Val Ile Pro Met Phe Thr Ala Leu 1 5 10 15 Ser
Glu Gly Ala Thr Pro Gln Asp Leu Asn Met Met Leu Asn 20 25 30
6121PRTArtificial Sequencesynthetic peptide construct 61Pro Arg Gly
Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln Glu Gln 1 5 10 15 Ile
Gly Trp Met Thr 20 6221PRTArtificial Sequencesynthetic peptide
construct 62Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Thr Leu Gln
Glu Gln 1 5 10 15 Ile Ala Trp Met Thr 20 6320PRTArtificial
Sequencesynthetic peptide construct 63Ser Ile Leu Asp Ile Arg Gln
Gly Pro Lys Glu Pro Phe Arg Asp Tyr 1 5 10 15 Val Asp Arg Phe 20
6420PRTArtificial Sequencesynthetic peptide construct 64Ser Ile Leu
Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr 1 5 10 15 Val
Asp Arg Phe 20 6515PRTArtificial Sequencesynthetic peptide
construct 65Gln Asn Ser Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu
Gly 1 5 10 15 6615PRTArtificial Sequencesynthetic peptide construct
66Gln Asn Ala Asn Pro Asp Cys Lys Thr Ile Leu Lys Ala Leu Gly 1 5
10 15 6725PRTArtificial Sequencesynthetic peptide construct 67Leu
Glu Glu Met Met Thr Ala Cys Gln Gly Val Gly Gly Pro Gly His 1 5 10
15 Lys Ala Arg Ile Leu Ala Glu Ala Met 20 25 6825PRTArtificial
Sequencesynthetic peptide construct 68Leu Glu Glu Met Met Thr Ala
Cys Gln Gly Val Gly Gly Pro Ser His 1 5 10 15 Lys Ala Arg Val Leu
Ala Glu Ala Met 20 25 69401PRTArtificial Sequencesynthetic peptide
construct 69Met Arg Val Thr Glu Ile Arg Lys Ser Tyr Gln His Trp Trp
Arg Trp 1 5 10 15 Gly Ile Met Leu Leu Gly Ile Leu Met Ile Cys Asn
Ala Glu Glu Lys 20 25 30 Leu Trp Val Thr Val Tyr Tyr Gly Val Pro
Val Trp Lys Glu Ala Thr 35 40 45 Thr Thr Leu Phe Cys Ala Ser Asp
Arg Lys Ala Tyr Asp Thr Glu Val 50 55 60 His Asn Val Trp Ala Thr
His Ala Cys Val Pro Thr Asp Pro Asn Pro 65 70 75 80 Gln Glu Val Glu
Leu Lys Asn Val Thr Glu Asn Phe Asn Met Trp Lys 85 90 95 Asn Asn
Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110
Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115
120 125 Asn Cys Thr Asp Leu Arg Asn Ala Thr Asn Gly Asn Asp Thr Asn
Thr 130 135 140 Thr Ser Ser Ser Arg Gly Met Val Gly Gly Gly Glu Met
Lys Asn Cys 145 150 155 160 Ser Phe Asn Ile Thr Thr Asn Ile Arg Gly
Lys Val Gln Lys Glu Tyr 165 170 175 Ala Leu Phe Tyr Lys Leu Asp Ile
Ala Pro Ile Asp Asn Asn Ser Asn 180 185 190 Asn Arg Tyr Arg Leu Ile
Ser Cys Gly Ala Lys Phe Glu Pro Ile Pro 195 200 205 Ile His Tyr Cys
Thr Pro Ala Gly Phe Ala Gly Ala Lys Val Gln Cys 210 215 220 Thr His
Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly 225 230 235
240 Ser Leu Ala Glu Ala Ala Asp Ser Gly Gly Asp Pro Glu Ile Val Met
245 250 255 His Ser Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Gly Ala
Lys Asp 260 265 270 Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val
Ala Ala Lys Ala 275 280 285 Arg Arg Arg Val Val Gln Arg Glu Lys Arg
Ala Gly Ala Val Gly Phe 290 295 300 Leu Gly Thr Ala Gly Ser Thr Met
Gly Ala Ala Ser Val Ala Val Leu 305 310 315 320 Thr Val Gln Ala Arg
Leu Leu Leu Ser Gly Ile Val Gln Gln Gln Asn 325 330 335 Asn Leu Leu
Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr 340 345 350 Val
Trp Gly Ile Lys Gln Leu Gln Ala Arg Ala Ala Asp Trp Leu Trp 355 360
365 Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val Gly Leu Arg
370 375 380 Ile Ala Ala Phe Arg Val Arg Lys Gly Tyr Ser Pro Leu Ser
Leu Gln 385 390 395 400 Thr 7017PRTArtificial Sequencesynthetic
peptide construct 70Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe
Ala Ile Leu Lys 1 5 10 15 Cys 7117PRTArtificial Sequencesynthetic
peptide construct 71Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr
Ala Ile Leu Lys 1 5 10 15 Cys 7227PRTArtificial Sequencesynthetic
peptide construct 72Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg
Pro Val Val Ser 1 5 10 15 Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala
Glu 20 25 7327PRTArtificial Sequencesynthetic peptide construct
73Asn Val Ser Ser Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser 1
5 10 15 Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu 20 25
7420PRTArtificial Sequencesynthetic peptide construct 74Gly Gly Asp
Pro Glu Ile Val Met His Thr Phe Asn Cys Gly Gly Glu 1 5 10 15 Phe
Phe Tyr Cys 20 7520PRTArtificial Sequencesynthetic peptide
construct 75Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys Arg
Gly Glu 1 5 10 15 Phe Phe Tyr Cys 20 7611PRTArtificial
Sequencesynthetic peptide construct 76Cys Arg Ile Lys Gln Ile Ile
Asn Met Trp Gln 1 5 10 7711PRTArtificial Sequencesynthetic peptide
construct 77Cys Lys Ile Arg Gln Ile Val Asn Arg Trp Gln 1 5 10
7818PRTArtificial Sequencesynthetic peptide construct 78Gly Gly Asp
Met Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 1 5 10 15 Val
Val 7918PRTArtificial Sequencesynthetic peptide construct 79Gly Gly
Asn Met Lys Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys 1 5 10 15
Val Val 8046PRTArtificial Sequencesynthetic peptide construct 80Leu
Thr Val Gln Ala Arg Leu Leu Leu Ser Gly Ile Val Gln Gln Gln 1 5 10
15 Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu
20 25 30 Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg Val Leu Ala 35
40 45 8146PRTArtificial Sequencesynthetic peptide construct 81Leu
Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln 1 5 10
15 Ser Asn Leu Leu Lys Ala Ile Glu Ala Gln Gln His Met Leu Gln Leu
20 25 30 Thr Val Trp Gly Ile Lys Gln Leu Gln Thr Arg Val Leu Ala 35
40 45 8216PRTArtificial Sequencesynthetic peptide construct 82Asp
Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys 1 5 10
15 8316PRTArtificial Sequencesynthetic peptide construct 83Asp Gln
Gln Leu Leu Gly Leu Trp Gly Cys Ser Gly Lys Leu Ile Cys 1 5 10 15
8420PRTArtificial Sequencesynthetic peptide construct 84Trp Leu Trp
Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu Val 1 5 10 15 Gly
Leu Arg Ile 20 8520PRTArtificial Sequencesynthetic peptide
construct 85Trp Leu Trp Tyr Ile Arg Ile Phe Ile Met Ile Val Gly Gly
Leu Ile 1 5 10 15 Gly Leu Arg Ile 20 864PRTArtificial
Sequencesynthetic peptide construct 86Ala Ala Ala Glu 1
874PRTArtificial Sequencesynthetic peptide construct 87Ala Ala Ala
Ala 1 885PRTArtificial Sequencesynthetic peptide construct 88Ala
Ala Ala Ala Leu 1 5 891209DNAArtificial Sequencesynthetic
nucleotide sequence Env-CE1_V1V2 BaL 89atgcgggtga cggagatccg
gaagtcgtat cagcactggt ggcggtgggg catcatgctc 60cttgggatac tgatgatctg
caacgccgag gagaagctgt gggtcacggt gtactacggg 120gtcccggtct
ggaaggaggc caccacgacc ctgttctgcg cgagcgaccg caaggcctac
180gacaccgagg tgcacaacgt gtgggctacg cacgcgtgcg tgccaacgga
ccctaacccg 240caggaggtgg aactgaagaa cgtgaccgag aacttcaaca
tgtggaagaa caacatggtg 300gagcagatgc acgaggacat catctccctg
tgggaccagt cgctgaagcc gtgcgtcaag 360ctgacgccgc tctgcgtcac
gctgaactgc accgacctgc gcaacgccac gaacggcaac 420gacaccaaca
ccactagtag ctcgcgcggc atggtgggcg gcggcgagat gaagaactgc
480agcttcaaca tcacgaccaa catccgcggc aaggtgcaga aggagtacgc
cctgttctac 540aagctggaca tcgcccctat cgacaacaac agcaacaacc
gctaccggct gatcagctgc 600ggcgctaagt tcgagccgat ccctatccac
tactgcacgc cagctggatt cgcgggagcg 660aaggtgcagt gcacgcacgg
catccgacct gtggtctcga cgcagctgct cctgaacgga 720agcctggcag
aggcagctga cagcggcggg gacccggaga tcgtcatgca ctccttcaac
780tgcgggggcg agttcttcta ctgcggagcc aaggacaact ggcggtctga
gctgtacaag 840tacaaggtcg tggctgcgaa ggcgcgacgg agggtggtcc
agcgcgagaa gcgagccggt 900gcagtgggct tccttgggac ggccggctcg
acgatgggag cggcatcggt cgcggtgctg 960acggtccagg cgaggctgct
gctcagcggg atcgtccagc agcagaacaa cctgctccgg 1020gcgatcgagg
cgcagcagca cctgctccag ttaacggtct ggggcatcaa gcagctgcag
1080gctcgagccg cggactggct ctggtacatc aagatcttca tcatgatcgt
gggcggactg 1140gtcggcctgc gaatcgctgc attccgcgtc cggaaggggt
actcgcctct cagcctccaa 1200acgtgataa 1209
* * * * *
References