U.S. patent application number 17/471573 was filed with the patent office on 2022-01-06 for broadly neutralizing anti-hiv antibodies and epitope therefor.
This patent application is currently assigned to The Rockefeller University. The applicant listed for this patent is California Institute of Technology, The Rockefeller University. Invention is credited to Pamela J. Bjorkman, Michel Nussenzweig, Louise Scharf, Johannes Scheid.
Application Number | 20220002391 17/471573 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-06 |
United States Patent
Application |
20220002391 |
Kind Code |
A1 |
Nussenzweig; Michel ; et
al. |
January 6, 2022 |
Broadly Neutralizing Anti-HIV Antibodies and Epitope Therefor
Abstract
The present invention relates to broadly neutralizing anti-HIV-1
antibodies and isolated antigens. Also disclosed are related
methods and compositions.
Inventors: |
Nussenzweig; Michel; (New
York, NY) ; Bjorkman; Pamela J.; (La Canada
Flintridge, CA) ; Scharf; Louise; (Porter Ranch,
CA) ; Scheid; Johannes; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Rockefeller University
California Institute of Technology |
New York
Pasadena |
NY
CA |
US
US |
|
|
Assignee: |
The Rockefeller University
New York
NY
California Institute of Technology
Pasadena
CA
|
Appl. No.: |
17/471573 |
Filed: |
September 10, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16578833 |
Sep 23, 2019 |
11155605 |
|
|
17471573 |
|
|
|
|
15115547 |
Jul 29, 2016 |
10421803 |
|
|
PCT/US2015/013924 |
Jan 30, 2015 |
|
|
|
16578833 |
|
|
|
|
61934359 |
Jan 31, 2014 |
|
|
|
International
Class: |
C07K 16/10 20060101
C07K016/10; A61K 39/42 20060101 A61K039/42; A61P 31/18 20060101
A61P031/18; A61K 39/21 20060101 A61K039/21; C07K 14/005 20060101
C07K014/005; G01N 33/569 20060101 G01N033/569 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. AI100148 and AI100663 awarded by the National Institutes of
Health. The government has certain rights in the invention.
Claims
1.-10. (canceled)
11. An isolated antigen comprising an epitope-scaffold that mimics
the HIV-1 envelope spike epitope of broadly neutralizing antibody
8ANC195.
12. The antigen of claim 11 wherein the epitope-scaffold comprises
a discontinous epitope and a scaffold, wherein the epitope is
derived from HIV-1 gp120 and gp41, and wherein at least part of the
scaffold is not derived from gp120 or gp41.
13. The antigen of claim 12 wherein the discontinuous epitope
comprises amino acids corresponding to amino acid numbers 44-47,
90-94, 97, 234, 236-238, 240, 274-278, 352-354, 357, 456, 463, 466,
487, and 625-641 of gp140 from HIV strain 93TH057 numbered using
standard numbering for HIV strain HXBC2.
14. The antigen of claim 13 wherein the amino acids corresponding
to amino acid numbers 234 and 276 are glycosylated.
15. An isolated nucleic acid encoding the antigen of claim 11.
16. A vector comprising the isolated nucleic acid of claim 15.
17. A cultured cell comprising the vector of claim 16.
18. A composition comprising the antigen of claim 11.
19. A method for inducing an HIV antigen-specific immune response
in a subject in need thereof, comprising administering to said
subject the composition of claim 18 in an amount effective to
generate an immune response.
20. A method of preventing or treating an HIV-1 infection in a
subject in need thereof comprising administering to said subject
the composition of claim 19 in an amount effective to generate an
immune response.
21. A method for detecting or isolating an HIV-1 binding antibody
in a subject comprising obtaining a biological sample from said
subject, contacting said sample with the antigen of claim 11, and
conducting an assay to detect or isolate an HIV-1 binding antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Divisional of U.S. patent application
Ser. No. 16/578,833, filed Sep. 23, 2019, which is a Continuation
of U.S. patent application Ser. No. 15/115,547, filed Jul. 29,
2016, now U.S. Pat. No. 10,421,803, which is the U.S. National
Phase of International Patent Application No. PCT/US2015/013924,
filed Jan. 30, 2015, which claims priority to U.S. Provisional
Application No. 61/934,359 filed Jan. 31, 2014, the contents of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The only target of neutralizing anti-HIV-1 antibodies is the
envelope (Env) spike, a heterotrimer of gp120 and gp41 subunits.
Single cell-based antibody cloning techniques have recently
uncovered a large number of antibodies that can potently neutralize
highly diverse HIV-1 variants by targeting Env (Klein et al.,
Science 341, 1199 (2013)). When transferred passively, broadly
neutralizing antibodies (bNAbs) can prevent infection by HIV-1 or
SHIV in humanized mice and macaques, respectively. Moreover,
combinations of bNAbs can also suppress established HIV-1 and SHIV
infections (Klein et al., Nature 492, 118 (2012); Barouch et al.,
Nature 503, 224 (2013); Shingai et al., Nature 503, 277
(2013)).
[0004] Most of the bNAbs characterized to date target one of four
major sites of vulnerability on HIV-1 Env: on gp120, the CD4
binding site, the V2 loop, and the base of the V3 loop, and on
gp41, the membrane proximal region (Klein et al., Science 341, 1199
(2013); Burton et al., Science 337, 183 (2012); Mascola et al.,
Immunological Reviews 254, 225 (2013)). 8ANC195 is among a small
group of bNAbs that does not appear to target any of these sites.
Although only two of the B cells originally isolated from the
8ANC195 donor, an HIV-1 elite controller, belonged to the 8ANC195
clone, the antibodies produced by this clone complemented the
neutralizing activity of antibodies produced by a more expanded B
cell clone that targeted the CD4 binding site (Scheid et al.,
Science 333, 1633 (2011)).
[0005] 8ANC195 is classified as a bNAb because it neutralized 66%
of viruses in a diverse viral panel (Scheid et al., Science 333,
1633 (2011)). Like other anti-HIV-1 bNAbs, 8ANC195 is highly
somatically mutated, including insertions and deletions in the
complementarity determining regions (CDRs) and framework regions
(FWRs) of its heavy chain (HC) and light chain (LC). Although
initial efforts to map the 8ANC195 epitope were unsuccessful
(Ibid.) computational analyses of neutralization data predicted
that intact potential N-linked glycosylation sites (PNGSs) at
positions 234.sub.gp120 and 276.sub.gp120 were essential for its
activity. These predictions were confirmed by evaluating the
neutralization potency of 8ANC195 against mutant HIV-1 strains in
vitro and in vivo (West, Jr. et al., Proceedings of the National
Academy of Sciences of the United States of America 110, 10598
(2013); Chuang et al., Journal of Virology 87, 10047 (2013)).
However, the precise 8ANC195 epitope on HIV-1 Env has heretofore
remained elusive.
SUMMARY OF INVENTION
[0006] This invention relates, in part, to the isolation of
broadly-neutralizing antibodies (bNAbs) directed at an epitope on
the HIV-1 envelope spike that spans the gp120 and gp41
subunits.
[0007] In one embodiment, the antibody comprises a heavy chain
having one of the following amino acid sequences:
TABLE-US-00001 (g52; SEQ ID NO: 1)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g23; SEQ ID NO: 2)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTS
DYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g8; SEQ ID NO: 3)
QIHLVQSGTGVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPITSLEIKNVTSDDTAVYFCTTTSTY
DKWSGLYHDGVMAFSSWGQGTLISVSAASTKG; (g20; SEQ ID NO: 4)
QIHLVQSGTEVKKPGSSVAVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHDFRGRVIIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTATSTP
DYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g59; SEQ ID NO: 5)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTY
DEWSDLHHDGVMAFSSWGQGTLISVSAASTKG; (g62; SEQ ID NO: 6)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DKWSGLHHDGVMAFSSRGQGTLISVSAASTKG; (g22; SEQ ID NO: 7)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGPSPPISSLEIKNLTSDDTAVYFCTTTSTY
DKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g15; SEQ ID NO: 8)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTASTY
DKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g4; SEQ ID NO: 9)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIIS
AVDLTGSSPPISPLEIKNLTSDDTAVYFCTTTSTS
DRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g46; SEQ ID NO: 10)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTY
DKWSGLHHDGVVAFSSWGQGTLISVSAASTKG; (g44; SEQ ID NO: 11)
QIHLVQSGTEVKKPGSSVTVSCKAYEVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNVTSDDTAVYFCTTTSTH
DKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g50; SEQ ID NO: 12)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQGLEYIGQIWRWKSSASHHFRGRVLIS
AIDLTGSSPPISSLEIKNVTSDDTAVYFCTTMSTY
DKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (g3; SEQ ID NO: 13)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
SWVRQAPGQRLEYIGQIRRWKSSASHHFRGRVTVS
AVDPTGSSPPISSLEIRDLTTDDTAVYFCTTTSTS
DYWSGLHNERGTAFSSWGQGTLISVSAASTKG; (3040HC; SEQ ID NO: 14)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DQWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3430HC; SEQ ID NO: 15)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVIIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTS
DYWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3484HC; SEQ ID NO: 16)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DRWSGLHHDGVMAFSSWGQGTLISVSAASTKG; (3044HC: SEQ ID NO: 17)
QIHLVQSGTEVRKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DKWSGLHHDGVMAFSSWGQGTLISVSAASTKG; and (3630HC: SEQ ID NO: 18)
QIHLVQSGTEVKKPGSSVTVSCKAYGVNTFGLYAV
NWVRQAPGQSLEYIGQIWRWKSSASHHFRGRVLIS
AVDLTGSSPPISSLEIKNLTSDDTAVYFCTTTSTY
DRWSGLHHDGVMAFSSWGQGTLISVSAASTKG.
[0008] In one embodiment, the antibody comprises a light chain
having one of the following amino acid sequences:
TABLE-US-00002 (k3; SEQ ID NO: 19)
DIQMTQSPSTLSASIGDTVRISCRASQSITGNWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k5; SEQ ID NO:
20) DIQMTQSPSTLSASTGDTVRISCRASQSITGNWVA
WYQQRPGKAPRLLIYRGAALLGGVPSRFRGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k59; SEQ ID NO:
21) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSKFSGSAAGT
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k62; SEQ ID NO:
22) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLVGGVPSRFSGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k18; SEQ ID NO:
23) DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGA
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k53; SEQ ID NO:
24) DIQMTQSPSTLSASIGDTVMISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSKLLGGVPSRFSGSAAGT
GFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k61; SEQ ID NO:
25) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVA
WYHQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k11; SEQ ID NO:
26) DIQMTQSPSTLSASVGGTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k19; SEQ ID NO:
27) DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
GFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (k81; SEQ ID NO:
28) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWVA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (3040LC; SEQ ID
NO: 29) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVA
WYQQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (3430LC; SEQ ID
NO: 30) DIQMTQSPSTLSASVGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (3484LC; SEQ ID
NO: 31) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVA
WYQQRPGKAPRLLIYRGAALLGGVPSRFRGSAAGT
DFTLTIGNLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; (3044LC; SEQ ID
NO: 32) DIQMTQSPSTLSASIGDTVRISCRASQSITGNWVA
WYQQRPGKAPRLLIYRGAALLGGVPSRFSGSAAGT
DFTLTIGNLQTEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF; and (3630LC; SEQ
ID NO: 33) DIQMTQSPSTLSASIGDTVRISCRASQSITGGWLA
WYHQRPGKAPRLLIYRGSRLLGGVPSRFSGSAAGT
DFTLTIANLQAEDFGTFYCQQYDTYPGTFGQGTKV EVKRTVAAPSVF.
[0009] Accordingly, one aspect of this invention features an
isolated polypeptide comprising the sequence of any one of SEQ ID
NOs: 1-33. The invention also provides an isolated anti-HIV
antibody comprising one or both of a heavy chain comprising the
sequence of any one of SEQ ID NOs: 1-18 and a light chain
comprising the sequence any one of SEQ ID NOs: 19-33.
[0010] The above-mentioned antibody can be a human antibody, a
chimeric antibody, or a humanized antibody. It can be an IgG1,
IgG2, IgG3, or IgG4. The antibodies of the invention recognize the
epitope on the HIV-1 envelope spike recognized by 8ANC195 and are
broadly neutralizing.
[0011] In another aspect, the invention provides an isolated
nucleic acid encoding the isolated polypeptide or anti-HIV-1
antibody described above. Also provided are a vector comprising the
nucleic acid and a cultured cell comprising the nucleic acid.
[0012] In another aspect, the invention provides a composition
comprising at least one of the above-described isolated polypeptide
or anti-HIV-1 antibody or a fragment thereof. In one embodiment,
the composition comprises a pharmaceutically acceptable
carrier.
[0013] In another aspect, the invention provides a method of
preventing or treating an HIV-1 infection or an HIV-related
disease. The method includes steps of identifying a patient in need
of such prevention or treatment, and administering to the patient a
first therapeutic agent comprising a therapeutically effective
amount of at least one of the above-described isolated polypeptide
or anti-HIV-1 antibody. The method can further comprise
administering a second therapeutic agent, such as an antiviral
agent.
[0014] In another embodiment, the present invention provides an
isolated antigen comprising an epitope-scaffold that mimics the
HIV-1 envelope spike epitope of broadly neutralizing antibody
8ANC195. In one aspect, the epitope-scaffold comprises a
discontinous epitope and a scaffold. In another aspect, the epitope
is derived from HIV-1 gp120 and gp41, and at least part of the
scaffold is not derived from gp120 or gp41. In another aspect, the
discontinuous epitope comprises amino acids corresponding to amino
acid numbers 44-47, 90-94, 97, 234, 236-238, 240, 274-278, 352-354,
357, 456, 463, 466, 487, and 625-641 of gp140 from HIV strain
93TH057 numbered using standard numbering for HIV strain HXBC2. The
amino acids corresponding to amino acid numbers 234 and 276 may be
glycosylated.
[0015] In another aspect, the invention provides an isolated
nucleic acid encoding the isolated antigen described above. Also
provided are a vector comprising the nucleic acid and a cultured
cell comprising the nucleic acid.
[0016] In another aspect, the invention provides a composition
comprising the isolated antigen. In one embodiment, the composition
further comprises a pharmaceutically acceptable carrier. In one
embodiment, the composition further comprises an adjuvant.
[0017] In another aspect, the present invention provides a method
for generating an immune response in a subject in need thereof,
comprising administering to said subject a composition comprising
the above-described isolated antigen in an amount effective to
generate an immune response.
[0018] In another aspect, the invention provides a method of
preventing or treating an HIV-1 infection or an HIV-related
disease. The method includes steps of identifying a patient in need
of such prevention or treatment, and administering to the patient a
first therapeutic agent comprising a therapeutically effective
amount of the above-described antigen. The method can further
comprise administering a second therapeutic agent, such as an
antiviral agent.
[0019] In another aspect, the present invention provides a method
for detecting or isolating an HIV-1 binding antibody in a subject
comprising obtaining a biological sample from the subject,
contacting the sample with the above-described antigen, and
conducting an assay to detect or isolate an HIV-1 binding
antibody.
[0020] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B illustrates alignments of (A) VH and (B) VL
sequences of mature 8ANC195 and its putative germ-line progenitor
(GL). 8ANC195 HC has the sequence of SEQ ID NO:34. 8ANC195 LC has
the sequence of SEQ ID NO:35. GL HC has the sequence of SEQ ID
NO:36. GL LC has the sequence of SEQ ID NO:37. Residues forming the
CDR loops are labeled (CDR1), (CDR2) and (CDR3). 8ANC195 is one of
the most heavily mutated bNAbs isolated to date, with 49 of 103
amino acid mutations in the HC and 25 of 90 in the LC. The HC was
too highly somatically mutated to accurately assign D and J gene
segments, but the LC showed sufficient homology to assign its J
segment as IGKJ5*01.
[0022] FIGS. 2A, 2B and 2C illustrates conformations of 8ANC195
CDRH1 and CDRH3 loops. (A) The hook-like conformation of CDRH1 is
stabilized by burial of the hydrophobic Phe30HC side chain and
hydrogen bonds within CDRH1 and with CDRH3 and FWR1 and FWR3
residues (Ala24HC and Asp73HC, respectively). (B) The complexed
CDRH3 conformation consists of a protruding loop (residues
95HC-100HC) and a small (3-sheet subdomain (residues 100dHC-100kHC)
stabilized by multiple hydrogen bonds within CDRH3 as well as with
CDRH1 and CDRL3. A hydrogen bond between Tyr92LC and Gly100cHC
stabilized the bifurcation of CDRH3 into its two subdomains. CDRH1
and CDRH3 loop backbone atoms are shown as sticks and side chains
of residues important for stabilizing the loop conformations are
shown as sticks (involved in direct contacts) or lines (backbone
involved in contacts) (other side chains Tyr98HC, Lys100HC and
Trp100aHC, are shown for clarity). (C) Comparison of CDRH3 loops in
8ANC195 and other anti-HIV-1 bNAbs. CDRH3 residues corresponding to
8ANC195HC residues 90-105 of NIH45-46 (PDB 3U7Y), PG16 (PDB 4DQO),
PGT121 (PDB 4FQC) and PGT128 (PDB 3TYG) are shown as Ca traces.
[0023] FIGS. 3A, 3B, 3C, 3D and 3E illustrates CD4 interactions
with 8ANC195. (A) Superimposition of sCD4 D1D2/gp120 structures
(ribbon diagrams) from complexes with 8ANC195, 17b (PDB 1GCI) and
21c PDB 3LQA). (B) Competition ELISA of 8ANC195 IgG binding to
93TH057 gp120 in the presence of increasing concentrations of
potential competitors (sCD4, diamonds; J3 VHH, triangles; 3BNC60
Fab, squares; NIH45-46 Fab, circles). No competition was observed
with small, single-Ig domain CD4-binding site ligands (sCD4, J3
VHH), but larger Fab fragments of CD4 binding site antibodies
(3BNC60, NIH45-46) competed for binding. (C) In vitro assay
comparing neutralization of YU2 by sCD4 (squares), 8ANC195 IgG
(triangles), and an equimolar mixture of 8ANC195 and sCD4
(circles). (D) Packing of 8ANC195/sCD4/gp120 crystals. Several
symmetry mates are shown as surface representations (8ANC195 HC;
8ANC195 LC; 93TH057 gp120; sCD4 D1D2). Areas where two complexes
form crystal contacts are indicated. (E) In vitro assay comparing
neutralization of YU2 by 8ANC195 IgG (squares), 3BNC60 IgG
(triangles), and an 8ANC195 IgG mutant that lacks the FWR3
insertion (Ser77a-Pro77b-Pro77c-Ile77d) that results in the
protruding "FWR3.sub.HC thumb" (circles).
[0024] FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H and 4I illustrates
surface area buried at interface of 8ANC195 Fab and gp120. Left
panels, surface area buried on 8ANC195 Fab by (A) gp120 protein
residues, (C) Asn276gp120 glycan or (E) Asn234gp120 glycan; right
panels, surface area buried by 8ANC195 Fab on (B) gp120 protein
residues, (D) Asn234gp120 glycan or (F) Asn276gp120 glycan. Atoms
buried at these interfaces are shown as surface representations
overlaid onto ribbon diagrams of 8ANC195 Fab and gp120 or stick
representations of glycans. 8ANC195 Fab: HC; LC; CDRH1; CDRH2;
CDRH3; gp120: inner domain; outer domain; loop D; loop V5;
Asn234gp120 glycan: Asn276gp120 glycan. 2Fo-Fc annealed omit
electron density maps (grey mesh, o'=1) used to build (E)
Asn234.sub.gp120 glycan and (H) Asn276.sub.gp120 glycan. (I)
Modeled fucose residue .alpha.1-6-linked to the first
N-acetylglucosamine residue of the Asn276.sub.gp120 glycan shows
that the core fucose of a complex-type N-glycan could be
accommodated by the 8ANC195. Glycan residues are shown as sticks,
and gp120, 8ANC195 HC and CD4 are shown as surface
representations.
[0025] FIGS. 5A and 5B illustrates a comparison of glycan-dependent
bNAbs. 8ANC195 is "bracketed" by two glycans (Asn234.sub.gp120
glycan; Asn276.sub.gp120 glycan) in the 8ANC195 Fab/gp120/sCD4
complex structure (left panels). For comparison, crystal structures
of PG16 (middle panels, PDB 4DQO) bound to a V1/V2 loop scaffold
and PGT128 (right panels, PDB 3TYG) bound to a V3 loop scaffold are
shown with (A) the antibody HCs aligned to the 8ANC195 HC or (B) an
alternative view showing their interactions with bracketing glycans
(for PG16: Asn160.sub.gp120 glycan/Asn172 glycan; for PGT128:
Asn301.sub.gp120 glycan/Asn332.sub.gp120 glycan). The proteins are
shown as ribbon diagrams and the glycans as stick
representations.
[0026] FIGS. 6A, 6B and 6C illustrates green EM refinement
statistics. (A) Electron micrograph at 52,000.times. magnification
and -0.8 .mu.m defocus. (B) Reference-free 2D class averages of the
SOSIP trimer in complex with 8ANC195 Fab showing various
orientations. (C) Fourier Shell Correlation (FSC) graph resulting
from refinement. The resolution was determined as 18.7 .ANG. at an
FSC cut-off of 0.5.
[0027] FIGS. 7A and 7B illustrates negative stain EM reconstruction
of BG505 SOSIP.664 in complex with 8ANC195 Fab fit two ways. (A)
When the gp120-8ANC195 Fab structure was fit into the EM density,
the gp120 from the complex structure was displaced slightly
outwards in comparison to the gp120 in the SOSIP trimer structure.
The HC and LC of the Fab are shown. The Asn234.sub.gp120 and
Asn276.sub.gp120 glycans are shown as spheres. (B) Close up of the
Fab-Env interface. The position of Asn637.sub.gp120 can be deduced
from the position of the C-terminus of HR2, which corresponds to
residue Gly664.sub.gp41. This residue is in close proximity to the
LC and the glycan at this position could interact with the 8ANC195
Fab.
[0028] FIGS. 8A, 8B, 8C and 8D illustrates EM reconstruction of
8ANC195 Fab/BG505 SOSIP.664 showing gp41 contacts. Top view of EM
density with the X-ray structures of BG505 SOSIP.664 (PDB ID 4NCO;
gp120, grey; gp41) and 8ANC195 Fab (HC; LC with a map contour level
of 0.0176 (A) and 0.030 (B). Areas of contact between 8ANC195 and
gp41 are marked with circles, those between 8ANC195 and gp120 with
black circles. (C,D) Close-up of 8ANC195 LC and HR2 region in EM
complex structure (HR2 coordinates in PDB 4NCO with presumptive
sidechains for strain YU2 added to the polyalanine coordinates).
(C) Fab is shown as a surface representation with highlights
(CDRL1; CDRL2; CDRH3, and gp41 HR2 is shown as a ribbon diagram.
The position of Asn637gp41 was deduced from the position of the
C-terminus of the SOSIP.664 trimer (Gly664gp41). (D) 8ANC195 HC and
LC residues (sticks) positioned to contact HR2, with side chains of
surface-exposed residues that vary between newly isolated 8ANC195
(T/K) variants shown as sticks.
[0029] FIGS. 9A, 9B and 9C relate to Single Cell Variants of
8ANC195. (A) Strategy of large scale single cell sorting. (B) IgH
and IgL chain genes from isolated single cell variants of 8ANC195.
Identical members are grouped together. The HC CDR3 of 8ANC195 has
the sequence of SEQ ID NO:38. The HC CDR3 of 8ANC142 has the
sequence of SEQ ID NO:39. The HC CDR3 of 8ANC3430 has the sequence
of SEQ ID NO:40. The HC CDR3 of 8ANC3484 has the sequence of SEQ ID
NO:41. The HC CDR3 of 8ANC3044 has the sequence of SEQ ID NO:42.
The HC CDR3 of 8ANC3630 has the sequence of SEQ ID NO:43. The LC
CDR3 of 8ANC195 has the sequence of SEQ ID NO:44. The LC CDR3 of
8ANC142 has the sequence of SEQ ID NO:45. The LC CDR3 of 8ANC3430
has the sequence of SEQ ID NO:46. The LC CDR3 of 8ANC3484 has the
sequence of SEQ ID NO:47. The LC CDR3 of 8ANC3044 has the sequence
of SEQ ID NO:48. The LC CDR3 of 8ANC3630 has the sequence of SEQ ID
NO:49. (C) IC.sub.50 neutralization titers of distinct single cell
versions of the 8ANC195 clone compared to 8ANC195 against a 15
virus Tier 2 panel.
[0030] FIGS. 10A and 10B depict the alignment of amino acid
sequences of all distinct single cell versions of the 8ANC195
clone. HC (A) and LC (B) sequences were aligned with the respective
germline genes. Mutations introduced by somatic hypermutation are
indicated.
[0031] FIGS. 11A, 11B and 11C are directed to Bulk Sorted Variants
of 8ANC195. (A) Strategy of bulk memory B cell sorting without
antigen. (B) PCR strategy for the amplification of 8ANC195 HC and
LC clone members. Shown are the priming sites aligned with the
original nucleotide sequence of 8ANC195 at the respective sites.
Mismatches with the respective germline genes are indicated.
Primers 1 and 2 for the 8ANC195 HC FWR1 have SEQ ID Nos: 50 and 51,
respectively. Primers 1 and 2 for the 8ANC195 LC FWR1 have SEQ ID
Nos: 52 and 53, respectively. The primer for the 8ANC195 HC J-gene
has SEQ ID NO:54. Primers 1 and 2 for the 8ANC195 LC J-gene have
SEQ ID Nos: 55 and 56, respectively. (C) Phylogenetic tree of 128
isolated HC and 100 LC sequences. Representative members chosen for
alignment are indicated.
[0032] FIGS. 12A and 12B depict alignment of amino acid sequences
of selected bulk sorted versions of the 8ANC195 clone. HC (A) and
LC (B) sequences were aligned with the respective germline genes as
well as the original 8ANC195 sequence. All mutations introduced by
somatic hypermutation are indicated.
[0033] FIGS. 13A and 13B show that 52.sub.HC .kappa.5.sub.LC is
more potent than 8ANC195. IC.sub.50 values of 52.sub.HC .kappa.5LC
and 8ANC195 against Tier 2 15 virus panel shown as dot plot (A) and
Table (B). NT, not tested.
[0034] FIGS. 14A, 14B and 14C show that somatic mutations in the
8ANC195 LC CDRs and FWRs could affect contacts with gp41. (A)
Surface representation of 8ANC195 Fab (HC; LC; somatically mutated,
surface-exposed LC residues; residue 64.sub.LC). (B) Surface
representation of 8ANC195 Fab and BG505 gp41 HR2 with a modeled
Man6 sugar attached to Asn637gp41. (C) Surface representation of
8ANC195 Fab (CDRL1; CDRL2; CDRH3; residue 64.sub.LC) and BG505 gp41
HR2 with a modeled Man6 sugar attached to Asn637gp41.
[0035] FIG. 15 illustrates locations of bNAb epitopes on HIV-1 Env
Trimer. EM density map of Env trimer including MPER region showing
approximate epitope locations for antibodies targeting the 8ANC195
epitope, CD4 binding site, V3 loop/Asn332 glycan (332 glycan shown
as spheres), V1/V2 loop/Asn160 glycan (160 glycan shown as
spheres), and MPER
[0036] FIGS. 16A, 16B and 16C illustrate crystal structures of
8ANC195 Fab and 8ANC195/gp120/sCD4 complex. (A) Superimposition of
unbound and bound (HC and LC) structures of 8ANC195 Fab shown as
ribbon diagrams. CDR loops are highlighted (CDRH1/CDRL1;
CDRH2/CDRL2; CDRH3; CDRL3) and a "thumb"-like loop formed by an
insertion in FWR3 is indicated. Disordered loops are shown as
dashed lines. (B) Space-filling model (inset) and ribbon diagram of
ternary complex of 8ANC195 (HC and LC), sCD4, and 93TH057 gp120
core (inner domain; outer domain; bridging sheet; loop D; loop V5;
CD4 binding loop). Ordered glycans attached to Asn234.sub.gp120 and
Asn276.sub.gp120 are shown as sticks. Fab CDR loops are indicated
as in (A). sCD4 was omitted from the right panel for clarity. (C)
Approximate locations of bNAb epitopes on a surface representation
of the gp120 core. The epitopes of V3 and V1N2 antibodies include
regions of loops (dotted lines) not present in the gp120 core
structure. CD4 binding site and 8ANC195 epitopes are outlined by
black (CD4 binding site) and (8ANC195) dots. Glycans included in
the 8ANC195 epitope are indicated. Subdomains of gp120 are
indicated as in (B).
[0037] FIGS. 17A, 17B, 17C, 17D and 17E show contacts made by
8ANC195 HC with gp120 protein residues and glycans. Labels for
gp120 protein and glycan residues are italicized. Hydrogen bonds
are shown as dashed lines. (A) FWR3.sub.HC loop contacts with loop
D, loop V5, and outer domain loop. (B) 8ANC195 HC CDRH1 and CDRH3
contacts with gp120 inner domain. (C) Buried surface area between
the Asn234.sub.gp120 glycan (transparent surface with glycan
residues shown as sticks) and 8ANC195 (HC FWR residues and CDRH2
are indicated). Antibody atoms buried by glycan interactions are
shown as surfaces. (D) Buried surface area between the Asn276
glycan.sub.gp120 (transparent surface with glycan residues shown as
sticks) and 8ANC195 (HC FWR residues and CDRH1 are indicated).
Antibody atoms buried by glycan interactions are shown as surfaces.
(E) Top: Contacts made by 8ANC195 HC FWR residues and CDRH2 with
Asn234.sub.gp120 glycan. Glycan and protein residues involved in
hydrogen bonds are shown as sticks. Bottom: schematic of ordered
high mannose glycans on Asn234.sub.gp120 and Asn276.sub.gp120
(bottom).
[0038] FIGS. 18A and 18B show the EM structure of 8ANC195/Env
trimer complex and model of 8ANC195 LC interactions with gp41 HR2.
(A) EM reconstruction of 8ANC195 Fab/BG505 SOSIP.664. Side (left)
and top (right) views of EM density with the X-ray structures of
BG505 SOSIP.664 (PDB ID 4NCO; gp120, gp41) and 8ANC195 Fab fit in
two ways: (i) fitting 8ANC195 Fab independently of gp140
coordinates to the EM density (best fit/independently placed), and
(ii) by aligning the gp120 of the gp120/8ANC195 complex structure
onto the gp120 of PDB 4NCO fit to the EM density. (B) Close-up of
8ANC195 LC/HR2 region of EM complex structure (Fab placement is
best fit/independently placed as in (A)). Left: Fab is shown as a
surface representation with highlights (CDRL1; CDRL2; CDRH1;
CDRH3), and gp140 is shown as a ribbon diagram (gp120; gp41). The
position of Asn637.sub.gp41 was deduced from the position of the
C-terminus of the SOSIP.664 trimer (Gly664.sub.gp41). Right:
8ANC195 HC and LC residues (sticks) positioned to contact HR2,
which is shown as a surface representation calculated from HR2
coordinates in PDB 4NCO with presumptive sidechains added to the
polyalanine coordinates.
[0039] FIGS. 19A, 19B and 19C show effects of LC sequence changes
on 8ANC195 neutralization potency. (A) Sequences of LC CDRs in
constructs used with 8ANC195 HC to make chimeric IgGs (left) and
location of CDRs on 8ANC195 structure (right). Sequences derived
from the mature antibody are shown and those derived from the
germline precursor are shown on a grey background. The mutations
introduced into CDRL3 in glCDRL3Ala are shown on a white
background. (B) Effects of changes in 8ANC195 LC on binding to
93TH057 and YU2 gp120s and neutralization of viral strains,
expressed as fold changes over results for 8ANC195 IgG. KD and
IC.sub.50 values for these experiments are shown in table S3. (C)
Heat map showing the expression and neutralization of randomly
paired HCs and LCs from the bulk sort on a Tier 2 15-virus panel.
Average IC.sub.50 values (arithmetic means) between 0.1 and 2
.mu.g/ml; between 2.1 and 10 .mu.g/ml, between 10.1 and 14.9
.mu.g/ml, and above 15 .mu.g/ml are indicated with varying degrees
of shaded squares. Empty squares represent insufficient antibody
expression.
[0040] FIG. 20 depicts the alignment of gp140 sequences from HIV
strains HXBC2 and 93TH057 using standard HXBC2 numbering of amino
acid residues. Amino acid residues contacted by 8ANC195 in the
complex crystal structure with 93TH057 gp120 core are indicated on
the 93TH057 sequence. Glycans contacted by 8ANC195 in the complex
crystal structure with 93TH057 gp120 core are shown as the
asparagine residues to which they are attached, highlighted in cyan
on the 93TH057 sequence. The region of gp41 contacted by 8ANC195
based on the EM complex structure is indicated. Select glycans are
shown as diagrams on the asparagine residues to which they are
attached.
DETAILED DESCRIPTION OF THE INVENTION
[0041] The present invention is based, at least in part, on the
identification of the epitope recognized by 8ANC195, a broadly
neutralizing antibody to the HIV-1 envelope glycoprotein. The
present invention, in one embodiment, provides an isolated antigen
comprising the epitope, compositions comprising the antigen, and
methods of using the antigen. In other embodiments, the present
invention provides isolated ant-HIV-1 antibodies that recognize the
epitope on the HIV-1 envelope spike recognized by 8ANC195,
compositions comprising the antibodies, and methods of using the
antibodies.
[0042] In one embodiment, the present invention is directed to an
isolated anti-HIV antibody comprising one or both of a heavy chain
comprising the sequence of any one of SEQ ID NOs: 1-18 and a light
chain comprising the sequence any one of of SEQ ID NOs: 19-33. In
one preferred embodiment, the heavy chain comprises the sequence of
SEQ ID NO:1 and the light chain comprises the sequence of SEQ ID
NO:20. In another embodiment, the present invention provides an
isolated polypeptide comprising the sequence of any one of SEQ ID
NOs: 1-33.
[0043] The above-mentioned antibody can be a human antibody, a
chimeric antibody, or a humanized antibody. It can be an IgG1,
IgG2, IgG3, or IgG4. The antibodies of the invention recognize the
epitope on the HIV-1 envelope spike recognized by 8ANC195 and are
broadly neutralizing. 8ANC195 is known in the art and disclosed,
for example, by Scheid et al., Science, 333, 1633 (2011). The heavy
chain of 8ANC195 has the sequence of SEQ ID NO:34 and the light
chain of 8ANC195 has the sequence of SEQ ID NO:35.
[0044] The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multispecific antibodies (for
example, bispecific antibodies and polyreactive antibodies), and
antibody fragments. Thus, the term "antibody" as used in any
context within this specification is meant to include, but not be
limited to, any specific binding member, immunoglobulin class
and/or isotype (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE
and IgM); and biologically relevant fragment or specific binding
member thereof, including but not limited to Fab, F(ab')2, Fv, and
scFv (single chain or related entity). It is understood in the art
that an antibody is a glycoprotein comprising at least two heavy
(H) chains and two light (L) chains inter-connected by disulfide
bonds, or an antigen binding portion thereof. A heavy chain is
comprised of a heavy chain variable region (VH) and a heavy chain
constant region (CH1, CH2 and CH3). A light chain is comprised of a
light chain variable region (VL) and a light chain constant region
(CL). The variable regions of both the heavy and light chains
comprise framework regions (FWR) and complementarity determining
regions (CDR). The four FWR regions are relatively conserved while
CDR regions (CDR1, CDR2 and CDR3) represent hypervariable regions
and are arranged from NH2 terminus to the COOH terminus as follows:
FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, and FWR4. The variable regions
of the heavy and light chains contain a binding domain that
interacts with an antigen while, depending of the isotype, the
constant region(s) may mediate the binding of the immunoglobulin to
host tissues or factors.
[0045] Also included in the definition of "antibody" as used herein
are chimeric antibodies, humanized antibodies, and recombinant
antibodies, human antibodies generated from a transgenic non-human
animal, as well as antibodies selected from libraries using
enrichment technologies available to the artisan.
[0046] The term "variable" refers to the fact that certain segments
of the variable (V) domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable regions. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable regions of native heavy and light chains
each comprise four FRs, largely adopting a beta sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the beta sheet
structure. The hypervariable regions in each chain are held
together in close proximity by the FRs and, with the hypervariable
regions from the other chain, contribute to the formation of the
antigen-binding site of antibodies (see, for example, Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)).
[0047] The term "hypervariable region" as used herein refers to the
amino acid residues of an antibody that are responsible for antigen
binding. The hypervariable region generally comprises amino acid
residues from a "complementarity determining region" ("CDR").
[0048] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. The term
"polyclonal antibody" refers to preparations that include different
antibodies directed against different determinants
("epitopes").
[0049] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with, or homologous to, corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with, or homologous to, corresponding
sequences in antibodies derived from another species or belonging
to another antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see, for example, U.S. Pat. No. 4,816,567; and Morrison et al.,
Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric
antibodies include antibodies having one or more human antigen
binding sequences (for example, CDRs) and containing one or more
sequences derived from a non-human antibody, for example, an FR or
C region sequence. In addition, chimeric antibodies included herein
are those comprising a human variable region antigen binding
sequence of one antibody class or subclass and another sequence,
for example, FR or C region sequence, derived from another antibody
class or subclass.
[0050] A "humanized antibody" generally is considered to be a human
antibody that has one or more amino acid residues introduced into
it from a source that is non-human. These non-human amino acid
residues often are referred to as "import" residues, which
typically are taken from an "import" variable region. Humanization
may be performed following the method of Winter and co-workers
(see, for example, Jones et al., Nature 321:522-525 (1986);
Reichmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science 239:1534-1536 (1988)), by substituting import hypervariable
region sequences for the corresponding sequences of a human
antibody. Accordingly, such "humanized" antibodies are chimeric
antibodies (see, for example, U.S. Pat. No. 4,816,567), where
substantially less than an intact human variable region has been
substituted by the corresponding sequence from a non-human
species.
[0051] An "antibody fragment" comprises a portion of an intact
antibody, such as the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include, but are
not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies;
linear antibodies (see, for example, U.S. Pat. No. 5,641,870;
Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0052] "Fv" is the minimum antibody fragment that contains a
complete antigen-recognition and antigen-binding site. This
fragment contains a dimer of one heavy- and one light-chain
variable region domain in tight, non-covalent association. From the
folding of these two domains emanate six hypervariable loops (three
loops each from the H and L chain) that contribute the amino acid
residues for antigen binding and confer antigen binding specificity
to the antibody. However, even a single variable region (or half of
an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity
than the entire binding site.
[0053] "Single-chain Fv" ("sFv" or "scFv") are antibody fragments
that comprise the VH and VL antibody domains connected into a
single polypeptide chain. The sFv polypeptide can further comprise
a polypeptide linker between the VH and VL domains that enables the
sFv to form the desired structure for antigen binding. For a review
of sFv, see, for example, Pluckthun in The Pharmacology of
Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995,
infra.
[0054] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments with short linkers (about
5-10 residues) between the VH and VL domains such that inter-chain
but not intra-chain pairing of the V domains is achieved, resulting
in a bivalent fragment, i.e., fragment having two antigen-binding
sites. Bispecific diabodies are heterodimers of two "crossover" sFv
fragments in which the VH and VL domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0055] Domain antibodies (dAbs), which can be produced in fully
human form, are the smallest known antigen-binding fragments of
antibodies, ranging from about 11 kDa to about 15 kDa. DAbs are the
robust variable regions of the heavy and light chains of
immunoglobulins (VH and VL, respectively). They are highly
expressed in microbial cell culture, show favorable biophysical
properties including, for example, but not limited to, solubility
and temperature stability, and are well suited to selection and
affinity maturation by in vitro selection systems such as, for
example, phage display. DAbs are bioactive as monomers and, owing
to their small size and inherent stability, can be formatted into
larger molecules to create drugs with prolonged serum half-lives or
other pharmacological activities. Examples of this technology have
been described in, for example, WO9425591 for antibodies derived
from Camelidae heavy chain Ig, as well in US20030130496 describing
the isolation of single domain fully human antibodies from phage
libraries.
[0056] Fv and sFv are the only species with intact combining sites
that are devoid of constant regions. Thus, they are suitable for
reduced nonspecific binding during in vivo use. sFv fusion proteins
can be constructed to yield fusion of an effector protein at either
the amino or the carboxy terminus of an sFv. See, for example,
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
also can be a "linear antibody", for example, as described in U.S.
Pat. No. 5,641,870 for example. Such linear antibody fragments can
be monospecific or bispecific.
[0057] In certain embodiments, antibodies of the described
invention are bispecific or multispecific. Bispecific antibodies
are antibodies that have binding specificities for at least two
different epitopes. Exemplary bispecific antibodies can bind to two
different epitopes of a single antigen. Other such antibodies can
combine a first antigen binding site with a binding site for a
second antigen. Alternatively, an anti-HIV arm can be combined with
an arm that binds to a triggering molecule on a leukocyte, such as
a T-cell receptor molecule (for example, CD3), or Fc receptors for
IgG (Fc gamma R), such as Fc gamma RI (CD64), Fc gamma RH (CD32)
and Fc gamma RIII (CD16), so as to focus and localize cellular
defense mechanisms to the infected cell. Bispecific antibodies also
can be used to localize cytotoxic agents to infected cells.
Bispecific antibodies can be prepared as full length antibodies or
antibody fragments (for example, F(ab')2 bispecific antibodies).
For example, WO 96/16673 describes a bispecific anti-ErbB2/anti-Fc
gamma RIII antibody and U.S. Pat. No. 5,837,234 discloses a
bispecific anti-ErbB2/anti-Fc gamma RI antibody. For example, a
bispecific anti-ErbB2/Fc alpha antibody is reported in WO98/02463;
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody. See also, for example, Mouquet et al., Polyreactivity
Increases The Apparent Affinity Of Anti-HIV Antibodies By
Heteroligation. NATURE. 467, 591-5 (2010).
[0058] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(see, for example, Millstein et al., Nature, 305:537-539 (1983)).
Similar procedures are disclosed in, for example, WO 93/08829,
Traunecker et al., EMBO J., 10:3655-3659 (1991) and see also;
Mouquet et al., Polyreactivity Increases The Apparent Affinity Of
Anti-HIV Antibodies By Heteroligation. NATURE. 467, 591-5
(2010).
[0059] Alternatively, antibody variable regions with the desired
binding specificities (antibody-antigen combining sites) are fused
to immunoglobulin constant domain sequences. The fusion is with an
Ig heavy chain constant domain, comprising at least part of the
hinge, CH2, and CH3 regions. According to some embodiments, the
first heavy-chain constant region (CH1) containing the site
necessary for light chain bonding, is present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host cell. This provides for greater flexibility in adjusting the
mutual proportions of the three polypeptide fragments in
embodiments when unequal ratios of the three polypeptide chains
used in the construction provide the optimum yield of the desired
bispecific antibody. It is, however, possible to insert the coding
sequences for two or all three polypeptide chains into a single
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
have no significant affect on the yield of the desired chain
combination.
[0060] Techniques for generating bispecific antibodies from
antibody fragments also have been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. For example, Brennan et al., Science, 229: 81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab')2 fragments. These fragments are reduced
in the presence of the dithiol complexing agent, sodium arsenite,
to stabilize vicinal dithiols and prevent intermolecular disulfide
formation. The Fab' fragments generated then are converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives then is reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0061] Other modifications of the antibody are contemplated herein.
For example, the antibody can be linked to one of a variety of
nonproteinaceous polymers, for example, polyethylene glycol,
polypropylene glycol, polyoxyalkylenes, or copolymers of
polyethylene glycol and polypropylene glycol. The antibody also can
be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization (for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate)microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), or
in macroemulsions. Such techniques are disclosed in, for example,
Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,
(1980).
[0062] Typically, the antibodies of the described invention are
produced recombinantly, using vectors and methods available in the
art. Human antibodies also can be generated by in vitro activated B
cells (see, for example, U.S. Pat. Nos. 5,567,610 and 5,229,275).
General methods in molecular genetics and genetic engineering
useful in the present invention are described in the current
editions of Molecular Cloning: A Laboratory Manual (Sambrook, et
al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression
Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel,
1991. Academic Press, San Diego, Calif.), "Guide to Protein
Purification" in Methods in Enzymology (M. P. Deutshcer, ed.,
(1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego,
Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd
Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), and Gene
Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,
The Humana Press Inc., Clifton, N.J.). Reagents, cloning vectors,
and kits for genetic manipulation are available from commercial
vendors such as BioRad, Stratagene, Invitrogen, ClonTech and
Sigma-Aldrich Co.
[0063] Human antibodies also can be produced in transgenic animals
(for example, mice) that are capable of producing a full repertoire
of human antibodies in the absence of endogenous immunoglobulin
production. For example, it has been described that the homozygous
deletion of the antibody heavy-chain joining region (JH) gene in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of the human germ-line
immunoglobulin gene array into such germ-line mutant mice results
in the production of human antibodies upon antigen challenge. See,
for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551
(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann
et al., Year in Immuno., 7:33 (1993); U.S. Pat. Nos. 5,545,806,
5,569,825, 5,591,669 (all of GenPharm); U.S. Pat. No. 5,545,807;
and WO 97/17852. Such animals can be genetically engineered to
produce human antibodies comprising a polypeptide of the described
invention.
[0064] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, for example,
Morimoto et al., Journal of Biochemical and Biophysical Methods
24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)).
However, these fragments can now be produced directly by
recombinant host cells. Fab, Fv and ScFv antibody fragments can all
be expressed in and secreted from E. coli, thus allowing the facile
production of large amounts of these fragments. Fab'-SH fragments
can be directly recovered from E. coli and chemically coupled to
form F(ab')2 fragments (see, for example, Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab')2 fragments can be isolated directly from recombinant host
cell culture. Fab and F(ab')2 fragment with increased in vivo
half-life comprising a salvage receptor binding epitope residues
are described in U.S. Pat. No. 5,869,046. Other techniques for the
production of antibody fragments will be apparent to the skilled
practitioner.
[0065] Other techniques that are known in the art for the selection
of antibody fragments from libraries using enrichment technologies,
including but not limited to phage display, ribosome display (Hanes
and Pluckthun, 1997, Proc. Nat. Acad. Sci. 94: 4937-4942),
bacterial display (Georgiou, et al., 1997, Nature Biotechnology 15:
29-34) and/or yeast display (Kieke, et al., 1997, Protein
Engineering 10: 1303-1310) may be utilized as alternatives to
previously discussed technologies to select single chain
antibodies. Single-chain antibodies are selected from a library of
single chain antibodies produced directly utilizing filamentous
phage technology. Phage display technology is known in the art
(e.g., see technology from Cambridge Antibody Technology (CAT)) as
disclosed in U.S. Pat. Nos. 5,565,332; 5,733,743; 5,871,907;
5,872,215; 5,885,793; 5,962,255; 6,140,471; 6,225,447; 6,291650;
6,492,160; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081,
as well as other U.S. family members, or applications which rely on
priority filing GB 9206318, filed 24 May 1992; see also Vaughn, et
al. 1996, Nature Biotechnology 14: 309-314). Single chain
antibodies may also be designed and constructed using available
recombinant DNA technology, such as a DNA amplification method
(e.g., PCR), or possibly by using a respective hybridoma cDNA as a
template.
[0066] Variant antibodies also are included within the scope of the
invention. Thus, variants of the sequences recited in the
application also are included within the scope of the invention.
Further variants of the antibody sequences having improved affinity
can be obtained using methods known in the art and are included
within the scope of the invention. For example, amino acid
substitutions can be used to obtain antibodies with further
improved affinity. Alternatively, codon optimization of the
nucleotide sequence can be used to improve the efficiency of
translation in expression systems for the production of the
antibody.
[0067] The present invention provides for antibodies, either alone
or in combination with other antibodies, such as, but not limited
to, VRC01, anti-V3 loop, CD4bs, and CD4i antibodies as well as
PG9/PG16-like antibodies, that have broad neutralizing activity in
serum.
[0068] The present invention also relates to isolated polypeptides
comprising the amino acid sequences of the light chains and heavy
chains of the antibodies of the invention. In one embodiment, the
isolated polypeptide comprises the sequence of any one of SEQ ID
NOs: 1-33.
[0069] The term "polypeptide" is used in its conventional meaning,
i.e., as a sequence of amino acids. The polypeptides are not
limited to a specific length of the product. Peptides,
oligopeptides, and proteins are included within the definition of
polypeptide, and such terms can be used interchangeably herein
unless specifically indicated otherwise. This term also includes
post-expression modifications of the polypeptide, for example,
glycosylations, acetylations, phosphorylations and the like, as
well as other modifications known in the art, both naturally
occurring and non-naturally occurring. A polypeptide can be an
entire protein, or a subsequence thereof.
[0070] A polypeptide "variant," as the term is used herein, is a
polypeptide that typically differs from a polypeptide specifically
disclosed herein in one or more substitutions, deletions, additions
and/or insertions. Such variants can be naturally occurring or can
be synthetically generated, for example, by modifying one or more
of the above polypeptide sequences of the invention and evaluating
one or more biological activities of the polypeptide as described
herein and/or using any of a number of techniques well known in the
art.
[0071] For example, certain amino acids can be substituted for
other amino acids in a protein structure without appreciable loss
of its ability to bind other polypeptides (for example, antigens)
or cells. Since it is the binding capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein
sequence, and, accordingly, its underlying DNA coding sequence,
whereby a protein with like properties is obtained. It is thus
contemplated that various changes can be made in the peptide
sequences of the disclosed compositions, or corresponding DNA
sequences that encode said peptides without appreciable loss of
their biological utility or activity.
[0072] In many instances, a polypeptide variant will contain one or
more conservative substitutions. A "conservative substitution" is
one in which an amino acid is substituted for another amino acid
that has similar properties, such that one skilled in the art of
peptide chemistry would expect the secondary structure and
hydropathic nature of the polypeptide to be substantially
unchanged.
[0073] Amino acid substitutions generally are based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like.
Exemplary substitutions that take various of the foregoing
characteristics into consideration are well known to those of skill
in the art and include: arginine and lysine; glutamate and
aspartate; serine and threonine; glutamine and asparagine; and
valine, leucine and isoleucine.
[0074] In another embodiment, the present invention provides an
isolated antigen comprising an epitope-scaffold that mimics the
HIV-1 envelope spike epitope of broadly neutralizing antibody
8ANC195. On one embodiment, the epitope-scaffold comprises a
discontinous epitope and a scaffold, wherein the epitope is derived
from HIV-1 gp120 and gp41, and wherein at least part of the
scaffold is not derived from gp120 or gp41. In one embodiment, the
discontinuous epitope comprises amino acids corresponding to amino
acid numbers 44-47, 90-94, 97, 234, 236-238, 240, 274-278, 352-354,
357, 456, 463, 466, 487, and 625-641 of gp140 from HIV strain
93TH057 numbered using standard numbering for HIV strain HXBC2 as
depicted in FIG. 20 and disclosed by Korber et al. (1998, Numbering
positions in HIV relative to HXBc2, p. III-102-IV-103. In B.
Korber, C. L. Kuiken, B. Foley, B. Hahn, F. McCutchan, J. W.
Mellors, and J. Sodroski (ed.), Human retroviruses and AIDS. Los
Alamos National Laboratories, Los Alamos, N. Mex.). In another
embodiment, the amino acids corresponding to amino acid numbers 234
and 276 are glycosylated.
[0075] Methods of making epitope-scaffolds are known in the art and
disclosed, for example, by Correia et al. Journal of Molecular
Biology 405, 284 (2011), Correia et al. Structure 18, 1116 (2010),
Ofek et al. Proc Natl Acas Sci USA 107, 17780 (2010), McLellan et
al. J Mol Biol 409, 853 (2011), Azoitei et al. Science 334, 373
(2011) and in US2010/0068217. Briefly, information obtained from
the crystallographic analysis disclosed herein is used to design
epitope-scaffolds that mimic the 8ANC195 epitope on the HIV-1
envelope spike. First, computational methods are utilized to
identify non-HIV scaffold proteins capable of supporting the
discontinuous epitope identified herein. Epitope-scaffolds are then
designed and produced, and their immunological properties are
characterized. For example, in the method of Azoitei et al., the
Protein Data Bank (www.pdb.org) is searched for suitable scaffolds
for the discontinuous epitope, for example by using an algorithm
such as Multigraft Match. An algorithm such as Multigraft Design
disclosed by Azoitei et al. is used for scaffold design in which
regions of the scaffold are deleted and new segments are built to
connect the epitope to the scaffold. Candidate epitope-scaffolds
may be expressed in a host cell and purified, and tested for
binding to 8ANC195 or another antibody that binds to the epitope
recognized by 8ANC195.
[0076] The invention also includes isolated nucleic acid sequences
encoding part or all of the light and heavy chains of the described
inventive antibodies, and fragments thereof. Due to redundancy of
the genetic code, variants of these sequences will exist that
encode the same amino acid sequences. In one embodiment, the
present invention provides an isolated nucleic acid encoding a
polypeptide having the sequence of any one of SEQ ID Nos:1-33. In
another embodiment, the isolated nucleic acid encodes an antibody
comprising a heavy chain comprising the sequence of any one of SEQ
ID Nos: 1-18 and a light chain comprising the sequence of any one
of SEQ ID Nos:19-33.
[0077] The invention also includes isolated nucleic acid sequences
that encode the antigen comprising the epitope-scaffold of the
invention.
[0078] The terms "nucleic acid" and "polynucleotide" are used
interchangeably herein to refer to single-stranded or
double-stranded RNA, DNA, or mixed polymers. Polynucleotides can
include genomic sequences, extra-genomic and plasmid sequences, and
smaller engineered gene segments that express, or can be adapted to
express polypeptides.
[0079] An "isolated nucleic acid" is a nucleic acid that is
substantially separated from other genome DNA sequences as well as
proteins or complexes such as ribosomes and polymerases, which
naturally accompany a native sequence. The term encompasses a
nucleic acid sequence that has been removed from its naturally
occurring environment, and includes recombinant or cloned DNA
isolates and chemically synthesized analogues or analogues
biologically synthesized by heterologous systems. A substantially
pure nucleic acid includes isolated forms of the nucleic acid.
Accordingly, this refers to the nucleic acid as originally isolated
and does not exclude genes or sequences later added to the isolated
nucleic acid by the hand of man.
[0080] A polynucleotide "variant," as the term is used herein, is a
polynucleotide that typically differs from a polynucleotide
specifically disclosed herein in one or more substitutions,
deletions, additions and/or insertions. Such variants can be
naturally occurring or can be synthetically generated, for example,
by modifying one or more of the polynucleotide sequences of the
invention and evaluating one or more biological activities of the
encoded polypeptide as described herein and/or using any of a
number of techniques well known in the art.
[0081] Modifications can be made in the structure of the
polynucleotides of the described invention and still obtain a
functional molecule that encodes a variant or derivative
polypeptide with desirable characteristics. When it is desired to
alter the amino acid sequence of a polypeptide to create an
equivalent, or even an improved, variant or portion of a
polypeptide of the invention, one skilled in the art typically will
change one or more of the codons of the encoding DNA sequence.
[0082] Typically, polynucleotide variants contain one or more
substitutions, additions, deletions and/or insertions, such that
the immunogenic binding properties of the polypeptide encoded by
the variant polynucleotide is not substantially diminished relative
to a polypeptide encoded by a polynucleotide sequence specifically
set forth herein.
[0083] In some embodiments, the polypeptide encoded by the
polynucleotide variant or fragment has the same binding specificity
(i.e., specifically or preferentially binds to the same epitope or
HIV strain) as the polypeptide encoded by the native
polynucleotide. In some embodiments, the described polynucleotides,
polynucleotide variants, fragments and hybridizing sequences,
encode polypeptides that have a level of binding activity of at
least about 50%, at least about 70%, and at least about 90% of that
for a polypeptide sequence specifically set forth herein.
[0084] The polynucleotides of the described invention, or fragments
thereof, regardless of the length of the coding sequence itself,
can be combined with other DNA sequences, such as promoters,
polyadenylation signals, additional restriction enzyme sites,
multiple cloning sites, other coding segments, and the like, such
that their overall length can vary considerably. A nucleic acid
fragment of almost any length is employed. For example,
illustrative polynucleotide segments with total lengths of about
10000, about 5000, about 3000, about 2000, about 1000, about 500,
about 200, about 100, about 50 base pairs in length, and the like,
(including all intermediate lengths) are included in many
implementations of this invention.
[0085] Further included within the scope of the invention are
vectors such as expression vectors, comprising a nucleic acid
sequence according to the invention. Cells transformed with such
vectors also are included within the scope of the invention.
[0086] The present invention also provides vectors and host cells
comprising a nucleic acid of the invention, as well as recombinant
techniques for the production of a polypeptide of the invention.
Vectors of the invention include those capable of replication in
any type of cell or organism, including, for example, plasmids,
phage, cosmids, and mini chromosomes. In some embodiments, vectors
comprising a polynucleotide of the described invention are vectors
suitable for propagation or replication of the polynucleotide, or
vectors suitable for expressing a polypeptide of the described
invention. Such vectors are known in the art and commercially
available.
[0087] "Vector" includes shuttle and expression vectors. Typically,
the plasmid construct also will include an origin of replication
(for example, the ColE1 origin of replication) and a selectable
marker (for example, ampicillin or tetracycline resistance), for
replication and selection, respectively, of the plasmids in
bacteria. An "expression vector" refers to a vector that contains
the necessary control sequences or regulatory elements for
expression of the antibodies including antibody fragment of the
invention, in bacterial or eukaryotic cells.
[0088] As used herein, the term "cell" can be any cell, including,
but not limited to, that of a eukaryotic, multicellular species
(for example, as opposed to a unicellular yeast cell), such as, but
not limited to, a mammalian cell or a human cell. A cell can be
present as a single entity, or can be part of a larger collection
of cells. Such a "larger collection of cells" can comprise, for
example, a cell culture (either mixed or pure), a tissue (for
example, endothelial, epithelial, mucosa or other tissue), an organ
(for example, lung, liver, muscle and other organs), an organ
system (for example, circulatory system, respiratory system,
gastrointestinal system, urinary system, nervous system,
integumentary system or other organ system), or an organism (e.g.,
a bird, mammal, or the like).
[0089] Polynucleotides of the invention may synthesized, whole or
in parts that then are combined, and inserted into a vector using
routine molecular and cell biology techniques, including, for
example, subcloning the polynucleotide into a linearized vector
using appropriate restriction sites and restriction enzymes.
Polynucleotides of the described invention are amplified by
polymerase chain reaction using oligonucleotide primers
complementary to each strand of the polynucleotide. These primers
also include restriction enzyme cleavage sites to facilitate
subcloning into a vector. The replicable vector components
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, and one or
more marker or selectable genes.
[0090] In order to express a polypeptide of the invention, the
nucleotide sequences encoding the polypeptide, or functional
equivalents, may be inserted into an appropriate expression vector,
i.e., a vector that contains the necessary elements for the
transcription and translation of the inserted coding sequence.
Methods well known to those skilled in the art may be used to
construct expression vectors containing sequences encoding a
polypeptide of interest and appropriate transcriptional and
translational control elements. These methods include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Such techniques are described, for example,
in Sambrook, J., et al. (1989) Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.
M. et al. (1989) Current Protocols in Molecular Biology, John Wiley
& Sons, New York. N.Y.
[0091] According to another embodiment, the present invention
provides methods for the preparation and administration of an HIV
antibody composition that is suitable for administration to a human
or non-human primate patient having HIV infection, or at risk of
HIV infection, in an amount and according to a schedule sufficient
to induce a protective immune response against HIV, or reduction of
the HIV virus, in a human.
[0092] According to another embodiment, the present invention
provides methods for the preparation and administration of an HIV
antigen composition that is suitable for administration to a human
or non-human primate patient having HIV infection, or at risk of
HIV infection, in an amount and according to a schedule sufficient
to induce a protective immune response against HIV, or reduction of
the HIV virus, in a human.
[0093] According to another embodiment, the present invention
provides a composition comprising at least one antibody or
polypeptide of the invention and a pharmaceutically acceptable
carrier. The composition may include a plurality of the antibodies
having the characteristics described herein in any combination and
can further include antibodies neutralizing to HIV as are known in
the art. According to another embodiment, the present invention
provides a composition comprising at least one antigen of the
invention and a pharmaceutically acceptable carrier, can further
include antibodies neutralizing to HIV as are known in the art, and
can further include an adjuvant.
[0094] It is to be understood that compositions can be a single or
a combination of antibodies disclosed herein, which can be the same
or different, in order to prophylactically or therapeutically treat
the progression of various subtypes of HIV infection after
vaccination. Such combinations can be selected according to the
desired immunity. When an antibody or antigen is administered to an
animal or a human, it can be combined with one or more
pharmaceutically acceptable carriers, excipients or adjuvants as
are known to one of ordinary skilled in the art. The composition
can further include broadly neutralizing antibodies known in the
art, including but not limited to, VRC01, b12, anti-V3 loop, CD4bs,
and CD4i antibodies as well as PG9/PG16-like antibodies.
[0095] Further, with respect to determining the effective level in
a patient for treatment of HIV, in particular, suitable animal
models are available and have been widely implemented for
evaluating the in vivo efficacy against HIV of various gene therapy
protocols (Sarver et al. (1993b), supra). These models include
mice, monkeys and cats. Even though these animals are not naturally
susceptible to HIV disease, chimeric mice models (for example,
SCID, bg/nu/xid, NOD/SCID, SCID-hu, immunocompetent SCID-hu, bone
marrow-ablated BALB/c) reconstituted with human peripheral blood
mononuclear cells (PBMCs), lymph nodes, fetal liver/thymus or other
tissues can be infected with lentiviral vector or HIV, and employed
as models for HIV pathogenesis. Similarly, the simian immune
deficiency virus (SIV)/monkey model can be employed, as can the
feline immune deficiency virus (FIV)/cat model. The pharmaceutical
composition can contain other pharmaceuticals, in conjunction with
a vector according to the invention, when used to therapeutically
treat AIDS. These other pharmaceuticals can be used in their
traditional fashion (i.e., as agents to treat HIV infection).
[0096] According to another embodiment, the present invention
provides an antibody-based pharmaceutical composition comprising an
effective amount of an isolated antibody of the invention, or an
affinity matured version, which provides a prophylactic or
therapeutic treatment choice to reduce infection of the HIV virus.
According to another embodiment, the present invention provides an
antigen-based pharmaceutical composition comprising an effective
amount of an isolated antigen of the invention, which provides a
prophylactic or therapeutic treatment choice to reduce infection of
the HIV virus. The pharmaceutical compositions of the present
invention may be formulated by any number of strategies known in
the art (e.g., see McGoff and Scher, 2000, Solution Formulation of
Proteins/Peptides: In McNally, E. J., ed. Protein Formulation and
Delivery. New York, N.Y.: Marcel Dekker; pp. 139-158; Akers and
Defilippis, 2000, Peptides and Proteins as Parenteral Solutions.
In: Pharmaceutical Formulation Development of Peptides and
Proteins. Philadelphia, Pa.: Talyor and Francis; pp. 145-177;
Akers, et al., 2002, Pharm. Biotechnol. 14:47-127). A
pharmaceutically acceptable composition suitable for patient
administration will contain an effective amount of the antibody in
a formulation which both retains biological activity while also
promoting maximal stability during storage within an acceptable
temperature range. The pharmaceutical compositions can also
include, depending on the formulation desired, pharmaceutically
acceptable diluents, pharmaceutically acceptable carriers and/or
pharmaceutically acceptable excipients, or any such vehicle
commonly used to formulate pharmaceutical compositions for animal
or human administration. The diluent is selected so as not to
affect the biological activity of the combination. Examples of such
diluents are distilled water, physiological phosphate-buffered
saline, Ringer's solutions, dextrose solution, and Hank's solution.
The amount of an excipient that is useful in the pharmaceutical
composition or formulation of this invention is an amount that
serves to uniformly distribute the antibody throughout the
composition so that it can be uniformly dispersed when it is to be
delivered to a subject in need thereof. It may serve to dilute the
antibody or antigen to a concentration which provides the desired
beneficial palliative or curative results while at the same time
minimizing any adverse side effects that might occur from too high
a concentration. It may also have a preservative effect. Thus, for
an active ingredient having a high physiological activity, more of
the excipient will be employed. On the other hand, for any active
ingredient(s) that exhibit a lower physiological activity, a lesser
quantity of the excipient will be employed. Compositions comprising
an antigen of the invention may further comprise one or more
adjuvants.
[0097] The above described antibodies and antibody compositions,
comprising at least one or a combination of the antibodies
described herein, can be administered for the prophylactic and
therapeutic treatment of HIV viral infection.
[0098] The above described antigens and antigen compositions,
comprising at least one or a combination of the antigens described
herein, can be administered for the prophylactic and therapeutic
treatment of HIV viral infection.
[0099] The present invention also provides kits useful in
performing diagnostic and prognostic assays using the antibodies,
polypeptides and nucleic acids of the present invention. Kits of
the present invention include a suitable container comprising an
HIV antibody, an antigen, a polypeptide or a nucleic acid of the
invention in either labeled or unlabeled form. In addition, when
the antibody, antigen, polypeptide or nucleic acid is supplied in a
labeled form suitable for an indirect binding assay, the kit
further includes reagents for performing the appropriate indirect
assay. For example, the kit may include one or more suitable
containers including enzyme substrates or derivatizing agents,
depending on the nature of the label. Control samples and/or
instructions may also be included. The present invention also
provides kits for detecting the presence of the HIV antibodies or
the nucleotide sequence of the HIV antibody of the present
invention in a biological sample by PCR or mass spectrometry.
[0100] "Label" as used herein refers to a detectable compound or
composition that is conjugated directly or indirectly to the
antibody so as to generate a "labeled" antibody. A label can also
be conjugated to a polypeptide and/or a nucleic acid sequence
disclosed herein. The label can be detectable by itself (for
example, radioisotope labels or fluorescent labels) or, in the case
of an enzymatic label, can catalyze chemical alteration of a
substrate compound or composition that is detectable. Antibodies
and polypeptides of the described invention also can be modified to
include an epitope tag or label, for example, for use in
purification or diagnostic applications. Suitable detection means
include the use of labels such as, but not limited to,
radionucleotides, enzymes, coenzymes, fluorescers,
chemiluminescers, chromogens, enzyme substrates or co-factors,
enzyme inhibitors, prosthetic group complexes, free radicals,
particles, dyes, and the like.
[0101] Methods for reducing an increase in HIV virus titer, virus
replication, virus proliferation or an amount of an HIV viral
protein in a subject are further provided. According to another
aspect, a method includes administering to the subject an amount of
an HIV antibody of the invention effective to reduce an increase in
HIV titer, virus replication or an amount of an HIV protein of one
or more HIV strains or isolates in the subject. According to
another aspect, a method includes administering to the subject an
amount of an HIV antigen of the invention effective to reduce an
increase in HIV titer, virus replication or an amount of an HIV
protein of one or more HIV strains or isolates in the subject.
[0102] According to another embodiment, the present invention
provides a method of reducing viral replication or spread of HIV
infection to additional host cells or tissues comprising contacting
a mammalian cell with the antibody, or a portion thereof, which
binds to the 8ANC195 antigenic epitope on gp120. According to
another embodiment, the present invention provides a method of
reducing viral replication or spread of HIV infection to additional
host cells or tissues comprising contacting a mammalian cell with
the antigen that mimics the 8ANC195 antigenic epitope on gp120.
[0103] According to another embodiment, the present invention
provides a method for treating a mammal infected with a virus
infection, such as, for example, HIV, comprising administering to
said mammal a pharmaceutical composition comprising the HIV
antibodies disclosed herein. According to one embodiment, the
method for treating a mammal infected with HIV comprises
administering to said mammal a pharmaceutical composition that
comprises an antibody of the present invention, or a fragment
thereof. The compositions of the invention can include more than
one antibody having the characteristics disclosed (for example, a
plurality or pool of antibodies). It also can include other HIV
neutralizing antibodies as are known in the art, for example, but
not limited to, VRC01, PG9 and b12.
[0104] Passive immunization has proven to be an effective and safe
strategy for the prevention and treatment of viral diseases. (See,
for example, Keller et al., Clin. Microbiol. Rev. 13:602-14 (2000);
Casadevall, Nat. Biotechnol. 20:114 (2002); Shibata et al., Nat.
Med. 5:204-10 (1999); and Igarashi et al., Nat. Med. 5:211-16
(1999). Passive immunization using human monoclonal antibodies
provides an immediate treatment strategy for emergency prophylaxis
and treatment of HIV.
[0105] According to another embodiment, the present invention
provides a method of inducing an HIV antigen-specific immune
response in a mammal infected with HIV or at risk of infection with
HIV comprising administering to the mammal a pharmaceutical
composition comprising the antigen of the invention.
[0106] According to another embodiment, the present invention
provides a method of inducing an HIV antigen-specific immune
response in a mammal infected with HIV or at risk of infection with
HIV comprising administering to the mammal a pharmaceutical
composition comprising a nucleic acid encoding the antigen of the
invention.
[0107] Subjects at risk for HIV-related diseases or disorders
include patients who have come into contact with an infected person
or who have been exposed to HIV in some other way. Administration
of a prophylactic agent can occur prior to the manifestation of
symptoms characteristic of HIV-related disease or disorder, such
that a disease or disorder is prevented or, alternatively, delayed
in its progression.
[0108] For in vivo treatment of human and non-human patients, the
patient is administered or provided a pharmaceutical formulation
including an HIV antibody or antigen of the invention. When used
for in vivo therapy, the antibodies and antigens of the invention
are administered to the patient in therapeutically effective
amounts (i.e., amounts that eliminate or reduce the patient's viral
burden). The antibodies or antigens are administered to a human
patient, in accord with known methods, such as intravenous
administration, for example, as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The antibodies
can be administered parenterally, when possible, at the target cell
site, or intravenously. In some embodiments, antibody is
administered by intravenous or subcutaneous administration.
Therapeutic compositions of the invention may be administered to a
patient or subject systemically, parenterally, or locally. The
above parameters for assessing successful treatment and improvement
in the disease are readily measurable by routine procedures
familiar to a physician.
[0109] For parenteral administration, the antibodies or antigens
may be formulated in a unit dosage injectable form (solution,
suspension, emulsion) in association with a pharmaceutically
acceptable, parenteral vehicle. Examples of such vehicles include,
but are not limited, water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Nonaqueous vehicles include,
but are not limited to, fixed oils and ethyl oleate. Liposomes can
be used as carriers. The vehicle may contain minor amounts of
additives such as substances that enhance isotonicity and chemical
stability, such as, for example, buffers and preservatives. The
antibodies can be formulated in such vehicles at concentrations of
about 1 mg/ml to 10 mg/ml.
[0110] The dose and dosage regimen depends upon a variety of
factors readily determined by a physician, such as the nature of
the infection, for example, its therapeutic index, the patient, and
the patient's history. Generally, a therapeutically effective
amount of an antibody or antigen is administered to a patient. In
some embodiments, the amount of antibody or antigen administered is
in the range of about 0.1 mg/kg to about 50 mg/kg of patient body
weight. Depending on the type and severity of the infection, about
0.1 mg/kg to about 50 mg/kg body weight (for example, about 0.1-15
mg/kg/dose) of antibody is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. The progress
of this therapy is readily monitored by conventional methods and
assays and based on criteria known to the physician or other
persons of skill in the art. The above parameters for assessing
successful treatment and improvement in the disease are readily
measurable by routine procedures familiar to a physician.
[0111] A dosage regimen for administration of an antigen to a
patient may be a suitable immunization regimen, including for
example at least three separate inoculations. The second
inoculation may be administered more than at least two weeks after
the first inoculation. The third inoculation may be administered at
least several months after the second administration.
[0112] Other therapeutic regimens may be combined with the
administration of the HIV antibody or antigen of the present
invention. The combined administration includes co-administration,
using separate formulations or a single pharmaceutical formulation,
and consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Such combined
therapy can result in a synergistic therapeutic effect. The above
parameters for assessing successful treatment and improvement in
the disease are readily measurable by routine procedures familiar
to a physician.
[0113] The terms "treating" or "treatment" or "alleviation" are
used interchangeably and refer to both therapeutic treatment and
prophylactic or preventative measures; wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. A subject or mammal is
successfully "treated" for an infection if, after receiving a
therapeutic amount of an antibody or antigen according to the
methods of the present invention, the patient shows observable
and/or measurable reduction in or absence of one or more of the
following: reduction in the number of infected cells or absence of
the infected cells; reduction in the percent of total cells that
are infected; and/or relief to some extent, one or more of the
symptoms associated with the specific infection; reduced morbidity
and mortality, and improvement in quality of life issues. The above
parameters for assessing successful treatment and improvement in
the disease are readily measurable by routine procedures familiar
to a physician.
[0114] The term "therapeutically effective amount" refers to an
amount of an antibody or a drug effective to treat a disease or
disorder in a subject or mammal.
[0115] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0116] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers that are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include, but not limited to,
buffers such as phosphate, citrate, and other organic acids;
antioxidants including, but not limited to, ascorbic acid; low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as, but not limited to, serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as, but not limited to,
polyvinylpyrrolidone; amino acids such as, but not limited to,
glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides, and other carbohydrates including,
but not limited to, glucose, mannose, or dextrins; chelating agents
such as, but not limited to, EDTA; sugar alcohols such as, but not
limited to, mannitol or sorbitol; salt-forming counterions such as,
but not limited to, sodium; and/or nonionic surfactants such as,
but not limited to, TWEEN; polyethylene glycol (PEG), and
PLURONICS.
[0117] According to another embodiment, the present invention
provides diagnostic methods. Diagnostic methods generally involve
contacting a biological sample obtained from a patient, such as,
for example, blood, serum, saliva, urine, sputum, a cell swab
sample, or a tissue biopsy, with an HIV antibody and determining
whether the antibody preferentially binds to the sample as compared
to a control sample or predetermined cut-off value, thereby
indicating the presence of the HIV virus.
[0118] According to another embodiment, the present invention
provides methods to detect the presence of the HIV antibodies of
the present invention in a biological sample from a patient.
Detection methods generally involve obtaining a biological sample
from a patient, such as, for example, blood, serum, saliva, urine,
sputum, a cell swab sample, or a tissue biopsy and isolating HIV
antibodies or fragments thereof, or the nucleic acids that encode
an HIV antibody, and assaying for the presence of an HIV antibody
in the biological sample. Also, the present invention provides
methods to detect the nucleotide sequence of an HIV antibody in a
cell. The nucleotide sequence of an HIV antibody may also be
detected using the primers disclosed herein. The presence of the
HIV antibody in a biological sample from a patient may be
determined utilizing known recombinant techniques and/or the use of
a mass spectrometer.
[0119] According to another embodiment, the present invention
provides methods for detecting or isolating an HIV-1 binding
antibody in a subject comprising obtaining a biological sample from
the subject, contacting said sample with the antigen of the
invention, and conducting an assay to detect or isolate an HIV-1
binding antibody.
[0120] The term "assessing" includes any form of measurement, and
includes determining if an element is present or not. The terms
"determining", "measuring", "evaluating", "assessing" and
"assaying" are used interchangeably and include quantitative and
qualitative determinations. Assessing may be relative or absolute.
"Assessing the presence of" includes determining the amount of
something present, and/or determining whether it is present or
absent. As used herein, the terms "determining," "measuring," and
"assessing," and "assaying" are used interchangeably and include
both quantitative and qualitative determinations.
[0121] Where a value of ranges is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range is encompassed within the invention. The
upper and lower limits of these smaller ranges which may
independently be included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either both of those included limits
are also included in the invention.
Example 1
[0122] This example describes materials and methods used in
EXAMPLES 2-5 below.
Protein Expression and Purification
[0123] The antibodies used in this study were produced and purified
as in previously-described studies (Diskin et al., Science 334,
1289 (2011).) Briefly, 8ANC195, 3BNC60 and chimeric antibody
(mature HC/various LC; /.kappa. combinations of newly isolated
8ANC195 variants) IgGs were expressed by transiently transfecting
HEK293-6E cells with vectors containing the appropriate heavy and
light chain genes. Secreted IgGs were purified from cell
supernatants using protein A affinity chromatography (GE
Healthcare). For neutralization assays, IgGs were diluted to 1
mg/mL stocks in 20 mM Tris pH 8.0, 150 mM sodium chloride (TBS
buffer). 8ANC195 Fab was expressed with a 6.times.-His tag on the
C-terminus of CH1 as described for IgGs and purified using
Ni.sup.2+-NTA affinity chromatography (GE Healthcare) and Superdex
200 16/60 size exclusion chromatography (GE Healthcare).
[0124] A truncated gp120 from the HIV-1 strain 93TH057 containing
mutations Asn88Gln.sub.gp120, Asn289Gln.sub.gp120,
Asn334Gln.sub.gp120, Asn392Gln.sub.gp120, Asn448Gln.sub.gp120 was
produced by transiently transfecting HEK293-S (GnTI-/-) cells
adapted for growth in suspension by the Caltech Protein Expression
Center with a pTT5 vector encoding His-tagged gp120. Secreted gp120
was captured on Ni.sup.2+-NTA resin (GE Healthcare) and further
purified using Superdex 200 16/60 size exclusion chromatography (GE
Healthcare).
[0125] Soluble CD4 domains 1 and 2 (sCD4) and sCD4.sub.K75T were
produced as described previously (Diskin et al. Nat Struct Mol Biol
17, 608 (2010)). Briefly, the pACgp67b vector encoding
6.times.-His-tagged sCD4 or sCD4.sub.K75T (residues 1-186 of mature
CD4) was used to make infectious baculovirus particles using
BaculoGold (BD Biosynthesis). Protein was expressed in Hi5 cells,
captured on a Ni.sup.2+-NTA column (GE Healthcare) and further
purified using Superdex 200 16/60 size exclusion chromatography (GE
Healthcare). To remove an N-linked glycan introduced by mutation in
sCD4.sub.K75T, the protein was treated with Endoglycosidase H (New
England Biolabs) for 16 hours at 25.degree. C. and then purified by
Superdex 200 16/60 size exclusion chromatography (GE
Healthcare).
[0126] For complex crystallization trials, purified 8ANC195 Fab,
93TH057 gp120 and EndoH-treated sCD4.sub.K75T were incubated at a
1:1:1 molar ratio for 2 hours at 25.degree. C. The complex was
purified by Superdex 200 10/300 size exclusion chromatography (GE
Healthcare) and the peak corresponding to 8ANC195
Fab/gp120/sCD4.sub.K75T complex concentrated to 16 mg/mL in TBS
buffer. For crystallization of 8ANC195 Fab alone, the protein was
concentrated to 20 mg/mL in TBS buffer.
[0127] Purified BG505 SOSIP trimers (Julien et al., PLoS pathogens
9, e1003342 (2013); Lyumkis et al., Science 342, 1484 (2013);
Sanders et al., PLoS pathogens 9, e1003618 (2013)) for EM studies
were the gift of Dr. John P. Moore (Weill Cornell Medical
College).
Crystallization
[0128] Crystallization conditions were screened using vapor
diffusion in sitting drops set using a Mosquito.RTM.
crystallization robot (TTP labs) in a final volume of 200 nL per
drop (1:1 protein to reservoir ratio) utilizing commercially
available crystallization screens (Hampton Research, Microlytic).
Initial crystallization hits for 8ANC195 Fab and for 8ANC195
Fab/93TH057 gp120/sCD4.sub.K75T complex were identified using the
MCSG-1 (Microlytic) and PEGRx (Hampton) screens and then manually
optimized. Crystals of 8ANC195 Fab (space group P41212, a=66.5
.ANG., b=66.5 .ANG., c=219.0 .ANG.; one molecule per asymmetric
unit) were obtained upon mixing a protein solution at 11 mg/mL with
0.1M Hepes pH 7, 20% PEG 6,000, 10 mM zinc chloride at 20.degree.
C. Crystals were briefly soaked in mother liquor solution
supplemented with 20% ethylene glycol before flash cooling in
liquid nitrogen. Crystals of the 8ANC195 Fab/93TH057
gp120/sCD4.sub.K75T complex (space group P212121, a=66.5 .ANG.,
b=132.5 .ANG., c=142.8 .ANG.; one molecule per asymmetric unit)
were obtained upon mixing a protein solution at 16 mg/mL with 14%
polyethylene glycol 3,350, 0.1 M HEPES pH 7.3, 2% benzamidine HCl
at 20.degree. C. Crystals were briefly soaked in mother liquor
solution supplemented with 30% ethylene glycol before flash cooling
in liquid nitrogen.
Crystallographic Data Collection, Structure Solution and
Refinement
[0129] X-ray diffraction data for 8ANC195 Fab crystals were
collected at the Argonne National Laboratory Advanced Photon Source
(APS) beamline 23-ID-D using a MAR 300 CCD detector. X-ray
diffraction data for 8ANC195 Fab/93TH057 gp120/sCD4.sub.K75T
complex crystals were collected at the Stanford Synchrotron
Radiation Lightsource (SSRL) beamline 12-2 using a Pilatus 6M pixel
detector (Dectris). The data were indexed, integrated and scaled
using XDS (Kabsch, Acta Crystallogr D Biol Crystallogr 66, 133
(2010)).
[0130] The 8ANC195 Fab structure was solved by molecular
replacement using Phenix (Adams et al., Acta Crystallogr D Biol
Crystallogr 66, 213 (2010)) and the V.sub.HV.sub.L and
C.sub.H1C.sub.L domains of NIH45-46 Fab (PDB code 3U7W) lacking all
CDR loops as two separate search models. The model was then refined
to 2.13 .ANG. resolution using an iterative approach involving
refinement and verification of model accuracy with simulated
annealing composite omit maps using the Phenix crystallography
package, and manually fitting models into electron density maps
using Coot (Emsley et al., Acta Crystallogr D Biol Crystallogr 60,
2126 (2004). The final model (R.sub.work=21.4%; R.sub.free=25.7%)
includes 3,279 protein atoms and 127 water molecules as shown in
Table 1.
TABLE-US-00003 TABLE 1 8ANC195 Fab/ gp120/sCD4 complex 8ANC195 Fab
Data collection Resolution range (.ANG.) 39.22-3.0 (3.22-3.0)
29.73-2.1 (2.21-2.1) Space group P 2.sub.1 2.sub.1 2.sub.1 P
4.sub.1 2.sub.1 2 Cell dimensions a, b, c (.ANG.) 66.53, 132.49,
142.77 66.48, 66.48, 219.03 .alpha., .beta., .gamma. (.degree.) 90,
90, 90 90, 90, 90 Total reflections 229212 (12539) 239217 (24708)
Unique reflections 36730 (3064) 28097 (2788) Multiplicity 6.2 (6.3)
8.4 (8.9) Completeness (%) 97.65 (99.80) 98.92 (91.00) Mean
I/.sigma.(I) 7.86 (2.1) 11.90 (3.16) Wilson B-factor 61.95 32.47
R.sub.merge 0.1747 (0.765) 0.1225 (0.5802) CC1/2 0.996 (0.854)
0.996 (0.876) CC* 0.999 (0.960) 0.999 (0.966) Refinement
R.sub.work/R.sub.free 0.2655/0.3149 0.2431/0.2772 Number of atoms
7272 3311 Protein 6881 3311 Ligands 391 0 Water 0 0 Protein
residues 939 437 RMS (bonds) 0.023 0.008 RMS (angles) 1.33 1.17
Clashscore 22.78 12.43 Average B-factor 81.5 36.5 Protein 81 36.5
Ligands 89 -- Water -- -- Statistics for the highest-resoluton
shell are shown in parentheses.
[0131] 96.75%, 2.78% and 0.0% of the residues were in the favored,
allowed and disallowed regions, respectively, of the Ramachandran
plot. Disordered residues that were not included in the model
include residues 146-153, 233-238 and the 6.times.-His tag of the
8ANC195 heavy chain, and residues 214-215 of the light chain.
[0132] The 8ANC195 Fab/93TH057 gp120/sCD4.sub.K75T complex
structure was solved by molecular replacement using Phaser (Adams
et al., Acta Crystallogr D Biol Crystallogr 66, 213 (2010)) and the
V.sub.HV.sub.L and C.sub.H1C.sub.L domains of 8ANC195 (lacking all
CDR loops), 93TH057 gp120 (taken from PDB code 3U7Y), and sCD4
(taken from PDB code 3LQA) as separate search models. The complex
structure was refined to 3.0 .ANG. resolution as described for the
Fab structure. In addition to considering l/.sigma..sub.l and
completeness of the highest resolution shell (2.1% and 99.9%,
respectively), CC.sub.1/2 statistic (Karplus et al., Science 336,
1030 (2012)) (correlation coefficient between two random halves of
the data set where CC.sub.1/2>10%) was used to determine the
high-resolution cutoff for the data. Phenix was used to compute
CC.sub.1/2 (85.4% for the highest resolution shell and 99.8% for
the entire data set), supporting our high-resolution cutoff
determination.
[0133] The final model (R.sub.work=23.4%; R.sub.free 28.6%)
includes x protein atoms and y atoms of carbohydrates (Table SI).
96.2%, 3.8% and 0.0% of the residues were in the favored, allowed
and disallowed regions, respectively, of the Ramachandran plot.
Disordered residues that were not included in the model include
residues 146-153, 206-208, 233-238 and the 6.times.-His tag of the
8ANC195 heavy chain, residues 213-215 of the light chain, residues
125-197 (V1/V2 substitution), 302-324 (V3 substitution), residues
397-409 (a total of 6 residues from V4), residues 492-494 and the
6.times.-His tag of 93TH057 gp120 and residues 106-111, 154-155,
177-186 of sCD4.sub.K75T.
[0134] Buried surface areas were calculated using PDBePISA
(Krissinel et al., Journal of molecular biology 372, 774 (2007))
and a 1.4 .ANG. probe. Superimposition calculations were done and
molecular representations were generated using PyMol (Schrodinger
(The PyMOL Molecular Graphics System, 2011). Pairwise Ca alignments
were performed using PDBeFold (Krissinel et al., Acta Crystallogr D
Biol Crystallogr 60, 2256 (2004)).
ELISAs
[0135] High-binding 96-well ELISA plates (Costar) were coated
overnight with 5 .mu.g/well of purified gp120 in 100 mM sodium
carbonate pH 9.6. After washing with TBS containing 0.05% Tween 20,
the plates were blocked for 2 h with 1% BSA, 0.05% Tween-TBS
(blocking buffer) and then incubated for 2 h with 8ANC195 IgG (1
.mu.g/mL) mixed with 1:2 serially diluted solutions of potential
antibody competitors (sCD4, J3 VHH, 3BNC60 Fab, NIH45-46 Fab) in
blocking buffer (competitor concentration range from 5 to 320
.mu.g/mL). After washing with TBS containing 0.05% Tween 20, the
plates were incubated with HRP-conjugated goat anti-human IgG
antibodies (Jackson ImmunoReseach) (at 0.8 .mu.g/ml in blocking
buffer) for 1 hour. The ELISAs were developed by addition of HRP
chromogenic substrate (TMB solution, BioLegend) and the color
development stopped by addition of 10% sulfuric acid. Experiments
were performed in duplicate.
Surface Plasmon Resonance
[0136] Experiments were performed using a Biacore T100 (Biacore)
using a standard single-cycle kinetics method. YU-2 and 93TH057
gp120 proteins were primary amine-coupled on CMS chips (Biacore) at
a coupling density of 1,000 RUs and one flow cell was mock coupled
using HBS-EP+ buffer. 8ANC195 and chimeric IgGs were injected over
flow cells at increasing concentrations (62.5 to 1,000 nM), at flow
rates of 20 .mu.l/min with 5 consecutive cycles of 2 min
association/1 min dissociation and a final 10 min dissociation
phase. Flow cells were regenerated with 3 pulses of 10 mM glycine
pH 2.5. Apparent binding constants (K.sub.D (M)) were calculated
from single-cycle kinetic analyses after subtraction of backgrounds
using a 1:1 binding model without a bulk reflective index (RI)
correction (Biacore T100 Evaluation software). Binding constants
for bivalent IgGs are referred to as "apparent" affinities to
emphasize that the K.sub.D values include potential avidity
effects.
Neutralization Assays
[0137] A TZM-bl/pseudovirus neutralization assay was used to
evaluate the neutralization potencies of the antibodies as
described (Montefiori, Current protocols in immunology edited by
John E. Coligan et al., Chapter 12, Unit 12 11 (2005)).
Pseudoviruses were generated by cotransfection of HEK 293T cells
with an Env expression plasmid and a replication-defective backbone
plasmid. Neutralization was determined by measuring the reduction
in luciferase reporter gene expression in the presence of antibody
following a single round of pseudovirus infection in TZM-bl cells.
Nonlinear regression analysis was used to calculate the
concentrations at which half-maximal inhibition was observed
(IC.sub.50 values).
Negative-Stain EM
[0138] The BG505 SOSIP.664/8ANC195 Fab complex and grids were
prepared as described previously (Kong et al., Nat Struct Mol Biol
20, 796 (2013). The data were collected on an FEI Tecnai T12
electron microscope coupled with a Tietz TemCam-F416 4 k.times.4 k
CMOS camera using the LEGINON interface. Images were collected in
10.degree. increments from 0.degree. to -40.degree. using a defocus
range of 0.6-0.9 .mu.m at a magnification of 52,000.times.,
resulting in a pixel size of 2.05 .ANG. at the specimen plane.
Particles were selected using DogPicker (Voss et al., Journal of
structural biology 166, 205 (2009)) within the Appion software
package (Lander et al., Journal of structural biology 166, 95
(2009)), and sorted from reference-free 2D class averages using the
SPARX package (Penczek et al., Ultramicroscopy 40, 33 (1992). An
initial model was generated by common lines from class averages
using the EMAN2 package (Tang et al., Journal of structural biology
157, 38 (2007) and was refined using 11,637 unbinned particles. The
refinement was carried out using the SPARX package (Penczek et al.,
Ultramicroscopy 53, 251 (1994)) with C3 symmetry applied. The
resulting resolution at a 0.5 Fourier Shell Correlation (FSC)
cut-off was 18.7 .ANG. (FIGS. 6A,B).
Human Samples
[0139] Human samples were collected after signed informed consent
in accordance with Institutional Review Board (IRB)-reviewed
protocols by all participating institutions. Patient 8 was selected
from a cohort of elite controllers that were followed at the Ragon
Institute in Boston.
Isolation of 8ANC195 Variants
[0140] Single Cell clonal variants of 8ANC195 were isolated by 2CC
core-specific single cell sorting, followed by reverse
transcription and immunoglobulin gene amplification as described
previously (Scheid et al., Science 333, 1633 (2011)).
Immunoglobulin genes were cloned into heavy and light chain
expression vectors and co-transfected for IgG production as
described previously (Tiller et al., Journal of immunological
methods 329, 112 (2008)).
[0141] IgG+CD19+ memory B cells were bulk sorted on a FACS AriaIII
cell sorter. Bulk mRNA was extracted using TRIzol (Invitrogen) and
reverse transcribed as previously described (Scheid et al., Science
333, 1633 (2011)). 8ANC195-related heavy and light chain genes were
PCR amplified using the following clone-specific primers:
TABLE-US-00004 For heavy chain amplification: (SEQ ID NO: 50) 5'
GGTGTACATTCTCAGATACACCTCGTACAA 3' and (SEQ ID NO: 51) 5'
CAGGTGTCCAGTCTCAGATACA 3' as forward primers and (SEQ ID NO: 57) 5'
GCGGAGACGGAGATGAGGGTT 3' as a reverse primer. For light chain
amplification: (SEQ ID NO: 52) 5' GACATCCAGATGACCCAGTCTCCTTCCA
CCCTGTCTGCATCTATAGGT 3' and (SEQ ID NO: 53) 5'
GACATCCAGATGACCCAGTCTCCTTCCA CCCTGTCTGCATCT 3' as forward and (SEQ
ID NO: 58) 5' GTTTCACCTCAACTTTAGTCCCTT 3' as well as (SEQ ID NO:
59) 5' GTTTCACCTCAACTTTAGTCCCTTGGCCGAAGGTC 3' as reverse
primers.
[0142] Amplification products were gel purified and cloned into
TOPO TA sequencing vectors (Invitrogen) and expression vectors as
described previously (T. Tiller et al., Journal of immunological
methods 329, 112 (2008)).
Phylogenetic Tree and Alignment Assembly.
[0143] Phylogenetic trees were assembled using Geneious
Neighbor-Joining Tree Software. Sequence Alignments were performed
using DNA Star Clustal W alignment software.
Computational Analysis.
[0144] The program AntibodyDatabase (West, Jr. et al., Proceedings
of the National Academy of Sciences of the United States of America
110, 10598 (2013)) was used to analyze 8ANC195 neutralization panel
data from Scheid et al., Science 333, 1633 (2011) and Chuang et
al., Journal of virology 87, 10047 (2013). This method attempts to
model the variation in neutralization potency across strains based
on a sum of terms ("rules") corresponding to specific residues or
potential N-linked glycosylation site (PNGS) positions. With the
free residual option deselected, the analysis finds a rule
corresponding to .about.3-fold better 8ANC195 neutralization for
strains with Glu632.sub.gp41. This correlation appears to hold
across clades based on neutralization data for strains having the
most favorable glycosylation pattern (PNGS at 234.sub.gp120 and
276.sub.gp120, and not at 230.sub.gp120) (22). For all clades, the
residue at 632.sub.gp41 versus geometric mean IC.sub.50s for
8ANC195 on strains with the most favorable glycosylation pattern
was as follows: Glu, 0.43 .mu.g/mL (n=53) versus Asp, 1.31 .mu.g/mL
(n=51). For separate clades, the correlations were Clade A: Glu,
0.47 .mu.g/mL (n=3); Asp, 1.30 .mu.g/mL (n=24); Clade B: Glu, 0.18
.mu.g/mL (n=15); Asp, 0.72 .mu.g/mL (n=6); Clade C: Glu, 0.32
.mu.g/mL (n=2); Asp, 1.31 .mu.g/mL (n=20).
Statistical Analysis of Neutralization Potencies of 8ANC195
Variants
[0145] IC.sub.50 values derived from neutralization assays with
8ANC195 and its 52.sub.HC/.kappa.5.sub.LC variant against 11
sensitive virus strains (IC.sub.50<50) were analyzed by G-test
for the relationship between the amino acid identity at position
636.sub.gp41 and the antibody IC.sub.50. For each antibody the
partition between "high" and "low" IC.sub.50s was chosen such that
approximately half of the strains had high IC.sub.50s (0.8 .mu.g/mL
for 8ANC195, 0.1 .mu.g/mL for 52.sub.HC/.kappa.5.sub.LC.
Example 2
[0146] Determination of the Crystal Structure of the Fab Fragment
of 8ANC195 Alone and Complexed with HIV-1 gp120 Core and CD4
Domains 1-2 (sCD4)
[0147] To determine the epitope recognized by 8ANC195 and
investigate its neutralization mechanism, crystal structures were
solved of the Fab fragment of 8ANC195 alone and complexed with an
HIV-1 clade A/E 93TH057 gp120 core and CD4 domains 1-2 (sCD4) at
1.9 .ANG. and 2.9 .ANG. resolution, respectively (FIG. 16A,B; Table
1). Five PNGSs on the core gp120 were removed by mutation
(Asn88Gln.sub.gp120, Asn289Gln.sub.gp120, Asn334Gln.sub.gp120,
Asn392Gln Asn448Gln.sub.gp120) to reduce glycan heterogeneity.
[0148] Comparison of 8ANC195 Fab in its free versus gp120-bound
states revealed high structural similarity (RMSD=0.7 .ANG. for 236
Ca atoms of V.sub.H-V.sub.L) except for a 3.5 .ANG. displacement of
the loop connecting strands D and E in HC FWR3 (FIG. 16A). The
CDRH1 and CDRH3 loops were folded into hook-like tertiary
structures in free and gp120-bound Fabs; therefore the
conformations were not induced upon binding to gp120 (FIG. 16A and
FIGS. 2A,B). The CDRH3 architecture differed from CDRH3s in other
antibodies including anti-HIV-1 antibodies with long CDR loops
(FIG. 2C). The CDRH1 loop conformation was stabilized by a hydrogen
bond network among backbone atoms of CDRH1, burial of Phe30.sub.HC,
and hydrogen bonds with Asp73.sub.HC and Thr104.sub.HC (FIG. 2A).
CDRH3 had a complex tertiary structure in which residues
102.sub.HC-110.sub.HC formed a loop protruding .about.10 .ANG. from
the antibody surface, and residues 111.sub.HC-118.sub.HC formed a
(3-sheet subdomain that was stabilized by hydrophobic stacking
between His113.sub.HC and Trp33.sub.LC and a hydrogen bond between
Met117.sub.HC and Gln90.sub.LC (FIG. 2B). The side chain of
Tyr92.sub.LC hydrogen bonded with the Gly110.sub.HC carbonyl
oxygen, stabilizing a kink in the loop that formed the transition
between these secondary structure elements (FIG. 2B).
[0149] The complex structure showed independent binding of sCD4 and
8ANC195 Fab to distinct sites on gp120 (FIG. 16B). sCD4 interacted
with the gp120 core as in other sCD4-gp120 structures (Kwong et
al., Nature 393, 648 (1998)) (FIG. 3A), thus its binding was not
altered by the presence of the adjacent antibody, consistent with
binding and neutralization experiments showing no effects of CD4
addition on 8ANC195 activity (FIG. 3B,C). sCD4 did, however,
contribute to crystal packing (FIG. 3D), rationalizing why
diffraction-quality crystals failed to grow in its absence. In the
ternary complex structure, 8ANC195 bound to a gp120 region adjacent
to the CD4 binding site, contacting mainly the gp120 inner domain,
loops D and V5, and a small patch of the gp120 outer domain
(His352.sub.gp120-Asn354.sub.gp120) (FIG. 16B,C).
[0150] 8ANC195 Fab bound gp120 core exclusively with its HC, using
residues in FWRs and its three CDR loops to form an extensive
interface (3,671 .ANG..sup.2 total buried surface area; 1287
.ANG..sup.2 HC-gp120 protein contacts; 2,384 .ANG..sup.2 HC-gp120
glycan contacts) (FIG. 16B, 2, FIG. 4; Table 2).
TABLE-US-00005 TABLE 2 Buried Surface Area (BSA) at interfaces
Hydrogen Bonds at Interfaces gp120 BSA (.ANG..sup.2) 8ANC195 HC BSA
(.ANG..sup.2) gp120 8ANC195 HC Distance (.ANG.) VAL 44 19.4 ASN 28
28.6 THR 278 O.gamma.1 THR 75 O 2.43 TRP 45 17.4 THR 29 22.9 ARG
456 NH2 GLY 76 O 3.37 LYS 46 35.1 GLY 31 26.3 ASN 354 N.delta.2 SER
77 O.gamma. 2.88 ASP 47 39.7 LEU 32 56.0 THR 278 O.gamma.1 SER 78 O
3.34 THR 95 39.1 ARG 54 54.5 ASN 92 N.delta.2 THR 104 O 2.88 GLU 91
5.7 TRP 55 4.2 ASN 92 N.delta.2 TYR 105 O 3.08 ASN 92 87.3 LYS 56
4.4 HIS 352 O THR 75 O.gamma.1 3.14 PHE 93 9.0 LEU 74 65.7 ASN 354
O.delta.1 THR 75 O.gamma.1 3.08 ASN 94 38.0 THR 75 44.3 ASP 47
O.delta.2 TYR 105 OH 3.09 LYS 97 7.2 GLY 76 81.0 LYS 487 N.zeta.
TYR 105 OH 3.49 THR 236 27.7 SER 77 33.9 GLY 237 21.1 SER 78 5.5
PRO 238 51.6 PRO 79 3.9 LYS 240 4.1 THR 104 11.7 SER 274 0.2 TYR
105 100.2 GLU 275 7.0 ASP 106 16.8 ASN 276 15.9 LYS 107 24.0 LEU
277 37.8 TRP 108 70.3 THR 278 69.6 Total 8ANC195 HC 653.9 HIS 352
17.0 PHE 353 10.0 ASN 354 48.1 LYS 357 3.5 ARG 456 13.8 THR 463 0.2
GLU 466 0.1 LYS 487 6.0 Total gp120 633.4 gp120 BSA (.ANG.2)
8ANC195 NC BSA (.ANG..sup.2) gly276 NAG.sup.1 121.2 TYR 25 12.8 GLY
26 36.7 VAL 27 6.5 ASN 28 15.3 LEU 74 21.2 PRO 79 4.2 gly276
NAG.sup.2 100.5 GLN 1 18.8 HIS 3 8.0 TYR 25 40.8 GLY 26 12.8 gly276
BMA.sup.3 65.5 HIS 3 35.3 VAL 5 6.5 TYR 25 19.4 gly275 MAN.sup.4
66.5 GLN 1 14.9 ILE 2 1.6 HIS 3 40.0 gly276 MAN.sup.5 45.2 VAL 5
18.4 TYR 25 18.7 Total gly276 398.8 Total 8ANC195 HC 331.8 Buried
Surface Area (BSA) at interfaces Hydrogen Bonds at Interfaces gp120
BSA (.ANG..sup.2) 8ANC195 HC BSA (.ANG..sup.2) gp120 8ANC195 HC
Distance (.ANG.) gly234 NAG.sup.1 108.4 ASN 28 1.5 gly234 NAG.sup.1
O4 TRP 55 N 1 2.99 THR 29 9.8 gly234 NAG.sup.1 O3 ASP 73 O.delta.2
3.80 TRP 55 24.9 gly234 NAG.sup.2 O6 ASP 73 N 3.13 ASP 73 29.7
gly234 MAN.sup.5 O6 VAL 72 N 2.76 LEU 74 16.8 gly234 MAN.sup.5 O6
ILE 81 O 2.64 gly234 NAG.sup.2 128.4 ARG 54 1.0 gly234 MAN.sup.5 O3
GLU 85 O 2 3.17 TRP 55 53.4 gly234 MAN.sup.5 O4 GLU 85 O 2 2.34 ALA
71 2 8 gly234 MAN.sup.10 O3 ALA 59 N 3.50 VAL 72 11.6 gly234
MAN.sup.10 O2 ALA 59 N 3.03 ASP 73 13.1 gly234 MAN.sup.10 O6 VAL 67
O 3.17 gly234 BMA.sup.3 100.6 ILE 52 9.5 gly234 MAN.sup.10 O6 GLY
65 N 2.58 TRP 55 24.5 gly234 MAN.sup.10 O2 SER 58 O 3.28 LYS 56 0.2
gly234 MAN.sup.10 O2 SEP 58 N 3.19 SEP 57 3.4 gly234 MAN.sup.10 O3
SER 57 O 2.79 ILE 69 0.8 SEP 70 10.5 ALA 71 11.4 VAL 72 10.5 gly234
MAN.sup.4 55.2 SER 70 8.8 ALA 71 5.2 VAL 72 31.0 gly234 MAN.sup.5
129.8 SEP 70 11.9 ALA 71 1.6 VAL 72 18.1 ILE 81 18.5 SER 83 18.6
gly234 MAN.sup.6 118.4 THR 19 16.7 LEU 68 24.3 SER 70 8.9 SER 83
7.6 GLU 85 25.7 gly234 MAN.sup.7 84.2 ILE 52 3.3 TRP 55 13.4 LYS 56
1.8 SER 57 20.2 LEU 88 18.2 ILE 69 3.6 SER 70 2 1 gly234 MAN.sup.8
68.3 SER 57 30.2 SEP 58 0.2 VAL 67 2.6 LEU 68 24.4 ILE 89 1.2
g1y234 MAN.sup.10 198.7 SEP 57 20.9 SER 58 11.0 ALA 59 21.8 ARG 64
14.3 GLY 6.5 14.2 VAL 67 13.3 LEU 68 10.9 ILE 69 3.5 Total gly234
992.0 Total 8ANC195 HC 661.2
[0151] A loop in FWR3.sub.HC, consisting of somatically-mutated
residues and extended by a four-residue insertion, reached like a
thumb into the pocket formed by loops D, V5 and outer domain
residues 352.sub.gp120-358.sub.gp120 (FIGS. 16A,B and 17A, FIG.
4B). CDRH1 and CDRH3 contacted the gp120 inner domain (FIG. 16B,
FIG. 4B), contributing to a 1287 .ANG..sup.2 interface between the
8ANC195 HC and gp120 protein residues. The CDRH1 and CDRH3 loop
conformations, conserved in the free Fab (FIG. 16A, FIG. 2A,B),
were necessary for binding gp120 since extending these loops would
result in clashes with gp120. The resulting antibody combining site
was exquisitely suited to contacting portions of the inner domain
of gp120 not targeted by other bNAbs (FIG. 16C).
[0152] The 8ANC195 Fab also made extensive interfaces with glycans
attached to Asn234.sub.gp120 (buried surface area=1,653
.ANG..sup.2) and Asn276.sub.gp120 (buried surface area=731
.ANG..sup.2), rationalizing its dependence on these PNGSs for
neutralization (West, Jr. et al., Proceedings of the National
Academy of Sciences of the United States of America 110, 10598
(2013); Chuang et al., Journal of virology 87, 10047 (2013)).
Together with CDRH2, somatically-mutated FWR residues in strands B,
C'', D and E contributed to an extensive interface with the
Asn234.sub.gp120-associated N-glycan (usually high mannose in
native HIV-1 Envs (Go et al., Journal of virology 85, 8270 (2011)
that involved 10 sugar moieties, including specific interactions
with terminal mannose residues (FIG. 17C,E, FIG. 4C,D). A
two-residue deletion at the CDRH2-FWR3.sub.HC boundary compared to
the germline sequence permitted these interactions, since the
longer loop would clash with inner domain residue Asn234.sub.gp120
and its neighbors. The Asn276.sub.gp120 glycan (a complex-type
N-glycan in native HIV-1 Envs (Go et al., Journal of virology 85,
8270 (2011); Binley et al., Journal of virology 84, 5637 (2010)),
but high mannose in the crystallized gp120) was wedged between
8ANC195 and sCD4, where it contacted FWR residues in strands A and
B and the N-terminal portion of CDRH1, forming an interface
involving only the core pentasaccharide common to both high mannose
and complex-type N-glycans (FIG. 17D, FIG. 4E,F).
[0153] The 8ANC195 HC was bracketed by the Asn234.sub.gp120 and
Asn276.sub.gp120 glycans in a manner analogous to interactions of
HIV-1 antibodies that penetrate the Env glycan shield, such as PG16
(interactions with Asn156.sub.gp120/Asn173.sub.gp120 and
Asn160.sub.gp120 glycans) (Pancera et al., Nature structural &
molecular biology 20, 804 (2013), PGT128 (with Asn301.sub.gp120 and
Asn332.sub.gp120 glycans) (Pejchal et al., Science 334, 1097
(2011)) and PGT121 (with Asn137.sub.gp120 and Asn332.sub.gp120
glycans) (Mouquet et al., Proceedings of the National Academy of
Sciences of the United States of America 109, E3268 (2012); Julien
et al., Science 342, 1477 (2013) Julien et al., PLoS pathogens 9,
e1003342 (2013)) (FIG. 5). However, in contrast to these
antibodies, 8ANC195 contacts with gp120 were made exclusively by
its HC; indeed, 33% of 8ANC195 V.sub.H domain residues not buried
at the LC interface contacted gp120. In summary, the 8ANC195-gp120
structure demonstrated that 8ANC195 recognizes a novel epitope
involving the Asn234.sub.gp120 and Asn276.sub.gp120 glycans, the
gp120 inner domain, loop D and loop V5, which would be adjacent to
gp41 in Env trimer (Julien et al., Science 342, 1477 (2013);
Lyumkis et al., Science 342, 1484 (2013)).
Example 3
Negative Stain Single Particle Electron Microscopy (EM) to
Determine the Structure of 8ANC195 Fab Bound to a Soluble HIV-1
SOSIP Trimer
[0154] To investigate portions of the 8ANC195 epitope beyond the
gp120 core, including potential contacts with gp41, negative stain
single particle EM was used to determine the structure of 8ANC195
Fab bound to a soluble HIV-1 SOSIP trimer derived from strain BG505
(FIG. 6) (Julien et al., Science 342, 1477 (2013); Lyumkis et al.,
Science 342, 1484 (2013); Sanders et al., PLoS pathogens 9,
e1003618 (2013)). Independent docking of the BG505 Env trimer
structure (PDB 4NCO) (Julien et al., Science 342, 1477 (2013)) and
8ANC195 Fab resulted in a model wherein the Fab contacted both
gp120 and gp41 within a single protomer (FIG. 18A, FIG. 7). The EM
model placed the CDRL1, CDRL2, and portions of FWR3.sub.LC and
CDRH3 in close proximity to the HR2 helix of gp41 (FIG. 18B).
Although gp41 residues were not definitively identified in the
trimer crystal structure (Julien et al., Science 342, 1477 (2013)),
based on the assignment of the HR2 C-terminus as Gly664.sub.gp41
(Lyumkis et al., Science 342, 1484 (2013), the kink in the HR2
helix was assigned as Asn637.sub.gp41 (FIG. 18B, FIG. 8), the
asparagine of a highly conserved PNGS. The EM model predicted that
the Asn637.sub.gp41-linked glycan and adjacent amino acid residues
on HR2 interacted with 8ANC195 CDRH3, CDRL1 and CDRL2.
[0155] Docking of the gp120-8ANC195 portion of the ternary crystal
structure onto the SOSIP trimer structure resulted in a slightly
different angular placement of the Fab in the EM density than when
the 8ANC195 Fab was fit independently (FIG. 18A, FIG. 7). The Fab,
especially the LC, was pushed further away from gp41 by comparison
to the placement suggested by the complex crystal structure. The LC
position in the EM model was more likely to be accurate since it
left space for bulky side chains at positions
625.sub.gp41-640.sub.gp41 that were modeled as alanines in the
trimer crystal structure (Julien et al., Science 342, 1477 (2013);
Lyumkis et al., Science 342, 1484 (2013)). The slightly different
placements could be due to crystal packing effects, spatial
restraints imposed by the gp41 glycans that were not present in the
8ANC195-gp120 complex, removal of the PNGS at Asn88.sub.gp120 in
the gp120 core, which may have allowed for a closer association of
8ANC195 and gp120 in the crystal structure, and/or a small
conformational change in the gp120 region of the trimer to
accommodate the Fab orientation trapped by crystallization.
Example 4
Neutralization and Binding Assays
[0156] The EM reconstruction highlighted a potential role for
8ANC195 LC contacts to gp41. To assess LC contacts with trimeric
Env, chimeras consisting of the 8ANC195 HC paired with different
Lcs were tested in neutralization and binding assays. The chimeras
included a full germline LC, a mature LC with individual CDR loops
reverted to their germline sequences or CDRL3 partially mutated to
alanines, or the LC from the CD4 binding site antibody 3BNC117
(FIG. 19A). As expected from the crystal structure in which all
gp120 contacts were made by the 8ANC195 HC, the chimeras bound
normally to gp120 core and to a full-length 93TH057 gp120 (FIG.
19B, table 3), thus changes in the LC did not disrupt the HC
portion of the antibody combining site.
TABLE-US-00006 TABLE 3 Kp (nM) IC50 (pg/mL) 93TH057 YU2 5C422
Antibody gp120 Gp120 YU2 Tro11 SF162 6535_3 6618 PV04 REJ04541
RHPA4259 8ANC195 IgG 33.1 82.0 0.4 0.31 0.30 0.43 0.69 0.52 0.248
0.17 8ANC195 38.0 56.1 6.76 >100 12.9 8.9 8.1 11.5 11.2 14.6
mHC/GILC 8ANC195 38.0 105.4 55 >100 65 24 97 >100 62.4 0.96
gICDRL1 8ANC195 40.5 119.5 6.26 18.9 44 13.3 69 97 82.9 0.82
gICDRL2 8ANC195 44.4 107.1 0.79 0.75 0.85 1.08 1.29 3.1 1.045 0.97
gICDRL3 8ANC195 ND ND 39 ND >100 >100 >100 >100 >100
0.81 CDRL3Ala 8ANC195HC/ ND ND 4.77 5.33 5.6 >100 22 ND 10.9
4.52 3BNC117LC 3BNC60 ND ND 0.027 0.04 0.05 0.335 0.07 0.06 0.063
0.02
[0157] In contrast to gp120 binding, neutralization potencies
assayed against native Env spike trimers were decreased by changes
in the 8ANC195 LC. For example, reverting CDRL1 and CDRL2 sequences
to germline precursor sequences (changing 3 of 7 and 3 of 3
residues, respectively) almost completely abrogated neutralization
of YU2, an 8ANC195-sensitive strain. Changes to CDRL3 led to a
moderate reduction in neutralization potency, as did substituting
the 3BNC117 LC for the cognate LC (FIG. 19B, table 3). A chimeric
IgG with one of the most conservatively-substituted LCs
(Thr-Gly-Asn, mature CDRL1 containing a one-residue insertion,
reverted to Ser-Ser, germline CDRL1) displayed unchanged binding to
gp120, yet showed reductions in neutralization potency of up to
250-fold. Similarly, conservative changes in CDRL2 (Arg-Gly-Ala,
the mature CDRL2, reverted to the germline Lys-Ala-Ser sequence)
caused large reductions in neutralization potencies but had little
effect on gp120 binding. Overall the data showed differential
sensitivities of the binding and neutralization assays to changes
in the 8ANC195 LC that were distant from the gp120 surface, which
supported the EM results suggesting that LC, and CDRL1 and CDRL2 in
particular, contacted gp41.
Example 5
Isolation of Antibodies
[0158] To further investigate Env recognition by 8ANC195,
additional members of this antibody clone were isolated from the
original donor by single cell sorting using gp120 stabilized in the
CD4-bound conformation (2CC core) as bait (FIG. 9). From 1536
single 2CC core-binding B cells, 10 (0.7%) were clonally related to
8ANC195, and of these, only four differed slightly from the two
previously-described members (1 to 3 and 1 to 7 residue differences
in the HCs and LCs, respectively) (FIG. 10). Consistent with the
limited sequence diversity, these antibodies exhibited similar
potencies to 8ANC195 in neutralization assays against a panel of 15
Tier 2 viruses (FIG. 9C and Table 4).
TABLE-US-00007 TABLE 4 Virus 8ANC3040 8ANC3484 8ANC3630 8ANC3044
8ANC 430 8ANG195 REJ04541.67 0.198 0.117 2.652 0.278 0.198 0.08
PVO.4 0.284 0.077 0.102 0.260 0.206 0.52 YU2.DG 0.617 0.461 0.468
0.747 0.545 0.79 3415.v1.c1 3.059 0.589 27.977 7.557 >23 2.404
3365.v2.c20 >25 >30 >30 >30 >23 >30 ZW153M.PB12
14.910 11.581 >30 15.164 >23 9.626 Z.M109F.PB4 NT >30
>30 >30 >23 >30 3016.v5.c45 0.427 0.131 0.136 0.242
0.271 0.195 231965.c1 1.174 0.294 0.375 1.332 1.190 0.514 X1254_c3
2.909 2.192 2.377 4.538 4.284 1.524 251-18 0.571 0.391 0.730 0.858
6.170 0.284 R1166.c1 2.370 1.027 1.453 2.381 3.642 0.986 H086.8 NT
0.394 0.300 3.830 >23 0.095 Du172.17 NT 4.011 >30 >30
>23 10.797 250-4 NT >30 >30 >30 >23 >50 MuLV
>30 >30 >30 >30 >23 >23
[0159] Reasoning that the 2CC core bait might fail to capture some
8ANC195 family members, clone-specific primers were used to amplify
8ANC195 variants from purified populations of CD19+ IgG+ memory B
cells (FIG. 11). 128 HC and 100 LC sequences were obtained that
were clonally related to 8ANC195 and displayed greater sequence
diversity than antibodies obtained using antigen-specific selection
(FIGS. 10, 12). Of the 13 HC and 6 LC genes exhibiting greatest
diversity, all combinations were co-transfected in order to
evaluate their neutralizing activity against a 15-member Tier 2
virus panel. 3 of 39 (7.7%) new antibodies were at least as broad
and potent as 8ANC195 (FIG. 19C and Table 5).
TABLE-US-00008 TABLE 5 .gamma.3 .gamma.4 .gamma.8 .gamma.15
.gamma.20 .gamma.22 .gamma.23 .gamma.44 .gamma.46 .gamma.52
.gamma.59 Virus .gamma.3.kappa.3 .gamma.4.kappa.3 .gamma.8.kappa.3
y15.kappa.3 .gamma.20.kappa.3 .gamma.22.kappa.3 .gamma.52.kappa.3
.gamma.59.kappa.3 REJ045451.67 0.260 >15 >15 >15 >15
>15 >15 8.543 PVO.4 0.170 >15 >15 5.316 >15 >15
1.918 >15 YU2.DG 0.420 >15 5.418 1.068 >15 >15 5.751
8.970 34>15.v1.c1 >15 >15 >15 >15 >15 >15
>15 >15 3365.v2.c20 >15 >15 >35 >15 >15 >15
>15 >15 ZM53M.PB12 >15 >15 >15 >15 >15 >15
>15 >15 ZM189F.PB4 >15 >15 >15 >15 >15 >15
>15 >15 31316.v5.c45 0.347 >15 2.383 1.265 >15 >15
3.050 >15 231965.c1 0.727 >15 3.477 1.611 >15 >15 6.594
>15 X1254_c3 1.822 18.047 4.609 3.487 14.702 >15 >15 3.392
251-18 >15 >15 >15 >15 215 >15 14.537 >15
R1166.c1 2.208 >15 7.596 3.943 >15 >15 23.408 24.226
H086.8 3.307 >15 >15 >15 >15 >15 >15 >15
Du172.17 >15 >15 >15 >15 >15 >15 >15 >15
250-4 >15 >15 >15 >15 >15 >15 >15 >15 MuLV
>30 NT >13 >30 >21 NT >30 >30 Virus
.gamma.3.kappa.5 .gamma.22.kappa.5 .gamma.23.kappa.5
.gamma.46.kappa.5 .gamma.52.kappa.5 .gamma.59.kappa.5 REJO4541.67
0.097 0.795 0.091 0.196 0.035 4.669 PVO.4 0.081 0.352 0.043 0.129
0.018 >15 YU2. DG 0.200 0.569 0.135 0.259 0.065 7.279 34
>15.v1.c1 >15 >15 1.479 >15 0.120 >15 3365.v2.C.20
>15 >15 >16 >15 >15 >15 ZM53M.PB12 >15 >15
5.676 12.482 3.134 24.057 ZM109F.PB4 >15 >15 >15 >15
>15 >15 3016.v5.c45 0.314 0.103 0.091 0.111 0.017 >15
231965.cl 0.697 0.680 0.291 0.526 0.094 >15 X1254_c3 1.717 1.521
0.919 1.792 0.584 9.416 251-18 0.721 1.696 0.176 0.609 0.048 6.969
R1166.c1 2.074 2.395 1 075 0.922 0.319 28.201 H086.8 0.434 >15
0.474 1.750 0.175 >15 Du172.11 3.728 >15 1.814 >15 NT
>15 250-4 >15 >15 >15 >15 >15 >15 MuLV >15
>30 >30 NT NT >30 Virus .gamma.3.kappa.11
.gamma.8.kappa.11 .gamma.15.kappa.11 .gamma.20.kappa.11
.gamma.22.kappa.11 .gamma.23.kappa.11 .gamma.44.kappa.11
.gamma.46.kappa.11 .gamma.52.kappa.11 .gamma.59.kappa.11
R8304541.67 0.091 >15 >15 >15 >15 0.140 >15 >15
3.473 1.572 PVO.4 0.074 8.153 5.704 >15 >15 0.103 >15
2.921 0.103 >15 YU2 00 0.276 9.349 4.122 >15 >15 0.340
>15 2.101 0.298 6.911 34>15.v1.cl >15 >15 >15 >15
>15 >15 >15 >15 >15 >15 3365.v2.c20 >15 >15
>15 >15 >15 >15 >15 >15 >16 >15 ZM53M.PB12
>15 >15 >15 >15 >15 >15 >15 >15 >15
>15 ZM109F.PB4 >15 >15 >15 >15 >15 >15 >35
>15 >15 >15 3016.v5.c45 0.143 2.463 1.325 >15 >15
0.159 >15 1.932 0.161 >15 231965.c1 0.455 2.490 5.103 >15
>15 0.543 >15 2.958 0.296 >15 X1254_c3 2.306 4.144 13.687
7.583 >15 1.695 >15 5.662 2.419 4.669 251-18 >15 >15
>15 >15 >15 2.490 >15 >15 0.647 >15 R1166.c1
1.792 3.883 9.813 20.237 >15 1.611 >15 3.390 1.159 12.580
H086.8 0.778 >15 >15 >15 >15 >15 >15 >15 1.638
>15 Du172.17 >15 >15 >15 >15 >15 11.298 >15
>15 >15 >15 250-4 >15 >15 >15 >15 >15
>15 >15 >15 >15 >15 MuLV >30 NT >30 >30
>30 >30 >19 >30 >30 >30 Virus .gamma.3.kappa.18
.gamma.4.kappa.18 .gamma.15.kappa.18 .gamma.20.kappa.18
.gamma.22.kappa.18 .gamma.23.kappa.18 .gamma.46.kappa.18
.gamma.52.kappa.18 .gamma.3.kappa.18 REJ04541.67 0.049 >15
>15 >15 >15 0.061 >15 2.132 1.227 PVO.4 0.028 >15
1.588 >15 >15 0.047 2.100 0.081 >15 YU2.DG 0.070 >15
1.169 >15 8.229 0.117 2.360 0.265 7.707 34>15.vl.l >15
>15 >15 >15 >15 >15 >15 >15 >15 3365.v2.c20
>15 >15 >15 >15 >15 >15 >15 >15 >15
ZM53M.PB12 >15 >15 >15 >15 >15 >15 >15 >15
>15 ZM109F.PB4 >15 >15 >15 >15 >15 >15 >15
>15 >15 3016.v5.c45 0.057 >15 5.554 >15 >15 >15
2.993 0.129 >15 231965.c1 0.163 >15 1.481 >15 13.350 0.047
4.157 0.264 >15 X1254_c3 0.616 16.224 2.849 5.221 25.988 0.128
4.202 1.567 4.066 251-18 >15 >15 >15 >15 1.563 0.648
>15 0.116 25.339 R1166.c1 0.578 >15 1.986 20.036 >15 1.678
4.096 1.351 11.701 H086.8 0.209 >15 >15 >15 7.311 0.708
>15 0.464 >15 Du172.17 11.518 >15 >15 >15 >15
>15 >15 >15 >15 250-4 >15 >15 >15 >15
>15 2.953 >15 >15 >15 MuLV >30 >30 NT >30
>15 >30 >15 >15 >15 Virus .gamma.20.kappa.19
REJO4541.67 >15 PYO.4 >15 YU2.DG >15 34>15.V1.c1 >15
3365.v2.c20 >15 ZM53M.PB12 >15 Z109F.PB4 >15 3016.v5.c45
>15 231965.c1 >15 X1254_c3 2.291 251-18 >15 R1166.c1
>15 H086.8 >15 Du172.17 >15 250-4 >15 MuLV >15 Virus
.gamma.15.kappa.61 .gamma.44.kappa.61 .gamma.46.kappa.61
.gamma.52.kappa.61 .gamma.59.kappa.61 REJO4541.67 2.029 >15
0.378 0.115 1.333 PVO.4 1.230 >15 0.244 0.078 >15 YU2.DG
3.446 >15 0.915 0.406 4.326 34>15.v1.cl >15 >15 >15
0.681 >15 3365.v2.c20 >15 >15 >15 >15 >15
ZM53M.PB12 >15 >15 27.493 8.435 13.301 ZM109F.PB4 >15
>15 >15 >15 >15 3016.v5.c45 1.214 >15 0.383 0.182
>15 231965.c1 5.231 >15 1.576 0.515 >15 X1254_c3 10.584
>15 4.468 2.565 4.018 251-18 4.656 >15 1.842 0.273 1.402
R1156 61 6.548 >15 2.678 1.635 7.461 H086.8 >15 >15 0.584
0.242 >15 Du172.17 >15 >15 26.083 7.275 >15 250-4
>15 >15 >15 >15 >15 MuLV >15 >15 >15 >15
>15
[0160] Of these, 52.sub.HC.kappa.5.sub.LC was 5-fold more potent
than 8ANC195 (neutralized 12 of 15 viruses with a mean IC.sub.50 of
0.45 .mu.g/ml as compared to 2.3 .mu.g/ml for 8ANC195) (FIG. 13), a
potency and breadth against this virus panel that was comparable to
those of other bNAbs, such as VRC01 (neutralized 12 of 15 viruses
with a 0.56 .mu.g/ml mean IC.sub.50) and 10-1074 (neutralized 6 of
15 viruses with a mean of 0.09 .mu.g/ml), that target
non-overlapping sites (Wu et al., Science 329, 856 (2010); Mouquet
et al., Proceedings of the National Academy of Sciences of the
United States of America 109, E3268 (2012)).
[0161] The LC was critical to the activity of more potent
52.sub.HC.kappa.5.sub.LC variant, as demonstrated by diminished
neutralization potencies when .kappa.5.sub.LC was swapped for
either .kappa.3.sub.LC or .kappa.11.sub.LC (FIG. 19C). The weaker
neutralization could be explained by differences between
.kappa.5.sub.LC and .kappa.3.sub.LC at solvent-exposed residues in
CDRL2 (53.sub.LC and 54.sub.LC) and FWRL3 (64.sub.LC), and a nearby
buried residue (34.sub.LC) that may affect the structural integrity
of CDRL1. Modeling of YU2 gp41 residues into the Env trimer
structure (Julien et al., Science 342, 1477 (2013)) suggested that
8ANC195 positions 53.sub.LC and 54.sub.LC were adjacent to the
Asn637.sub.gp41 PNGS (FIGS. 8, 14). The improved neutralizing
activity of .kappa.5.sub.LC compared with the other newly-isolated
LCs was associated with small side chains at positions 34.sub.LC
(Val), 53.sub.LC (Ala) and 54.sub.LC (Ala), whereas .kappa.3.sub.LC
or .kappa.11.sub.LC, which were less broadly neutralizing when
paired with identical HCs, included bulkier and/or charged side
chains that would clash with the nearby gp41 glycan.
.kappa.5.sub.LC was the only LC containing an S64R.sub.LC
substitution and this single change compared to the 8ANC195 LC may
account for the 5-fold improved potency of
52.sub.HC.kappa.5.sub.LC. Residues in the immediate vicinity of
Asn637.sub.gp41 might also modify neutralization; all six viral
strains that were potently neutralized by the
52.sub.HC.kappa.5.sub.LC variant had Asp636.sub.gp41 or
Asn636.sub.gp41 whereas the remaining eight strains had
Ser636.sub.gp41 (p<0.001 by G-test). The same association
between Asp636.sub.gp41/Asn636.sub.gp41 and neutralization potency
was also statistically significant for 8ANC195 (p<0.01 by
G-test), consistent an interaction between the N-terminal portion
of gp41 HR2 (residues .about.625 to 640) and 8ANC195 LC (FIG. 8).
Also consistent with changes in the gp41 HR2 region affecting
8ANC195 neutralization, a computational analysis of neutralization
panel data using the Antibody Database program (West, Jr. et al.,
Proceedings of the National Academy of Sciences of the United
States of America 110, 10598 (2013)) suggested that Glu632.sub.gp41
was associated with stronger neutralization.
[0162] In conclusion, 8ANC195 defines a novel site of HIV-1 Env
vulnerability to neutralizing antibodies that spans gp120 and gp41
(FIG. 15). Rather than penetrating the glycan shield using only a
single CDR loop, a strategy employed by antibodies such as PG9 and
PGT128 (Pejchal et al., Science 334, 1097 (2011); McLellan et al.,
Nature 480, 336 (2011)) 8ANC195 inserted its entire HC variable
region into a gap in the shield to form a large interface, of which
>50% involved contacts to gp120 glycans (FIG. 17).
[0163] Although it was not possible to obtain large numbers of
8ANC195 variants by standard single cell cloning techniques (Scheid
et al., J Immunol Methods 343, 65 (2009)), randomly combining HCs
and LCs obtained from memory B cells without antigen-specific
sorting demonstrated that the target of this antibody supported
neutralization activity comparable to that against the most
vulnerable sites on Env. Potent variants of 8ANC195 are
particularly since the epitope does not overlap with the targets of
CD4 binding site, V2 loop, V3 loop or MPER antibodies.
[0164] The foregoing examples and description of the preferred
embodiments should be taken as illustrating, rather than as
limiting the present invention as defined by the claims. As will be
readily appreciated, numerous variations and combinations of the
features set forth above can be utilized without departing from the
present invention as set forth in the claims. Such variations are
not regarded as a departure from the scope of the invention, and
all such variations are intended to be included within the scope of
the following claims. All references cited herein are incorporated
herein by reference in their entireties.
Sequence CWU 1
1
1231137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Tyr Asp Arg Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 1352137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
2Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5
10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Ile Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Ser
Asp Tyr Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120 125Ser Val Ser Ala Ala
Ser Thr Lys Gly 130 1353137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 3Gln Ile His Leu Val Gln
Ser Gly Thr Gly Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Leu Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Thr Ser Leu Glu Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu
Tyr 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
1354137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 4Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Ala Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His Asp Phe Arg 50 55 60Gly Arg Val Ile Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Ala Thr Ser Thr Pro Asp Tyr Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 1355137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5
10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Val Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Glu Trp Ser Asp Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120 125Ser Val Ser Ala Ala
Ser Thr Lys Gly 130 1356137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 6Gln Ile His Leu Val Gln
Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Leu Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Arg Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
1357137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 7Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Pro Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 1358137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
8Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5
10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Ile Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Ala Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120 125Ser Val Ser Ala Ala
Ser Thr Lys Gly 130 1359137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Gln Ile His Leu Val Gln
Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Ile Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Pro Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Ser Asp Arg Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
13510137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 10Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Val Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 13511137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
11Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Glu Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Val Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr His
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120 125Ser Val Ser Ala Ala
Ser Thr Lys Gly 130 13512137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Gln Ile His Leu Val Gln
Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Leu Ile Ser Ala Ile Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Met Ser Thr Tyr Asp Lys Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
13513137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Ser Trp Val Arg Gln Ala
Pro Gly Gln Arg Leu Glu Tyr 35 40 45Ile Gly Gln Ile Arg Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Thr Val Ser Ala
Val Asp Pro Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Arg Asp Leu Thr Thr Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu His 100 105 110Asn Glu
Arg Gly Thr Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 13514137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Gln Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120 125Ser Val Ser Ala Ala
Ser Thr Lys Gly 130 13515137PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 15Gln Ile His Leu Val Gln
Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Ile Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
13516137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 16Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Tyr Asp Arg Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 13517137PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
17Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Arg Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90
95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His
100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr
Leu Ile 115 120 125Ser Val Ser Ala Ala Ser Thr Lys Gly 130
13518137PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 18Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Tyr Asp Arg Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly Thr Leu Ile 115 120
125Ser Val Ser Ala Ala Ser Thr Lys Gly 130 13519117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly1
5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly
Asn 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro Gly Lys Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu Gly Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile
Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln
Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val
Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe
11520117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Thr Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Arg 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro
Ser Val Phe 11521117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 21Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys
Arg Ala Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His
Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser
Arg Leu Leu Gly Gly Val Pro Ser Lys Phe Ser 50 55 60Gly Ser Ala Ala
Gly Thr Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu
Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly
Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe 11522117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 22Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp
Tyr His Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg
Gly Ser Arg Leu Val Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser
Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75
80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro
85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe 11523117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly
Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro Gly Lys Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu Gly Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Ala Asp Phe Thr Leu Thr Ile
Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln
Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val
Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe
11524117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 24Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Met Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Lys Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Gly
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro
Ser Val Phe 11525117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys
Arg Ala Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr His
Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala
Ala Leu Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala
Gly Thr Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu
Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly
Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe 11526117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 26Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Gly Thr Val Arg Ile
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp
Tyr His Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg
Gly Ser Arg Leu Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser
Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75
80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro
85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe 11527117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
27Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1
5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly
Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro Gly Lys Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu Gly Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Gly Phe Thr Leu Thr Ile
Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln
Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val
Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe
11528117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 28Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Gly 20 25 30Trp Val Ala Trp Tyr His Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro
Ser Val Phe 11529117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 29Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys
Arg Ala Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln
Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala
Ala Leu Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala
Gly Thr Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu
Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly
Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe 11530117PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 30Asp Ile Gln Met Thr Gln
Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Thr Val Arg Ile
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp
Tyr His Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg
Gly Ser Arg Leu Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser
Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75
80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro
85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val
Ala 100 105 110Ala Pro Ser Val Phe 11531117PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
31Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly1
5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly
Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu Gly Gly Val Pro Ser
Arg Phe Arg 50 55 60Gly Ser Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile
Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln
Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val
Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro Ser Val Phe
11532117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Thr Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105 110Ala Pro
Ser Val Phe 11533117PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 33Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys
Arg Ala Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His
Gln Arg Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser
Arg Leu Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala
Gly Thr Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu
Asp Phe Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly
Thr Phe Gly Gln Gly Thr Lys Val Glu Val Lys Arg Thr Val Ala 100 105
110Ala Pro Ser Val Phe 11534133PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 34Gln Ile His Leu Val Gln
Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser
Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn
Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln
Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg
Val Leu Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln Gly
Thr Leu Ile 115 120 125Ser Val Ser Ala Ala 13035107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
35Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly1
5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly
Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Arg
Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu Gly Gly Val Pro Ser
Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp Phe Thr Leu Thr Ile
Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly Thr Phe Tyr Cys Gln
Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly Gln Gly Thr Lys Val
Glu Val 100 1053697PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 36Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Thr Ile Ser Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Arg Ile Ile Pro
Ile Leu Gly Ile Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Thr37106PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 37Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Pro Ile 85 90 95Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 1053820PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Thr
Ser Thr Tyr Asp Lys Trp Ser Gly Leu His His Asp Gly Val Met1 5 10
15Ala Phe Ser Ser 203920PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Thr Ser Thr Tyr Asp Gln Trp
Ser Gly Leu His His Asp Gly Val Met1 5 10 15Ala Phe Ser Ser
204020PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 40Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu His His
Asp Gly Val Met1 5 10 15Ala Phe Ser Ser 204120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 41Thr
Ser Thr Tyr Asp Arg Trp Ser Gly Leu His His Asp Gly Val Met1 5 10
15Ala Phe Ser Ser 204220PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 42Thr Ser Thr Tyr Asp Lys Trp
Ser Gly Leu His His Asp Gly Val Met1 5 10 15Ala Phe Ser Ser
204320PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 43Thr Ser Thr Tyr Asp Arg Trp Ser Gly Leu His His
Asp Gly Val Met1 5 10 15Ala Phe Ser Ser 20449PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 5459PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 5469PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 46Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 5479PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 47Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 5489PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 5499PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 49Gln
Gln Tyr Asp Thr Tyr Pro Gly Thr1 55030DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
50ggtgtacatt ctcagataca cctcgtacaa 305122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
51caggtgtcca gtctcagata ca 225248DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 52gacatccaga tgacccagtc
tccttccacc ctgtctgcat ctataggt 485342DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
53gacatccaga tgacccagtc tccttccacc ctgtctgcat ct
425421DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 54cgcctctgcc tctactccca a 215524DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
55caaagtggag ttgaaatcag ggaa 245635DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
56caaagtggag ttgaaatcag ggaaccggct tccag 355721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
57gcggagacgg agatgagggt t 215824DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 58gtttcacctc aactttagtc
cctt 245935DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 59gtttcacctc aactttagtc ccttggccga aggtc
35606PRTArtificial SequenceDescription of Artificial Sequence
Synthetic 6xHis tag 60His His His His His His1 56198PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
61Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg62121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 12063121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 63Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu
Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser 115 12064121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
64Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 12065121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 65Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu
Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser 115 12066121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
66Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Gln Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 12067121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 67Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu
Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Thr Ser Thr Tyr Asp Gln Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser 115 12068121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
68Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Arg Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 12069121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 69Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Ile
Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser 115 12070121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
70Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Arg Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 12071121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 71Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Arg Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu
Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser 115 12072121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
72Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
Pro Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser 115 1207395PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 73Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser 85 90
9574100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 74Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10075100PRTArtificial SequenceDescription of Artificial
Sequence
Synthetic polypeptide 75Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10076100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 76Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10077100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10078100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 78Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10079100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10080100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 80Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10081100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 81Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10082100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 82Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Arg 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10083100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Thr Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
10084100PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala Ser
Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg Pro
Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu Leu
Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr Asp
Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe Gly
Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr Phe Gly
1008532DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 85caggtgccca gtctcagata cacctcgtac aa
328648DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 86gacatccaga tgacccagtc tccttccacc
ctggctgcat ctataggt 488725DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 87ccggcgcctc
tgcctctact cccaa 258840DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 88caaagtggag
ttgaaatcag ggaaccggct tccagggacc 4089124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
89Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln 115 12090124PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 90Gln Ile His Leu Val
Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val
Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val
Ser Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Tyr 35 40 45Ile Gly
Gln Ile Arg Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly
Arg Val Thr Val Ser Ala Val Asp Pro Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Arg Asp Leu Thr Thr Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu
His 100 105 110Asn Glu Arg Gly Thr Ala Phe Ser Ser Trp Gly Gln 115
12091124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 91Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Ile Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Pro Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr Ser Asp Arg Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115 12092124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
92Gln Ile His Leu Val Gln Ser Gly Thr Gly Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Ser Ser Pro Pro65 70 75 80Ile Thr Ser Leu Glu Ile Lys Asn Val Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu Tyr 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln 115 12093124PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 93Gln Ile His Leu Val
Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val
Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val
Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly
Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly
Arg Val Ile Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Ala Ser Thr Tyr Asp Lys Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115
12094124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 94Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Ala Val Ser Cys Lys Ala Tyr Gly
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His Asp Phe Arg 50 55 60Gly Arg Val Ile Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Ala Thr Ser Thr Pro Asp Tyr Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115 12095124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
95Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu
Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His
His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly
Pro Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr
Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr
Asp Lys Trp Ser Gly Leu His 100 105 110His Asp Gly Val Met Ala Phe
Ser Ser Trp Gly Gln 115 12096124PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 96Gln Ile His Leu Val
Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val
Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val
Asn Trp Val Arg Gln Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly
Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly
Arg Val Ile Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75
80Ile Ser Ser Leu Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val
85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Ser Asp Tyr Trp Ser Gly Leu
His 100 105 110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115
12097124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 97Gln Ile His Leu Val Gln Ser Gly Thr Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Glu
Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln Ala
Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp Lys
Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser Ala
Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu Glu
Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys Thr
Thr Thr Ser Thr His Asp Lys Trp Ser Gly Leu His 100 105 110His Asp
Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115 12098124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
98Gln Ile His Leu Val Gln Ser Gly Thr Glu Val Lys Lys Pro Gly Ser1
5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr Gly Val Asn Thr Phe Gly
Leu 20 25 30Tyr
Ala Val Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr 35 40
45Ile Gly Gln Ile Trp Arg Trp Lys Ser Ser Ala Ser His His Phe Arg
50 55 60Gly Arg Val Leu Ile Ser Ala Val Asp Leu Thr Gly Ser Ser Pro
Pro65 70 75 80Ile Ser Ser Leu Glu Ile Lys Asn Val Thr Ser Asp Asp
Thr Ala Val 85 90 95Tyr Phe Cys Thr Thr Thr Ser Thr Tyr Asp Lys Trp
Ser Gly Leu His 100 105 110His Asp Gly Val Val Ala Phe Ser Ser Trp
Gly Gln 115 12099124PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 99Gln Ile His Leu Val Gln Ser Gly
Thr Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys
Ala Tyr Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp
Arg Trp Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu
Ile Ser Ala Ile Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser
Ser Leu Glu Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr
Phe Cys Thr Thr Met Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105
110His Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115
120100124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 100Gln Ile His Leu Val Gln Ser Gly Thr Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr
Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln
Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp
Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser
Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu
Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys
Thr Thr Thr Ser Thr Tyr Asp Arg Trp Ser Gly Leu His 100 105 110His
Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115
120101124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 101Gln Ile His Leu Val Gln Ser Gly Thr Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr
Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp
Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser
Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu
Glu Ile Lys Asn Val Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys
Thr Thr Thr Ser Thr Tyr Asp Glu Trp Ser Asp Leu His 100 105 110His
Asp Gly Val Met Ala Phe Ser Ser Trp Gly Gln 115
120102124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 102Gln Ile His Leu Val Gln Ser Gly Thr Glu
Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Thr Val Ser Cys Lys Ala Tyr
Gly Val Asn Thr Phe Gly Leu 20 25 30Tyr Ala Val Asn Trp Val Arg Gln
Ala Pro Gly Gln Ser Leu Glu Tyr 35 40 45Ile Gly Gln Ile Trp Arg Trp
Lys Ser Ser Ala Ser His His Phe Arg 50 55 60Gly Arg Val Leu Ile Ser
Ala Val Asp Leu Thr Gly Ser Ser Pro Pro65 70 75 80Ile Ser Ser Leu
Glu Ile Lys Asn Leu Thr Ser Asp Asp Thr Ala Val 85 90 95Tyr Phe Cys
Thr Thr Thr Ser Thr Tyr Asp Lys Trp Ser Gly Leu His 100 105 110His
Asp Gly Val Met Ala Phe Ser Ser Arg Gly Gln 115
12010396PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 103Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser Leu Glu
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Pro 85 90
9510499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 104Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe10599PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 105Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe10699PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 106Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Thr Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr Gln Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu
Leu Gly Gly Val Pro Ser Arg Phe Arg 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe10799PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 107Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Gly Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe10899PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 108Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Ala
Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe10999PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 109Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Val Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Gly Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe11099PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 110Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Met Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Lys Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Gly Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe11199PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 111Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Lys Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Ala Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe11299PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 112Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Asn 20 25 30Trp Val Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ala Ala Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe11399PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 113Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Leu Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Val Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe11499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 114Asp Ile Gln Met Thr Gln Ser Pro Ser Thr
Leu Ser Ala Ser Ile Gly1 5 10 15Asp Thr Val Arg Ile Ser Cys Arg Ala
Ser Gln Ser Ile Thr Gly Gly 20 25 30Trp Val Ala Trp Tyr His Gln Arg
Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45Ile Tyr Arg Gly Ser Arg Leu
Leu Gly Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Ala Ala Gly Thr
Asp Phe Thr Leu Thr Ile Gly Asn Leu Gln65 70 75 80Ala Glu Asp Phe
Gly Thr Phe Tyr Cys Gln Gln Tyr Asp Thr Tyr Pro 85 90 95Gly Thr
Phe1157PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 115Gln Ser Ile Thr Gly Asn Trp1
51168PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 116Gln Gln Tyr Asp Thr Tyr Pro Gly1
51176PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 117Gln Ser Ile Ser Ser Trp1 51188PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 118Gln
Gln Tyr Asn Thr Tyr Pro Ile1 51198PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 119Gln Gln Ala Ala Ala Ala
Pro Gly1 51206PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 120Gln Ser Ile Ser Ser Trp1
51218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 121Gln Gln Tyr Asn Thr Tyr Pro Ile1
5122685PRTHuman immunodeficiency virus 122Met Arg Val Lys Glu Lys
Tyr Gln His Leu Trp Arg Trp Gly Trp Arg1 5 10 15Trp Gly Thr Met Leu
Leu Gly Met Leu Met Ile Cys Ser Ala Thr Glu 20 25 30Lys Leu Trp Val
Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala 35 40 45Thr Thr Thr
Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Asp Thr Glu 50 55 60Val His
Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn65 70 75
80Pro Gln Glu Val Val Leu Val Asn Val Thr Glu Asn Phe Asn Met Trp
85 90 95Lys Asn Asp Met Val Glu Gln Met His Glu Asp Ile Ile Ser Leu
Trp 100 105 110Asp Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu
Cys Val Ser 115 120 125Leu Lys Cys Thr Asp Leu Lys Asn Asp Thr Asn
Thr Asn Ser Ser Ser 130 135 140Gly Arg Met Ile Met Glu Lys Gly Glu
Ile Lys Asn Cys Ser Phe Asn145 150 155 160Ile Ser Thr Ser Ile Arg
Gly Lys Val Gln Lys Glu Tyr Ala Phe Phe 165 170 175Tyr Lys Leu Asp
Ile Ile Pro Ile Asp Asn Asp Thr Thr Ser Tyr Lys 180 185 190Leu Thr
Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 195 200
205Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Phe Ala
210 215 220Ile Leu Lys Cys Asn Asn Lys Thr Phe Asn Gly Thr Gly Pro
Cys Thr225 230 235 240Asn Val Ser Thr Val Gln Cys Thr His Gly Ile
Arg Pro Val Val Ser 245 250 255Thr Gln Leu Leu Leu Asn Gly Ser Leu
Ala Glu Glu Glu Val Val Ile 260 265 270Arg Ser Val Asn Phe Thr Asp
Asn Ala Lys Thr Ile Ile Val Gln Leu 275 280 285Asn Thr Ser Val Glu
Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg 290 295 300Lys Arg Ile
Arg Ile Gln Arg Gly Pro Gly Arg Ala Phe Val Thr Ile305 310 315
320Gly Lys Ile Gly Asn Met Arg Gln Ala His Cys Asn Ile Ser Arg Ala
325 330 335Lys Trp Asn Asn Thr Leu Lys Gln Ile Ala Ser Lys Leu Arg
Glu Gln 340 345 350Phe Gly Asn Asn Lys Thr Ile Ile Phe Lys Gln Ser
Ser Gly Gly Asp 355 360 365Pro Glu Ile Val Thr His Ser Phe Asn Cys
Gly Gly Glu Phe Phe Tyr 370 375
380Cys Asn Ser Thr Gln Leu Phe Asn Ser Thr Trp Phe Asn Ser Thr
Trp385 390 395 400Ser Thr Glu Gly Ser Asn Asn Thr Glu Gly Ser Asp
Thr Ile Thr Leu 405 410 415Pro Cys Arg Ile Lys Gln Ile Ile Ile Asn
Met Trp Gln Lys Val Gly 420 425 430Lys Ala Met Tyr Ala Pro Pro Ile
Ser Gly Gln Ile Arg Cys Ser Ser 435 440 445Asn Ile Thr Gly Leu Leu
Leu Thr Arg Asp Gly Gly Asn Ser Asn Asn 450 455 460Glu Ser Glu Ile
Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp465 470 475 480Arg
Ser Glu Leu Tyr Lys Tyr Lys Val Val Lys Ile Glu Pro Leu Gly 485 490
495Val Ala Pro Thr Lys Ala Lys Arg Arg Val Val Gln Arg Glu Lys Arg
500 505 510Ala Val Gly Ile Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala
Ala Gly 515 520 525Ser Thr Met Gly Ala Ala Ser Met Thr Leu Thr Val
Gln Ala Arg Gln 530 535 540Leu Leu Ser Gly Ile Val Gln Gln Gln Asn
Asn Leu Leu Arg Ala Ile545 550 555 560Glu Ala Gln Gln His Leu Leu
Gln Leu Thr Val Trp Gly Ile Lys Gln 565 570 575Leu Gln Ala Arg Ile
Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln 580 585 590Leu Leu Gly
Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala 595 600 605Val
Pro Trp Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu Gln Ile Trp 610 615
620Asn His Thr Thr Trp Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr
Thr625 630 635 640Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn
Gln Gln Glu Lys 645 650 655Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu Trp Asn 660 665 670Trp Phe Asn Ile Thr Asn Trp Leu
Trp Tyr Ile Lys Leu 675 680 685123679PRTHuman immunodeficiency
virus 123Met Arg Val Lys Glu Thr Gln Met Asn Trp Pro Asn Leu Trp
Lys Trp1 5 10 15Gly Thr Leu Ile Leu Gly Leu Val Ile Ile Cys Ser Ala
Ser Asp Asn 20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
Lys Asp Ala Asp 35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala
His Glu Thr Glu Val 50 55 60His Asn Val Trp Ala Thr His Ala Cys Val
Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Ile His Leu Glu Asn Val
Thr Glu Asn Phe Asn Met Trp Lys 85 90 95Asn Asn Met Val Glu Gln Met
Gln Glu Asp Val Ile Ser Leu Trp Asp 100 105 110Gln Ser Leu Gln Pro
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125His Cys Thr
Thr Ala Lys Leu Thr Asn Val Thr Asn Ile Thr Asn Val 130 135 140Pro
Asn Ile Gly Asn Ile Thr Asp Glu Val Arg Asn Cys Ser Phe Asn145 150
155 160Met Thr Thr Glu Ile Arg Asp Lys Lys Gln Lys Val His Ala Leu
Phe 165 170 175Tyr Lys Leu Asp Ile Val Gln Ile Glu Asp Lys Asn Asp
Ser Ser Lys 180 185 190Tyr Arg Leu Ile Asn Cys Asn Thr Ser Val Ile
Lys Gln Ala Cys Pro 195 200 205Lys Ile Ser Phe Asp Pro Ile Pro Ile
His Tyr Cys Thr Pro Ala Gly 210 215 220Tyr Val Ile Leu Lys Cys Asn
Asp Lys Asn Phe Asn Gly Thr Gly Pro225 230 235 240Cys Lys Asn Val
Ser Ser Val Gln Cys Thr His Gly Ile Lys Pro Val 245 250 255Val Ser
Thr Gln Leu Leu Leu Asn Gly Ser Leu Ala Glu Glu Glu Ile 260 265
270Ile Ile Arg Ser Glu Asn Leu Thr Asn Asn Ala Lys Thr Ile Ile Val
275 280 285His Leu Asn Lys Ser Val Glu Ile Asn Cys Thr Arg Pro Ser
Asn Asn 290 295 300Met Arg Thr Ser Met Arg Ile Gly Pro Gly Gln Val
Phe Tyr Arg Thr305 310 315 320Gly Ser Ile Thr Gly Asp Ile Arg Lys
Ala Tyr Cys Glu Ile Asn Gly 325 330 335Thr Lys Trp Asn Lys Val Leu
Lys Gln Val Thr Glu Lys Leu Lys Glu 340 345 350His Phe Asn Asn Lys
Thr Ile Ile Phe Gln Pro Pro Ser Gly Gly Asp 355 360 365Leu Glu Ile
Thr Met His His Phe Asn Cys Arg Gly Glu Phe Phe Tyr 370 375 380Cys
Asn Thr Thr Gln Leu Phe Asn Asn Thr Cys Ile Gly Asn Glu Thr385 390
395 400Met Lys Gly Cys Asn Gly Thr Ile Thr Leu Pro Cys Lys Ile Lys
Gln 405 410 415Ile Ile Ile Asn Met Trp Gln Gly Thr Gly Gln Ala Met
Tyr Ala Pro 420 425 430Pro Ile Asp Gly Lys Ile Asn Cys Val Ser Asn
Ile Thr Gly Ile Leu 435 440 445Leu Thr Arg Asp Gly Gly Ala Asn Asn
Thr Ser Asn Glu Thr Phe Arg 450 455 460Pro Gly Gly Gly Asn Ile Lys
Asp Asn Trp Arg Ser Glu Leu Tyr Lys465 470 475 480Tyr Lys Val Val
Gln Ile Glu Pro Leu Gly Ile Ala Pro Thr Arg Ala 485 490 495Lys Arg
Arg Val Val Glu Arg Glu Lys Arg Ala Val Gly Ile Gly Ala 500 505
510Met Ile Phe Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala
515 520 525Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly
Ile Val 530 535 540Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala
Gln Gln His Leu545 550 555 560Leu Gln Leu Thr Val Trp Gly Ile Lys
Gln Leu Gln Ala Arg Val Leu 565 570 575Ala Val Glu Arg Tyr Leu Lys
Asp Gln Lys Phe Leu Gly Leu Trp Gly 580 585 590Cys Ser Gly Lys Ile
Ile Cys Thr Thr Ala Val Pro Trp Asn Ser Thr 595 600 605Trp Ser Asn
Lys Ser Phe Glu Glu Ile Trp Asn Asn Met Thr Trp Ile 610 615 620Glu
Trp Glu Arg Glu Ile Ser Asn Tyr Thr Asn Gln Ile Tyr Glu Ile625 630
635 640Leu Thr Glu Ser Gln Asn Gln Gln Asp Arg Asn Glu Lys Asp Leu
Leu 645 650 655Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp
Ile Thr Asn 660 665 670Trp Leu Trp Tyr Ile Lys Ile 675
* * * * *