U.S. patent application number 12/621992 was filed with the patent office on 2010-04-15 for modified hiv env polypeptides.
This patent application is currently assigned to NOVARTIS VACCINES AND DIAGNOSTICS, INC.. Invention is credited to Susan Barnett, Karin Hartog, Eric Martin.
Application Number | 20100092502 12/621992 |
Document ID | / |
Family ID | 26812260 |
Filed Date | 2010-04-15 |
United States Patent
Application |
20100092502 |
Kind Code |
A1 |
Barnett; Susan ; et
al. |
April 15, 2010 |
MODIFIED HIV ENV POLYPEPTIDES
Abstract
Polynucleotide encoding modified HIV Env polypeptides are
disclosed. The Env polypeptides are modified so as to expose at
least part of the CD4 binding region. Methods of diagnosis,
treatment and prevention using the polynucleotides and polypeptides
are also provided.
Inventors: |
Barnett; Susan; (San
Francisco, CA) ; Hartog; Karin; (Piedmont, CA)
; Martin; Eric; (El Cerrito, CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS INC.
INTELLECTUAL PROPERTY- X100B, P.O. BOX 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
NOVARTIS VACCINES AND DIAGNOSTICS,
INC.
EMERYVILLE
CA
|
Family ID: |
26812260 |
Appl. No.: |
12/621992 |
Filed: |
November 19, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10452018 |
Jul 25, 2003 |
7662916 |
|
|
12621992 |
|
|
|
|
09476242 |
Dec 30, 1999 |
6689879 |
|
|
10452018 |
|
|
|
|
60114495 |
Dec 31, 1998 |
|
|
|
60156670 |
Sep 29, 1999 |
|
|
|
Current U.S.
Class: |
424/188.1 ;
424/208.1; 435/320.1 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 2039/53 20130101; A61K 2039/54 20130101; C12N 7/00 20130101;
C12N 2740/16052 20130101; C12N 2740/16322 20130101; C12N 2740/16122
20130101; C12N 2770/24222 20130101; A61K 2039/5258 20130101; C07K
14/005 20130101; A61K 2039/5256 20130101; C12N 2740/16222 20130101;
A61K 39/00 20130101; A61P 31/18 20180101; C12N 2740/16062 20130101;
C12N 2740/16023 20130101; A61K 2039/5156 20130101 |
Class at
Publication: |
424/188.1 ;
435/320.1; 424/208.1 |
International
Class: |
A61K 39/21 20060101
A61K039/21; C12N 15/63 20060101 C12N015/63 |
Claims
1. A construct comprising a nucleotide sequence selected from the
group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID
NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20,
SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:25, and SEQ ID NO:26.
2. A vaccine composition comprising an immunogenic modified HIV Env
polypeptide of a selected variant of HIV, the modified HIV Env
polypeptide having at least one amino acid deleted relative to the
wild-type Env polypeptide of the selected variant in the region
corresponding to residues 420 to 436 numbered relative to HXB-2
(SEQ ID NO:1), such that epitopes that are not exposed in the
wild-type Env polypeptide of the selected variant are exposed in
the modified Env polypeptide.
3. The vaccine composition of claim 2 wherein one amino acid is
deleted.
4. The vaccine composition of claim 2 wherein more than one amino
acid is deleted.
5. The vaccine composition of claim 2 wherein at least one of amino
acid residues 427, 428, and 429 is deleted.
6. The vaccine composition of claim 2 wherein the V1 and V2 regions
of the polypeptide are truncated.
7. The vaccine composition of claim 5 wherein the V1 and V2 regions
of the polypeptide are truncated.
8. The vaccine composition of claim 2 wherein the selected variant
is HIV strain SF162 (SEQ ID NO:2).
9. The vaccine composition of claim 2 further comprising an
adjuvant.
10. The vaccine composition of claim 5 further comprising an
adjuvant.
11. A method of inducing an immune response in subject comprising
administering a polynucleotide in an amount sufficient to induce an
immune response in the subject, wherein the polynucleotide encodes
an immunogenic modified HIV Env polypeptide of a selected variant
of HIV, the modified HIV Env polypeptide having at least one amino
acid deleted relative to the wild-type Env polypeptide of the
selected variant in the region corresponding to residues 420 to 436
numbered relative to HXB-2 (SEQ ID NO:1), such that epitopes that
are not exposed in the wild-type Env polypeptide of the selected
variant are exposed in the modified Env polypeptide.
12. The method of claim 11 further comprising administering an
adjuvant to the subject.
13. A method of inducing an immune response in a subject comprising
steps of: (a) administering a first composition comprising a
polynucleotide encodes an immunogenic modified HIV Env polypeptide
of a selected variant of HIV, the modified HIV Env polypeptide
having at least one amino acid deleted relative to the wild-type
Env polypeptide of the selected variant in the region corresponding
to residues 420 to 436 numbered relative to HXB-2 (SEQ ID NO:1),
such that epitopes that are not exposed in the wild-type Env
polypeptide of the selected variant are exposed in the modified Env
polypeptide in a priming step; and (b) administering a second
composition comprising the modified HIV Env polypeptide as a
booster in an amount sufficient to induce an immune response in the
subject.
14. The method of claim 13 wherein the first composition or second
composition further comprise an adjuvant.
15. The method of claim 13 wherein the first and second
compositions further comprise an adjuvant.
Description
[0001] This application is a division of Ser. No. 10/452,018 filed
Jul. 25, 2003, which is a continuation of Ser. No. 09/476,242 filed
Dec. 30, 1999, now issued as U.S. Pat. No. 6,689,879, which claims
the benefit of Ser. No. 60/114,495 filed Dec. 31, 1998 and Ser. No.
60/156,670 filed Sep. 29, 1999. Each of these applications is
incorporated herein by reference in its entirety.
[0002] This application incorporates by reference an 87.1 kb text
file created on Nov. 8, 2009 and named
"PAT050994_sequencelisting.txt," which is the sequence listing for
this application.
TECHNICAL FIELD
[0003] The invention relates generally to modified HIV envelope
(Env) polypeptides which are useful as immunizing agents or for
generating an immune response in a subject, for example a cellular
immune response or a protective immune response. More particularly,
the invention relates Env polypeptides such as gp120, gp140 or
gp160, wherein at least one of the native .beta.-sheet
configurations has been modified. The invention also pertains to
methods of using these polypeptides to elicit an immune response
against a broad range of HIV subtypes.
BACKGROUND OF THE INVENTION
[0004] The human immunodeficiency virus (HIV-1, also referred to as
HTLV-III, LAV or HTLV-III/LAV) is the etiological agent of the
acquired immune deficiency syndrome (AIDS) and related disorders.
(see, e.g., Barre-Sinoussi, et al., (1983) Science 220:868-871;
Gallo et al. (1984) Science 224:500-503; Levy et al., (1984)
Science 225:840-842; Siegal et al., (1981) N. Engl. J. Med.
305:1439-1444). AIDS patients usually have a long asymptomatic
period followed by the progressive degeneration of the immune
system and the central nervous system. Replication of the virus is
highly regulated, and both latent and lytic infection of the CD4
positive helper subset of T-lymphocytes occur in tissue culture
(Zagury et al., (1986) Science 231:850-853). Molecular studies of
HIV-1 show that it encodes a number of genes (Ratner et al., (1985)
Nature 313:277-284; Sanchez-Pescador et al., (1985) Science
227:484-492), including three structural genes--gag, pol and
env--that are common to all retroviruses. Nucleotide sequences from
viral genomes of other retroviruses, particularly HIV-2 and simian
immunodeficiency viruses, SW (previously referred to as STLV-111),
also contain these structural genes. (Guyader et al., (1987) Nature
326:662-669; Chakrabarti et al., (1987) Nature
[0005] The envelope protein of HIV-1, HIV-2 and SN is a
glycoprotein of about 160 kd (gp160). During virus infection of the
host cell, gp160 is cleaved by host cell proteases to form gp120
and the integral membrane protein, gp41. The gp41 portion is
anchored in the membrane bilayer of virion, while the gp120 segment
protrudes into the surrounding environment. gp120 and gp41 are more
covalently associated and free gp120 can be released from the
surface of virions and infected cells.
[0006] As depicted in FIG. 1, crystallography studies of the gp120
core polypeptide indicate that this polypeptide is folded into two
major domains having certain emanating structures. The inner domain
(inner with respect to the N and C terminus) features a two-helix,
two-stranded bundle with a small five-stranded .beta.-sandwich at
its termini-proximal end and a projection at the distal end from
which the V1/V2 stem emanates. The outer domain is a staked double
barrel that lies along side the inner domain so that the outer
barrel and inner bundle axes are approximately parallel. Between
the distal inner domain and the distal outer domain is a
four-stranded bridging sheet which holds a peculiar minidomain in
contact with, but distinct from, the inner, the outer domain, and
the V1/V2 domain. The bridging sheet is composed of four
.beta.-strand structures (.beta.-3, .beta.-2, .beta.-21, .beta.-20,
shown in FIG. 1). The bridging region can be seen in FIG. 1 packing
primarily over the inner domain, although some surface residues of
the outer domain, such as Phe 382, reach into the bridging sheet to
form part of its hydrophobic core.
[0007] The basic unit of the .beta.-sheet conformation of the
bridging sheet region is the .beta.-strand which exists as a less
tightly coiled helix, with 2.0 residues per turn. The .beta.-strand
conformation is only stable when incorporated into a .beta.-sheet,
where hydrogen bonds with close to optimal geometry are formed
between the peptide groups on adjacent .beta.-strands; the dipole
moments of the strands are also aligned favorably. Side chains from
adjacent residues of the same strand protrude from opposite sides
of the sheet and do not interact with each other, but have
significant interactions with their backbone and with the side
chains of neighboring strands. For a general description of
.beta.-sheets, see, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); and A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., 1975).
[0008] The gp120 polypeptide is instrumental in mediating entry
into the host cell. Recent studies have indicated that binding of
CD4 to gp120 induces a conformational change in Env that allows for
binding to a co-receptor (e.g, a chemokine receptor) and subsequent
entry of the virus into the cell. (Wyatt, R., et al. (1998) Nature
393:705-711; Kwong, P., et al. (1998) Nature 393:648-659).
Referring again to FIG. 1, CD4 is bound into a depression formed at
the interface of the outer domain, the inner domain and the
bridging sheet of gp120.
[0009] Immunogenicity of the gp120 polypeptide has also been
studied. For example, individuals infected by HIV-1 usually develop
antibodies that can neutralize the virus in in vitro assays, and
this response is directed primarily against linear neutralizing
determinants in the third variable loop of gp120 glycoprotein
(Javaherian, K., et al. (1989) Proc. Natl. Acad. Sci. 86:6786-6772;
Matsushita, M., et al. (1988) J. Virol. 62:2107-2144; Putney, S.,
et al. (1986) Science 234:1392-1395; Rushe, J. R., et al. (1988)
Proc. Nat. Acad. Sci. USA 85: 3198-3202.). However, these
antibodies generally exhibit the ability to neutralize only a
limited number of HIV-1 strains (Matthews, T. (1986) Proc. Natl.
Acad. Sci. USA. 83:9709-9713; Nara, P. L., et al. (1988) J. Virol.
62:2622-2628; Palker, T. J., et al. (1988) Proc. Natl. Acad. Sci.
USA. 85:1932-1936). Later in the course of HIV infection in humans,
antibodies capable of neutralizing a wider range of HIV-1 isolates
appear (Barre-Sinoussi, F., et al. (1983) Science 220:868-871;
Robert-Guroff, M., et al. (1985) Nature (London) 316:72-74; Weis,
R., et al. (1985) Nature (London) 316:69-72; Weis, R., et al.
(1986) Nature (London) 324:572-575).
[0010] Recent work done by Stamatatos et al (1998) AIDS Res Hum
Retroviruses 14(13):1129-39, shows that a deletion of the variable
region 2 from a HIV-1.sub.SF162 virus, which utilizes the CCR-5
co-receptor for virus entry, rendered the virus highly susceptible
to serum-mediated neutralization. This V2 deleted virus was also
neutralized by sera obtained from patients infected not only with
Glade B HIV-1 isolates but also with Glade A, C, D and F HIV-1
isolates. However, deletion of the variable region 1 had no effect.
Deletion of the variable regions 1 and 2 from a LAI isolate
HIV-I.sub.IIIB also increased the susceptibility to neutralization
by monoclonal antibodies whose epitopes are located within the V3
loop, the CD4-binding site, and conserved gp120 regions (Wyatt, R.,
et al. (1995) J Virol. 69:5723-5733). Rabbit immunogenicity studies
done with the HIV-1 virus with deletions in the V1/V2 and V3 region
from the LAI strain, which uses the CXCR4 co-receptor for virus
entry, showed no improvement in the ability of Env to raise
neutralizing antibodies (Leu et al. (1998) AIDS Res. and Human
Retroviruses. 14:151-155).
[0011] Further, a subset of the broadly reactive antibodies, found
in most infected-individuals, interferes with the binding of gp120
and CD4 (Kang, C.-Y., et al. (1991) Proc. Natl. Acad. Sci. USA.
88:6171-6175; McDougal, J. S., et al. (1986) J. Immunol.
137:2937-2944). Other antibodies are believed to bind to the
chemokine receptor binding region after CD4 has bound to Env (Thali
et al. (1993) J. Virol. 67:3978-3988). The fact that neutralizing
antibodies generated during the course of HIV infection do not
provide permanent antiviral effect may in part be due to the
generation of "neutralization escapes" virus mutants and to the
general decline in the host immune system associated with
pathogenesis. In contrast, the presence of pre-existing
neutralizing antibodies upon initial HIV-1 exposure will likely
have a protective effect.
[0012] It is widely thought that a successful vaccine should be
able to induce a strong, broadly neutralizing antibody response
against diverse HIV-1 strains (Montefiori and Evans (1999) AIDS
Res. Hum. Ret. 15(8):689-698; Bolognesi, D. P., et al. (1994) Ann.
Int. Med. 8:603-611; Haynes, B., F., et al. (1996) Science; 271:
324-328.). Neutralizing antibodies, by attaching to the incoming
virions, can reduce or even prevent their infectivity for target
cells and prevent the cell-to-cell spread of virus in tissue
culture (Hu et al. (1992) Science 255:456-459; Burton, D., R. and
Montefiori, D. (1997) AIDS 11(suppl. A): 587-598). However as
described above, antibodies directed against gp120 do not generally
exhibit broad antibody responses against different HIV strains.
[0013] Currently, the focus of vaccine development, from the
perspective of humoral immunity, is on the neutralization of
primary isolates that utilize the CCR5 chemokine co-receptor
believed to be important in virus entry (Zhu, T., et al. (1993)
Science 261:1179-1181; Fiore, J., et al. (1994) Virology;
204:297-303). These viruses are generally much more resistant to
antibody neutralization than T-cell line adapted strains that use
the CXCR4 co-receptor, although both can be neutralized in vitro by
certain broadly and potent acting monoclonal antibodies, such as
IgG1b12, 2G12 and 2F5 (Trkola, A., et al. (1995) J. Virol.
69:6609-6617; D'Sousa P M., et al (1997) J. Infect. Dis.
175:1062-1075). These monoclonal antibodies are directed to the CD4
binding site, a glycosylation site and to the gp41 fusion domain,
respectively. The problem that remains, however, is that it is not
known how to induce antibodies of the appropriate specificity by
vaccination. Antibodies (Abs) elicited by gp120 glycoprotein from a
given isolate are usually only able to neutralize closely related
viruses generally from similar, usually from the same, HIV-1
subtype.
[0014] Despite the above approaches, there remains a need for Env
antigens that can elicit an immunological response (e.g.,
neutralizing and/or protective antibodies) in a subject against
multiple HIV strains and subtypes, for example when administered as
a vaccine. The present invention solves these and other problems by
providing modified Env polypeptides (e.g., gp120) to expose
epitopes in or near the CD4 binding site.
SUMMARY OF THE INVENTION
[0015] In accordance with the present invention, modified HIV Env
polypeptides are provided. In particular, deletions and/or
mutations are made in one or more of the
4-.beta.antiparallel-bridging sheet in the HIV Env polypeptide. In
this way, enough structure is left to allow correct folding of the
polypeptide, for example of gp120, yet enough of the bridging sheet
is removed to expose the CD4 groove, allowing an immune response to
be generated against epitopes in or near the CD4 binding site of
the Env polypeptide (e.g., gp120).
[0016] In one aspect, the invention includes a polynucleotide
encoding a modified HIV Env polypeptide wherein the polypeptide has
at least one modified (e.g., deleted or replaced) amino acid
residue deleted in the region corresponding to residues 421 to 436
relative to HXB-2, for example the constructs depicted in FIGS.
6-29 (SEQ ID NOs:3 to 26). In certain embodiments, the
polynucleotide also has the region corresponding to residues
124-198 of the polypeptide HXB-2 (e.g., V1/V2) deleted and at least
one amino acid deleted or replaced in the regions corresponding to
the residues 119 to 123 and 199 to 210, relative to HXB-2. In other
embodiments, these polynucleotides encode Env polypeptides having
at least one amino acid of the small loop of the bridging sheet
(e.g., amino acid residues 427 to 429 relative to HXB-2) deleted or
replaced. The amino acid sequences of the modified polypeptides
encoded by the polynucleotides of the present invention can be
based on any HIV variant, for example SF162.
[0017] In another aspect, the invention includes immunogenic
modified HIV Env polypeptides having at least one modified (e.g.,
deleted or replaced) amino acid residue deleted in the region
corresponding to residues 421 to 436 relative to HXB-2, for example
a deletion or replacement of one amino acids in the small loop
region (e.g., amino acid residues 427 to 429 relative to HXB-2).
These polypeptides may have modifications (e.g., a deletion or a
replacement) of at least one amino acid between about amino acid
residue 420 and amino acid residue 436, relative to HXB-2 and,
optionally, may have deletions or truncations of the V1 and/or V2
regions. The immunogenic, modified polypeptides of the present
invention can be based on any HIV variant, for example SF162.
[0018] In another aspect, the invention includes a vaccine
composition comprising any of the polynucleotides encoding modified
Env polypeptides described above. Vaccine compositions comprising
the modified Env polypeptides and, optionally, an adjuvant are also
included in the invention.
[0019] In yet another aspect, the invention includes a method of
inducing an immune response in subject comprising, administering
one or more of the polynucleotides or constructs described above in
an amount sufficient to induce an immune response in the subject.
In certain embodiments, the method further comprises administering
an adjuvant to the subject.
[0020] In another aspect, the invention includes a method of
inducing an immune response in a subject comprising administering a
composition comprising any of the modified Env polypeptides
described above and an adjuvant. The composition is administered in
an amount sufficient to induce an immune response in the
subject.
[0021] In another aspect, the invention includes a method of
inducing an immune response in a subject comprising
[0022] (a) administering a first composition comprising any of the
polynucleotides described above in a priming step and
[0023] (b) administering a second composition comprising any of the
modified Env polypeptides described above, as a booster, in an
amount sufficient to induce an immune response in the subject. In
certain embodiments, the first composition, the second composition
or both the first and second compositions further comprise an
adjuvant.
[0024] These and other embodiments of the subject invention will
readily occur to those of skill in the art in light of the
disclosure herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a schematic depiction of the tertiary structure of
the HIV-1.sub.HXB-2 Env gp120 polypeptide, as determined by
crystallography studies.
[0026] FIGS. 2A-C depict alignment of the amino acid sequence of
wild-type HIV-1.sub.HXB-2 Env gp160 polypeptide (SEQ ID NO:1) with
amino acid sequence of HIV variants SF162 (shown as "162") (SEQ ID
NO:2), SF2, CM236 and US4. Arrows indicate the regions that are
deleted or replaced in the modified polypeptides. Black dots
indicate conserved cysteine residues. The star indicates the
position of the last amino acid in gp120.
[0027] FIGS. 3A-J depict alignment of nucleotide sequences of
polynucleotides encoding modified Env polypeptides having V1/V2
deletions. The unmodified amino acid residues encoded by these
sequences correspond to wildtype SF162 residues but are numbered
relative to HXB-2.
[0028] FIGS. 4A-M depict alignment of nucleotide sequences of
polynucleotides encoding modified Env polypeptides having deletions
or replacements in the small loop. The unmodified amino acid
residues encoded by these sequences correspond to wildtype SF162
residues but are numbered relative to HXB-2.
[0029] FIGS. 5A-N depict alignment of nucleotide sequences of
polynucleotides encoding modified Env polypeptides having both
V1/V2 deletions and, in addition, deletions or replacements in the
small loop. The unmodified amino acid residues encoded by these
sequences correspond to wildtype SF162 residues but are numbered
relative to HXB-2.
[0030] FIG. 6 depicts the nucleotide sequence of the construct
designated Val120-Ala204 (SEQ ID NO:3).
[0031] FIG. 7 depicts the nucleotide sequence of the construct
designated Val120-Ile201 (SEQ ID NO:4).
[0032] FIG. 8 depicts the nucleotide sequence of the construct
designated Val120-Ile201B (SEQ ID NO:5).
[0033] FIG. 9 depicts the nucleotide sequence of the construct
designated Lys121-Val200 (SEQ ID NO:6).
[0034] FIG. 10 depicts the nucleotide sequence of the construct
designated Leu122-Ser199 (SEQ ID NO:7).
[0035] FIG. 11 depicts the nucleotide sequence of the construct
designated Val120-Thr202 (SEQ ID NO:8).
[0036] FIG. 12 depicts the nucleotide sequence of the construct
designated Trp427-Gly431 (SEQ ID NO:9).
[0037] FIG. 13 depicts the nucleotide sequence of the construct
designated Arg426-Gly431 (SEQ ID NO:10).
[0038] FIG. 14 depicts the nucleotide sequence of the construct
designated Arg426-Gly431B (SEQ ID NO:11).
[0039] FIG. 15 depicts the nucleotide sequence of the construct
designated Arg426-Lys432 (SEQ ID NO:12).
[0040] FIG. 16 depicts the nucleotide sequence of the construct
designated Asn425-Lys432 (SEQ ID NO:13).
[0041] FIG. 17 depicts the nucleotide sequence of the construct
designated Ile424-Ala433 (SEQ ID NO:14).
[0042] FIG. 18 depicts the nucleotide sequence of the construct
designated Ile423-Met434 (SEQ ID NO:15).
[0043] FIG. 19 depicts the nucleotide sequence of the construct
designated Gln422-Tyr435 (SEQ ID NO:16).
[0044] FIG. 20 depicts the nucleotide sequence of the construct
designated Gln422-Tyr435B (SEQ ID NO:17).
[0045] FIG. 21 depicts the nucleotide sequence of the construct
designated Leu122-Ser199; Arg426-Gly431 (SEQ ID NO:18).
[0046] FIG. 22 depicts the nucleotide sequence of the construct
designated Leu122-Ser199; Arg426-Lys432 (SEQ ID NO:19).
[0047] FIG. 23 depicts the nucleotide sequence of the construct
designated Leu122-Ser199; Trp427-Gly431 (SEQ ID NO:20).
[0048] FIG. 24 depicts the nucleotide sequence of the construct
designated Lys121-Val200; Asn425-Lys432 (SEQ ID NO:21).
[0049] FIG. 25 depicts the nucleotide sequence of the construct
designated Val120-Ile201; Ile424-Ala433 (SEQ ID NO:22).
[0050] FIG. 26 depicts the nucleotide sequence of the construct
designated Val120-Ile201B; Ile424-Ala433 (SEQ ID NO:23).
[0051] FIG. 27 depicts the nucleotide sequence of the construct
designated Val120-Thr202; Ile424-Ala433 (SEQ ID NO:24).
[0052] FIG. 28 depicts the nucleotide sequence of the construct
designated Val127-Asn195 (SEQ ID NO:25).
[0053] FIG. 29 depicts the nucleotide sequence of the construct
designated Val127-Asn195; Arg426-Gly431 (SEQ ID NO:26).
DETAILED DESCRIPTION OF THE INVENTION
[0054] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
viral immunobiology, molecular biology and recombinant DNA
techniques within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., T. E. Creighton,
Proteins: Structures and Molecular Properties (W.H. Freeman and
Company, 1993); Nelson L. M. and Jerome H. K. HIV Protocols in
Methods in Molecular Medicine, vol. 17, 1999; Sambrook, et al.,
Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Laboratory, 1989); F. M. Ausubel et al. Current Protocols in
Molecular Biology, Greene Publishing Associates & Wiley
Interscience New York; and Lipkowitz and Boyd, Reviews in
Computational Chemistry, volumes 1-present (Wiley-VCH, New York,
N.Y., 1999).
[0055] It must be noted that, as used in this specification and the
appended claims, the singular forms "a", "an" and "the" include
plural referents unless the content clearly dictates otherwise.
Thus, for example, reference to "a polypeptide" includes a mixture
of two or more polypeptides, and the like.
[0056] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0057] Definitions
[0058] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0059] The terms "polypeptide," and "protein" are used
interchangeably herein to denote any polymer of amino acid
residues. The terms encompass peptides, oligopeptides, dimers,
multimers, and the like. Such polypeptides can be derived from
natural sources or can be synthesized or recombinantly produced.
The terms also include postexpression modifications of the
polypeptide, for example, glycosylation, acetylation,
phosphorylation, etc.
[0060] A polypeptide as defined herein is generally made up of the
20 natural amino acids Ala (A), Arg (R), Asn (N), Asp (D), Cys (C),
Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met
(M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y) and Val
(V) and may also include any of the several known amino acid
analogs, both naturally occurring and synthesized analogs, such as
but not limited to homoisoleucine, asaleucine,
2-(methylenecyclopropyl)glycine, S-methylcysteine,
S-(prop-1-enyl)cysteine, homoserine, ornithine, norleucine,
norvaline, homoarginine, 3-(3-carboxyphenyl)alanine,
cyclohexylalanine, mimosine, pipecolic acid, 4-methylglutamic acid,
canavanine, 2,3-diaminopropionic acid, and the like. Further
examples of polypeptide agents which will find use in the present
invention are set forth below.
[0061] By "geometry" or "tertiary structure" of a polypeptide or
protein is meant the overall 3-D configuration of the protein. As
described herein, the geometry can be determined, for example, by
crystallography studies or by using various programs or algorithms
which predict the geometry based on interactions between the amino
acids making up the primary and secondary structures.
[0062] By "wild type" polypeptide, polypeptide agent or polypeptide
drug, is meant a naturally occurring polypeptide sequence, and its
corresponding secondary structure. An "isolated" or "purified"
protein or polypeptide is a protein which is separate and discrete
from a whole organism with which the protein is normally associated
in nature. It is apparent that the term denotes proteins of various
levels of purity. Typically, a composition containing a purified
protein will be one in which at least about 35%, preferably at
least about 40-50%, more preferably, at least about 75-85%, and
most preferably at least about 90% or more, of the total protein in
the composition will be the protein in question.
[0063] By "Env polypeptide" is meant a molecule derived from an
envelope protein, preferably from HIV Env. The envelope protein of
HIV-1 is a glycoprotein of about 160 kd (gp160). During virus
infection of the host cell, gp160 is cleaved by host cell proteases
to form gp120 and the integral membrane protein, gp41. The gp41
portion is anchored in (and spans) the membrane bilayer of virion,
while the gp120 segment protrudes into the surrounding environment.
As there is no covalent attachment between gp120 and gp41, free
gp120 is released from the surface of virions and infected cells.
Env polypeptides may also include gp140 polypeptides. Env
polypeptides can exist as monomers, dimers or multimers.
[0064] By a "gp120 polypeptide" is meant a molecule derived from a
gp120 region of the Env polypeptide. Preferably, the gp120
polypeptide is derived from HIV Env. The primary amino acid
sequence of gp120 is approximately 511 amino acids, with a
polypeptide core of about 60,000 daltons. The polypeptide is
extensively modified by N-linked glycosylation to increase the
apparent molecular weight of the molecule to 120,000 daltons. The
amino acid sequence of gp120 contains five relatively conserved
domains interspersed with five hypervariable domains. The positions
of the 18 cysteine residues in the gp120 primary sequence of the
HIV-1.sub.HXB-2 (hereinafter "HXB-2") strain, and the positions of
13 of the approximately 24 N-linked glycosylation sites in the
gp120 sequence are common to most, if not all, gp120 sequences. The
hypervariable domains contain extensive amino acid substitutions,
insertions and deletions. Despite this variation, most, if not all,
gp120 sequences preserve the virus's ability to bind to the viral
receptor CD4. A "gp120 polypeptide" includes both single subunits
or multimers.
[0065] Env polypeptides (e.g., gp120, gp140 and gp160) include a
"bridging sheet" comprised of 4 anti-parallel .beta.-strands
(.beta.-2, .beta.-3, .beta.-20 and (.beta.-21) that form a
.beta.-sheet. Extruding from one pair of the .beta.-strands
(.beta.-2 and .beta.-3) are two loops, V1 and V2. The .beta.-2
sheet occurs at approximately amino acid residue 119 (Cys) to amino
acid residue 123 (Thr) while .beta.-3 occurs at approximately amino
acid residue 199 (Ser) to amino acid residue 201 (He), relative to
HXB-2. The "V1/V2 region" occurs at approximately amino acid
positions 126 (Cys) to residue 196 (Cys), relative to HXB-2. (see,
e.g., Wyatt et al. (1995) J. Virol. 69:5723-5733; Stamatatos et al.
(1998) J. Virol. 72:7840-7845). Extruding from the second pair of
.beta.-strands (.beta.-20 and .beta.-21) is a "small-loop"
structure, also referred to herein as "the bridging sheet small
loop." In HXB-2, .beta.-20 extends from about amino acid residue
422 (Gln) to amino acid residue 426 (Met) while .beta.-21 extends
from about amino acid residue 430 (Val) to amino acid residue 435
(Tyr). In variant SF162, the Met-426 is an Arg (R) residue. The
"small loop" extends from about amino acid residue 427 (Tip)
through 429 (Lys), relative to HXB-2. A representative diagram of
gp120 showing the bridging sheet, the small loop, and V1/V2 is
shown in FIG. 1. In addition, alignment of the amino acid sequences
of Env polypeptide gp160 of selected variants is shown, relative to
HXB-2, in FIGS. 2A-C.
[0066] Furthermore, an "Env polypeptide" or "gp120 polypeptide" as
defined herein is not limited to a polypeptide having the exact
sequence described herein. Indeed, the HIV genome is in a state of
constant flux and contains several variable domains which exhibit
relatively high degrees of variability between isolates. It is
readily apparent that the terms encompass Env (e.g., gp120)
polypeptides from any of the identified HIV isolates, as well as
newly identified isolates, and subtypes of these isolates.
Descriptions of structural features are given herein with reference
to HXB-2. One of ordinary skill in the art in view of the teachings
of the present disclosure and the art can determine corresponding
regions in other HIV variants (e.g., isolates HIV.sub.IIIb,
HIV.sub.SF2, HIV-1.sub.SF162, HIV-1.sub.SF170, HIV.sub.LAV,
HIV.sub.LAI, HIV-1.sub.MN, HIV-1.sub.CM235, HIV-1.sub.US4, other
HIV-1 strains from diverse subtypes (e.g., subtypes, A through G,
and O), HIV-2 strains and diverse subtypes (e.g., HIV-2.sub.UC1 and
HIV-2.sub.UC2), and simian immunodeficiency virus (SIV). (See,
e.g., Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental
Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds. 1991);
Virology, 3rd Edition (Fields, B N, D M Knipe, P M Howley, Editors,
1996, Lippincott-Raven, Philadelphia, Pa.; for a description of
these and other related viruses), using for example, sequence
comparison programs (e.g., BLAST and others described herein) or
identification and alignment of structural features (e.g., a
program such as the "ALB" program described herein that can
identify .beta.-sheet regions). The actual amino acid sequences of
the modified Env polypeptides can be based on any HIV variant.
[0067] Additionally, the term "Env polypeptide" (e.g., "gp120
polypeptide") encompasses proteins which include additional
modifications to the native sequence, such as additional internal
deletions, additions and substitutions. These modifications may be
deliberate, as through site-directed mutagenesis, or may be
accidental, such as through naturally occurring mutational events.
Thus, for example, if the Env polypeptide is to be used in vaccine
compositions, the modifications must be such that immunological
activity (i.e., the ability to elicit an antibody response to the
polypeptide) is not lost. Similarly, if the polypeptides are to be
used for diagnostic purposes, such capability must be retained.
[0068] Thus, a "modified Env polypeptide" is an Env polypeptide
(e.g., gp120 as defined above), which has been manipulated to
delete or replace all or a part of the bridging sheet portion and,
optionally, the variable regions V1 and V2. Generally, modified Env
(e.g., gp120) polypeptides have enough of the bridging sheet
removed to expose the CD4 binding site, but leave enough of the
structure to allow correct folding (e.g., correct geometry). Thus,
modifications to the .beta.-20 and .beta.-21 regions (between about
amino acid residues 420 and 435 relative to HXB-2) are preferred.
Additionally, modifications to the .beta.-2 and .beta.-3 regions
(between about amino acid residues 119 (Cys) and 201 (Ile)) and
modifications (e.g., truncations) to the V1 and V2 loop regions may
also be made. Although not all possible .beta.-sheet and V1/V2
modifications have been exemplified herein, it is to be understood
that other disrupting modifications are also encompassed by the
present invention.
[0069] Normally, such a modified polypeptide is capable of
secretion into growth medium in which an organism expressing the
protein is cultured. However, for purposes of the present
invention, such polypeptides may also be recovered intracellularly.
Secretion into growth media is readily determined using a number of
detection techniques, including, e.g., polyacrylamide gel
electrophoresis and the like, and immunological techniques such as
Western blotting and immunoprecipitation assays as described in,
e.g., International Publication No. WO 96/04301, published Feb. 15,
1996.
[0070] A gp120 or other Env polypeptide is produced
"intracellularly" when it is found within the cell, either
associated with components of the cell, such as in association with
the endoplasmic reticulum (ER) or the Golgi Apparatus, or when it
is present in the soluble cellular fraction. The gp120 and other
Env polypeptides of the present invention may also be secreted into
growth medium so long as sufficient amounts of the polypeptides
remain present within the cell such that they can be purified from
cell lysates using techniques described herein.
[0071] An "immunogenic" gp120 or other Env protein is a molecule
that includes at least one epitope such that the molecule is
capable of either eliciting an immunological reaction in an
individual to which the protein is administered or, in the
diagnostic context, is capable of reacting with antibodies directed
against the HIV in question.
[0072] By "epitope" is meant a site on an antigen to which specific
B cells and/or T cells respond, rendering the molecule including
such an epitope capable of eliciting an immunological reaction or
capable of reacting with HIV antibodies present in a biological
sample. The term is also used interchangeably with "antigenic
determinant" or "antigenic determinant site." An epitope can
comprise 3 or more amino acids in a spatial conformation unique to
the epitope. Generally, an epitope consists of at least 5 such
amino acids and, more usually, consists of at least 8-10 such amino
acids. Methods of determining spatial conformation of amino acids
are known in the art and include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance.
Furthermore, the identification of epitopes in a given protein is
readily accomplished using techniques well known in the art, such
as by the use of hydrophobicity studies and by site-directed
serology. See, also, Geysen et al., Proc. Natl. Acad. Sci. USA
(1984) 81:3998-4002 (general method of rapidly synthesizing
peptides to determine the location of immunogenic epitopes in a
given antigen); U.S. Pat. No. 4,708,871 (procedures for identifying
and chemically synthesizing epitopes of antigens); and Geysen et
al., Molecular Immunology (1986) 23:709-715 (technique for
identifying peptides with high affinity for a given antibody).
Antibodies that recognize the same epitope can be identified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen.
[0073] An "immunological response" or "immune response" as used
herein is the development in the subject of a humoral and/or a
cellular immune response to the Env (e.g., gp120) polypeptide when
the polypeptide is present in a vaccine composition. These
antibodies may also neutralize infectivity, and/or mediate
antibody-complement or antibody dependent cell cytotoxicity to
provide protection to an immunized host. Immunological reactivity
may be determined in standard immunoassays, such as a competition
assays, well known in the art.
[0074] Techniques for determining amino acid sequence "similarity"
are well known in the art. In general, "similarity" means the exact
amino acid to amino acid comparison of two or more polypeptides at
the appropriate place, where amino acids are identical or possess
similar chemical and/or physical properties such as charge or
hydrophobicity. A so-termed "percent similarity" then can be
determined between the compared polypeptide sequences. Techniques
for determining nucleic acid and amino acid sequence identity also
are well known in the art and include determining the nucleotide
sequence of the mRNA for that gene (usually via a cDNA
intermediate) and determining the amino acid sequence encoded
thereby, and comparing this to a second amino acid sequence. In
general, "identity" refers to an exact nucleotide to nucleotide or
amino acid to amino acid correspondence of two polynucleotides or
polypeptide sequences, respectively.
[0075] Two or more polynucleotide sequences can be compared by
determining their "percent identity." Two or more amino acid
sequences likewise can be compared by determining their "percent
identity." The percent identity of two sequences, whether nucleic
acid or peptide sequences, is generally described as the number of
exact matches between two aligned sequences divided by the length
of the shorter sequence and multiplied by 100. An approximate
alignment for nucleic acid sequences is provided by the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981). This algorithm can be extended to use
with peptide sequences using the scoring matrix developed by
Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff
ed., 5 suppl. 3:353-358, National Biomedical Research Foundation,
Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res.
14(6):6745-6763 (1986). An implementation of this algorithm for
nucleic acid and peptide sequences is provided by the Genetics
Computer Group (Madison, Wis.) in their BestFit utility
application. The default parameters for this method are described
in the Wisconsin Sequence Analysis Package Program Manual, Version
8 (1995) (available from Genetics Computer Group, Madison, Wis.).
Other equally suitable programs for calculating the percent
identity or similarity between sequences are generally known in the
art.
[0076] For example, percent identity of a particular nucleotide
sequence to a reference sequence can be determined using the
homology algorithm of Smith and Waterman with a default scoring
table and a gap penalty of six nucleotide positions. Another method
of establishing percent identity in the context of the present
invention is to use the MPSRCH package of programs copyrighted by
the University of Edinburgh, developed by John F. Collins and Shane
S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain
View, Calif.). From this suite of packages, the Smith-Waterman
algorithm can be employed where default parameters are used for the
scoring table (for example, gap open penalty of 12, gap extension
penalty of one, and a gap of six). From the data generated, the
"Match" value reflects "sequence identity." Other suitable programs
for calculating the percent identity or similarity between
sequences are generally known in the art, such as the alignment
program BLAST, which can also be used with default parameters. For
example, BLASTN and BLASTP can be used with the following default
parameters: genetic code=standard; filter=none; strand=both;
cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences;
sort by =HIGH SCORE; Databases=non-redundant,
GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss
protein+Spupdate+PIR. Details of these programs can be found at the
following internet address:
http://www.ncbi.nlm.gov/cgi-bin/BLAST.
[0077] One of skill in the art can readily determine the proper
search parameters to use for a given sequence in the above
programs. For example, the search parameters may vary based on the
size of the sequence in question. Thus, for example, a
representative embodiment of the present invention would include an
isolated polynucleotide having X contiguous nucleotides, wherein
(i) the X contiguous nucleotides have at least about 50% identity
to Y contiguous nucleotides derived from any of the sequences
described herein, (ii) X equals Y, and (iii) X is greater than or
equal to 6 nucleotides and up to 5000 nucleotides, preferably
greater than or equal to 8 nucleotides and up to 5000 nucleotides,
more preferably 10-12 nucleotides and up to 5000 nucleotides, and
even more preferably 15-20 nucleotides, up to the number of
nucleotides present in the full-length sequences described herein
(e.g., see the Sequence Listing and claims), including all integer
values falling within the above-described ranges.
[0078] The synthetic expression cassettes (and purified
polynucleotides) of the present invention include related
polynucleotide sequences having about 80% to 100%, greater than
80-85%, preferably greater than 90-92%, more preferably greater
than 95%, and most preferably greater than 98% sequence (including
all integer values falling within these described ranges) identity
to the synthetic expression cassette sequences disclosed herein
(for example, to the claimed sequences or other sequences of the
present invention) when the sequences of the present invention are
used as the query sequence.
[0079] Computer programs are also available to determine the
likelihood of certain polypeptides to form structures such as
.beta.-sheets. One such program, described herein, is the "ALB"
program for protein and polypeptide secondary structure calculation
and predication. In addition, secondary protein structure can be
predicted from the primary amino acid sequence, for example using
protein crystal structure and aligning the protein sequence related
to the crystal structure (e.g., using Molecular Operating
Environment (MOE) programs available from the Chemical Computing
Group Inc., Montreal, P.Q., Canada). Other methods of predicting
secondary structures are described, for example, in Garnier et al.
(1996) Methods Enzymol. 266:540-553; Geourjon et al. (1995) Comput.
Applic. Biosci. 11:681-684; Levin (1997) Protein Eng. 10:771-776;
and Rost et al. (1993) J. Molec. Biol. 232:584-599.
[0080] Homology can also be determined by hybridization of
polynucleotides under conditions which form stable duplexes between
homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. Two DNA, or two polypeptide sequences are
"substantially homologous" to each other when the sequences exhibit
at least about 80%-85%, preferably at least about 90%, and most
preferably at least about 95%-98% sequence identity over a defined
length of the molecules, as determined using the methods above. As
used herein, substantially homologous also refers to sequences
showing complete identity to the specified DNA or polypeptide
sequence. DNA sequences that are substantially homologous can be
identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0081] A "coding sequence" or a sequence which "encodes" a selected
protein, is a nucleic acid sequence which is transcribed (in the
case of DNA) and translated (in the case of mRNA) into a
polypeptide in vitro or in vivo when placed under the control of
appropriate regulatory sequences. The boundaries of the coding
sequence are determined by a start codon at the 5' (amino) terminus
and a translation stop codon at the 3' (carboxy) terminus. A coding
sequence can include, but is not limited to cDNA from viral
nucleotide sequences as well as synthetic and semisynthetic DNA
sequences and sequences including base analogs. A transcription
termination sequence may be located 3' to the coding sequence.
[0082] "Control elements" refers collectively to promoter
sequences, ribosome binding sites, polyadenylation signals,
transcription termination sequences, upstream regulatory domains,
enhancers, and the like, which collectively provide for the
transcription and translation of a coding sequence in a host cell.
Not all of these control elements need always be present so long as
the desired gene is capable of being transcribed and
translated.
[0083] A control element "directs the transcription" of a coding
sequence in a cell when RNA polymerase will bind the promoter
sequence and transcribe the coding sequence into mRNA, which is
then translated into the polypeptide encoded by the coding
sequence.
[0084] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control elements operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence when RNA polymerase is present. The control
elements need not be contiguous with the coding sequence, so long
as they function to direct the expression thereof. Thus, for
example, intervening untranslated yet transcribed sequences can be
present between, e.g., a promoter sequence and the coding sequence
and the promoter sequence can still be considered "operably linked"
to the coding sequence.
[0085] "Recombinant" as used herein to describe a nucleic acid
molecule means a polynucleotide of genomic, cDNA, semisynthetic, or
synthetic origin which, by virtue of its origin or manipulation:
(1) is not associated with all or a portion of the polynucleotide
with which it is associated in nature; and/or (2) is linked to a
polynucleotide other than that to which it is linked in nature. The
term "recombinant" as used with respect to a protein or polypeptide
means a polypeptide produced by expression of a recombinant
polynucleotide. "Recombinant host cells," "host cells," "cells,"
"cell lines," "cell cultures," and other such terms denoting
pracaryotic microorganisms or eucaryotic cell lines cultured as
unicellular entities, are used interchangeably, and refer to cells
which can be, or have been, used as recipients for recombinant
vectors or other transfer DNA, and include the progeny of the
original cell which has been transfected. It is understood that the
progeny of a single parental cell may not necessarily be completely
identical in morphology or in genomic or total DNA complement to
the original parent, due to accidental or deliberate mutation.
Progeny of the parental cell which are sufficiently similar to the
parent to be characterized by the relevant property, such as the
presence of a nucleotide sequence encoding a desired peptide, are
included in the progeny intended by this definition, and are
covered by the above terms.
[0086] By "vertebrate subject" is meant any member of the subphylum
chordata, including, without limitation, humans and other primates,
including non-human primates such as chimpanzees and other apes and
monkey species; farm animals such as cattle, sheep, pigs, goats and
horses; domestic mammals such as dogs and cats; laboratory animals
including rodents such as mice, rats and guinea pigs; birds,
including domestic, wild and game birds such as chickens, turkeys
and other gallinaceous birds, ducks, geese, and the like. The term
does not denote a particular age. Thus, both adult and newborn
individuals are intended to be covered.
[0087] As used herein, a "biological sample" refers to a sample of
tissue or fluid isolated from an individual, including but not
limited to, for example, blood, plasma, serum, fecal matter, urine,
bone marrow, bile, spinal fluid, lymph fluid, samples of the skin,
external secretions of the skin, respiratory, intestinal, and
genitourinary tracts, samples derived from the gastric epithelium
and gastric mucosa, tears, saliva, milk, blood cells, organs,
biopsies and also samples of in vitro cell culture constituents
including but not limited to conditioned media resulting from the
growth of cells and tissues in culture medium, e.g., recombinant
cells, and cell components.
[0088] The terms "label" and "detectable label" refer to a molecule
capable of detection, including, but not limited to, radioactive
isotopes, fluorescers, chemiluminescers, enzymes, enzyme
substrates, enzyme cofactors, enzyme inhibitors, chromophores,
dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and
the like. The term "fluorescer" refers to a substance or a portion
thereof which is capable of exhibiting fluorescence in the
detectable range: Particular examples of labels which may be used
with the invention include, but are not limited to fluorescein,
rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum
esters, NADPH, .alpha.-.beta.-galactosidase, horseradish
peroxidase, glucose oxidase, alkaline phosphatase and urease.
[0089] Overview
[0090] The present invention concerns modified Env polypeptide
molecules (e.g., glycoprotein ("gp") 120). Without being bound by a
particular theory, it appears that it has been difficult to
generate immunological responses against Env because the CD4
binding site is buried between the outer domain, the inner domain
and the V1/V2 domains. Thus, although deletion of the V1/V2 domain
may render the virus more susceptible to neutralization by
monoclonal antibody directed to the CD4 site, the bridging sheet
covering most of the CD4 binding domain may prevent an antibody
response. Thus, the present invention provides Env polypeptides
that maintain their general overall structure yet expose the CD4
binding domain. This allows the generation of an immune response
(e.g., an antibody response) to epitopes in or near the CD4 binding
site.
[0091] Various forms of the different embodiments of the invention,
described herein, may be combined.
[0092] .beta.-Sheet Conformations
[0093] In the present invention, location of the .beta.-sheet
structures were identified relative to 3-D (crystal) structure of
an HXB-2 crystallized Env protein (see, Example 1A). Based on this
structure, constructs encoding polypeptides having replacements and
or excisions which maintain overall geometry while exposing the CD4
binding site were designed. In particular, the crystal structure of
HXB-2 was downloaded from the Brookhaven Database. Using the
default parameters of the Loop Search feature of the Biopolymer
module of the Sybyl molecular modeling package, homology and fit of
amino acids which could replace the native loops between
.beta.-strands yet maintain overall tertiary structure were
determined. Constructs encoding the modified Env polypeptides were
then designed (Example 1.B.).
[0094] Thus, the modified Env polypeptides typically have enough of
the bridging sheet removed to expose the CD4 groove, but have
enough of the structure to allow correct folding of the Env
glycoprotein. Exemplary constructs are described below.
[0095] Polypeptide Production
[0096] The polypeptides of the present invention can be produced in
any number of ways which are well known in the art.
[0097] In one embodiment, the polypeptides are generated using
recombinant techniques, well known in the art. In this regard,
oligonucleotide probes can be devised based on the known sequences
of the Env (e.g., gp120) polypeptide genome and used to probe
genomic or cDNA libraries for Env genes. The gene can then be
further isolated using standard techniques and, e.g., restriction
enzymes employed to truncate the gene at desired portions of the
full-length sequence. Similarly, the Env gene(s) can be isolated
directly from cells and tissues containing the same, using known
techniques, such as phenol extraction and the sequence further
manipulated to produce the desired truncations. See, e.g., Sambrook
et al., supra, for a description of techniques used to obtain and
isolate DNA.
[0098] The genes encoding the modified (e.g., truncated and/or
substituted) polypeptides can be produced synthetically, based on
the known sequences. The nucleotide sequence can be designed with
the appropriate codons for the particular amino acid sequence
desired. The complete sequence is generally assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge (1981)
Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al.
(1984) J. Biol. Chem. 259:6311; Stemmer et al. (1995) Gene
164:49-53.
[0099] Recombinant techniques are readily used to clone a gene
encoding an Env polypeptide gene which can then be mutagenized in
vitro by the replacement of the appropriate base pair(s) to result
in the codon for the desired amino acid. Such a change can include
as little as one base pair, effecting a change in a single amino
acid, or can encompass several base pair changes. Alternatively,
the mutations can be effected using a mismatched primer which
hybridizes to the parent nucleotide sequence (generally cDNA
corresponding to the RNA sequence), at a temperature below the
melting temperature of the mismatched duplex. The primer can be
made specific by keeping primer length and base composition within
relatively narrow limits and by keeping the mutant base centrally
located. See, e.g., Innis et al, (1990) PCR Applications: Protocols
for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983)
100:468. Primer extension is effected using DNA polymerase, the
product cloned and clones containing the mutated DNA, derived by
segregation of the primer extended strand, selected. Selection can
be accomplished using the mutant primer as a hybridization probe.
The technique is also applicable for generating multiple point
mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad.
Sci. USA (1982) 79:6409.
[0100] Once coding sequences for the desired proteins have been
isolated or synthesized, they can be cloned into any suitable
vector or replicon for expression. As will be apparent from the
teachings herein, a wide variety of vectors encoding modified
polypeptides can be generated by creating expression constructs
which operably link, in various combinations, polynucleotides
encoding Env polypeptides having deletions or mutation therein.
Thus, polynucleotides encoding a particular deleted V1/V2 region
can be operably linked with polynucleotides encoding polypeptides
having deletions or replacements in the small loop region and the
construct introduced into a host cell for polypeptide expression.
Non-limiting examples of such combinations are discussed in the
Examples.
[0101] Numerous cloning vectors are known to those of skill in the
art, and the selection of an appropriate cloning vector is a matter
of choice. Examples of recombinant DNA vectors for cloning and host
cells which they can transform include the bacteriophage .lamda.
(E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative
bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells).
See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et
al., supra; B. Perbal, supra.
[0102] Insect cell expression systems, such as baculovirus systems,
can also be used and are known to those of skill in the art and
described in, e.g., Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987). Materials and methods
for baculovirus/insect cell expression systems are commercially
available in kit form from, inter alia, Invitrogen, San Diego
Calif. ("MaxBac" kit).
[0103] Plant expression systems can also be used to produce the
modified Env proteins. Generally, such systems use virus-based
vectors to transfect plant cells with heterologous genes. For a
description of such systems see, e.g., Porta et al., Mol. Biotech.
(1996) 5:209-221; and Hackland et al., Arch. Virol. (1994)
139:1-22.
[0104] Viral systems, such as a vaccinia based
infection/transfection system, as described in Tomei et al., J.
Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)
74:1103-1113, will also find use with the present invention. In
this system, cells are first transfected in vitro with a vaccinia
virus recombinant that encodes the bacteriophage T7 RNA polymerase.
This polymerase displays exquisite specificity in that it only
transcribes templates bearing T7 promoters. Following infection,
cells are transfected with the DNA of interest, driven by a T7
promoter. The polymerase expressed in the cytoplasm from the
vaccinia virus recombinant transcribes the transfected DNA into RNA
which is then translated into protein by the host translational
machinery. The method provides for high level, transient,
cytoplasmic production of large quantities of RNA and its
translation product(s).
[0105] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator (collectively referred to herein as "control"
elements), so that the DNA sequence encoding the desired Env
polypeptide is transcribed into RNA in the host cell transformed by
a vector containing this expression construction. The coding
sequence may or may not contain a signal peptide or leader
sequence. With the present invention, both the naturally occurring
signal peptides or heterologous sequences can be used. Leader
sequences can be removed by the host in post-translational
processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437;
4,338,397. Such sequences include, but are not limited to, the TPA
leader, as well as the honey bee mellitin signal sequence.
[0106] Other regulatory sequences may also be desirable which allow
for regulation of expression of the protein sequences relative to
the growth of the host cell. Such regulatory sequences are known to
those of skill in the art, and examples include those which cause
the expression of a gene to be turned on or off in response to a
chemical or physical stimulus, including the presence of a
regulatory compound. Other types of regulatory elements may also be
present in the vector, for example, enhancer sequences.
[0107] The control sequences and other regulatory sequences may be
ligated to the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site.
[0108] In some cases it may be necessary to modify the coding
sequence so that it may be attached to the control sequences with
the appropriate orientation; i.e., to maintain the proper reading
frame. Mutants or analogs may be prepared by the deletion of a
portion of the sequence encoding the protein, by insertion of a
sequence, and/or by substitution of one or more nucleotides within
the sequence. Techniques for modifying nucleotide sequences, such
as site-directed mutagenesis, are well known to those skilled in
the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I
and II, supra; Nucleic Acid Hybridization, supra.
[0109] The expression vector is then used to transform an
appropriate host cell. A number of mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others.
Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and
Streptococcus spp., will find use with the present expression
constructs. Yeast hosts useful in the present invention include
inter alia, Saccharomyces cerevisiae, Candida albicans, Candida
maltosa, Hansenula polymorpha, Kluyveromyces fragilis,
Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,
Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for
use with baculovirus expression vectors include, inter alia, Aedes
aegypti, Autographa californica, Bombyx mori, Drosophila
melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
[0110] Depending on the expression system and host selected, the
proteins of the present invention are produced by growing host
cells transformed by an expression vector described above under
conditions whereby the protein of interest is expressed. The
selection of the appropriate growth conditions is within the skill
of the art.
[0111] In one embodiment, the transformed cells secrete the
polypeptide product into the surrounding media. Certain regulatory
sequences can be included in the vector to enhance secretion of the
protein product, for example using a tissue plasminogen activator
(TPA) leader sequence, a .gamma.-interferon signal sequence or
other signal peptide sequences from known secretory proteins. The
secreted polypeptide product can then be isolated by various
techniques described herein, for example, using standard
purification techniques such as but not limited to, hydroxyapatite
resins, column chromatography, ion-exchange chromatography,
size-exclusion chromatography, electrophoresis, HPLC,
immunoadsorbent techniques, affinity chromatography,
immunoprecipitation, and the like.
[0112] Alternatively, the transformed cells are disrupted, using
chemical, physical or mechanical means, which lyse the cells yet
keep the Env polypeptides substantially intact. Intracellular
proteins can also be obtained by removing components from the cell
wall or membrane, e.g., by the use of detergents or organic
solvents, such that leakage of the Env polypeptides occurs. Such
methods are known to those of skill in the art and are described
in, e.g., Protein Purification Applications: A Practical Approach,
(E. L. V. Harris and S. Angal, Eds., 1990)
[0113] For example, methods of disrupting cells for use with the
present invention include but are not limited to: sonication or
ultrasonication; agitation; liquid or solid extrusion; heat
treatment; freeze-thaw; desiccation; explosive decompression;
osmotic shock; treatment with lytic enzymes including proteases
such as trypsin, neuraminidase and lysozyme; alkali treatment; and
the use of detergents and solvents such as bile salts, sodium
dodecylsulphate, Triton, NP40 and CHAPS. The particular technique
used to disrupt the cells is largely a matter of choice and will
depend on the cell type in which the polypeptide is expressed,
culture conditions and any pre-treatment used.
[0114] Following disruption of the cells, cellular debris is
removed, generally by centrifugation, and the intracellularly
produced Env polypeptides are further purified, using standard
purification techniques such as but not limited to, column
chromatography, ion-exchange chromatography, size-exclusion
chromatography, electrophoresis, HPLC, immunoadsorbent techniques,
affinity chromatography, immunoprecipitation, and the like.
[0115] For example, one method for obtaining the intracellular Env
polypeptides of the present invention involves affinity
purification, such as by immunoaffinity chromatography using
anti-Env specific antibodies, or by lectin affinity chromatography.
Particularly preferred lectin resins are those that recognize
mannose moieties such as but not limited to resins derived from
Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA
or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin),
Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum
agglutinin (AUA). The choice of a suitable affinity resin is within
the skill in the art. After affinity purification, the Env
polypeptides can be further purified using conventional techniques
well known in the art, such as by any of the techniques described
above.
[0116] It may be desirable to produce Env (e.g., gp120) complexes,
either with itself or other proteins. Such complexes are readily
produced by e.g., co-transfecting host cells with constructs
encoding for the Env (e.g., gp120) and/or other polypeptides of the
desired complex. Co-transfection can be accomplished either in
trans or cis, i.e., by using separate vectors or by using a single
vector which bears both of the Env and other gene. If done using a
single vector, both genes can be driven by a single set of control
elements or, alternatively, the genes can be present on the vector
in individual expression cassettes, driven by individual control
elements. Following expression, the proteins will spontaneously
associate. Alternatively, the complexes can be formed by mixing the
individual proteins together which have been produced separately,
either in purified or semi-purified form, or even by mixing culture
media in which host cells expressing the proteins, have been
cultured. See, International Publication No. WO 96/04301, published
Feb. 15, 1996, for a description of such complexes.
[0117] Relatively small polypeptides, i.e., up to about 50 amino
acids in length, can be conveniently synthesized chemically, for
example by any of several techniques that are known to those
skilled in the peptide art. In general, these methods employ the
sequential addition of one or more amino acids to a growing peptide
chain. Normally, either the amino or carboxyl group of the first
amino acid is protected by a suitable protecting group. The
protected or derivatized amino acid can then be either attached to
an inert solid support or utilized in solution by adding the next
amino acid in the sequence having the complementary (amino or
carboxyl) group suitably protected, under conditions that allow for
the formation of an amide linkage. The protecting group is then
removed from the newly added amino acid residue and the next amino
acid (suitably protected) is then added, and so forth. After the
desired amino acids have been linked in the proper sequence, any
remaining protecting groups (and any solid support, if solid phase
synthesis techniques are used) are removed sequentially or
concurrently, to render the final polypeptide. By simple
modification of this general procedure, it is possible to add more
than one amino acid at a time to a growing chain, for example, by
coupling (under conditions which do not racemize chiral centers) a
protected tripeptide with a properly protected dipeptide to form,
after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co.,
Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis. Synthesis. Biology, editors E. Gross and J.
Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254,
for solid phase peptide synthesis techniques; and M. Bodansky,
Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984) and
E. Gross and J. Meienhofer, Eds., The Peptides: Analysis,
Synthesis, Biology, Vol. 1, for classical solution synthesis.
[0118] Typical protecting groups include t-butyloxycarbonyl (Boc),
9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);
p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);
biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,
isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl,
isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
[0119] Typical solid supports are cross-linked polymeric supports.
These can include divinylbenzene cross-linked-styrene-based
polymers, for example, divinylbenzene-hydroxymethylstyrene
copolymers, divinylbenzene-chloromethylstyrene copolymers and
divinylbenzene-benzhydrylaminopolystyrene copolymers.
[0120] The polypeptide analogs of the present invention can also be
chemically prepared by other methods such as by the method of
simultaneous multiple peptide synthesis. See, e.g., Houghten Proc.
Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Pat. No.
4,631,211.
[0121] Diagnostic and Vaccine Applications
[0122] The intracellularly produced Env polypeptides of the present
invention, complexes thereof, or the polynucleotides coding
therefor, can be used for a number of diagnostic and therapeutic
purposes. For example, the proteins and polynucleotides or
antibodies generated against the same, can be used in a variety of
assays, to determine the presence of reactive antibodies/and or Env
proteins in a biological sample to aid in the diagnosis of HIV
infection or disease status or as measure of response to
immunization.
[0123] The presence of antibodies reactive with the Env (e.g.,
gp120) polypeptides and, conversely, antigens reactive with
antibodies generated thereto, can be detected using standard
electrophoretic and immunodiagnostic techniques, including
immunoassays such as competition, direct reaction, or sandwich type
assays. Such assays include, but are not limited to, western blots;
agglutination tests; enzyme-labeled and mediated immunoassays, such
as ELISAs; biotin/avidin type assays; radioimmunoassays;
immunoelectrophoresis; immunoprecipitation, etc. The reactions
generally include revealing labels such as fluorescent,
chemiluminescent, radioactive, or enzymatic labels or dye
molecules, or other methods for detecting the formation of a
complex between the antigen and the antibody or antibodies reacted
therewith.
[0124] Solid supports can be used in the assays such as
nitrocellulose, in membrane or microtiter well form;
polyvinylchloride, in sheets or microtiter wells; polystyrene
latex, in beads or microtiter plates; polyvinylidine fluoride;
diazotized paper; nylon membranes; activated beads, and the
like.
[0125] Typically, the solid support is first reacted with the
biological sample (or the gp120 proteins), washed and then the
antibodies, (or a sample suspected of containing antibodies),
applied. After washing to remove any non-bound ligand, a secondary
binder moiety is added under suitable binding conditions, such that
the secondary binder is capable of associating selectively with the
bound ligand. The presence of the secondary binder can then be
detected using techniques well known in the art. Typically, the
secondary binder will comprise an antibody directed against the
antibody ligands. A number of anti-human immunoglobulin (Ig)
molecules are known in the art (e.g., commercially available goat
anti-human Ig or rabbit anti-human Ig). Ig molecules for use herein
will preferably be of the IgG or IgA type, however, IgM may also be
appropriate in some instances. The Ig molecules can be readily
conjugated to a detectable enzyme label, such as horseradish
peroxidase, glucose oxidase, Beta-galactosidase, alkaline
phosphatase and urease, among others, using methods known to those
of skill in the art. An appropriate enzyme substrate is then used
to generate a detectable signal.
[0126] Alternatively, a "two antibody sandwich" assay can be used
to detect the proteins of the present invention. In this technique,
the solid support is reacted first with one or more of the
antibodies directed against Env (e.g., gp120), washed and then
exposed to the test sample. Antibodies are again added and the
reaction visualized using either a direct color reaction or using a
labeled second antibody, such as an anti-immunoglobulin labeled
with horseradish peroxidase, alkaline phosphatase or urease.
[0127] Assays can also be conducted in solution, such that the
viral proteins and antibodies thereto form complexes under
precipitating conditions. The precipitated complexes can then be
separated from the test sample, for example, by centrifugation. The
reaction mixture can be analyzed to determine the presence or
absence of antibody-antigen complexes using any of a number of
standard methods, such as those immunodiagnostic methods described
above.
[0128] The modified Env proteins, produced as described above, or
antibodies to the proteins, can be provided in kits, with suitable
instructions and other necessary reagents, in order to conduct
immunoassays as described above. The kit can also contain,
depending on the particular immunoassay used, suitable labels and
other packaged reagents and materials (i.e. wash buffers and the
like). Standard immunoassays, such as those described above, can be
conducted using these kits.
[0129] The Env polypeptides and polynucleotides encoding the
polypeptides can also be used in vaccine compositions, individually
or in combination, in e.g., prophylactic (i.e., to prevent
infection) or therapeutic (to treat HIV following infection)
vaccines. The vaccines can comprise mixtures of one or more of the
modified Env proteins (or nucleotide sequences encoding the
proteins), such as Env (e.g., gp120) proteins derived from more
than one viral isolate. The vaccine may also be administered in
conjunction with other antigens and immunoregulatory agents, for
example, immunoglobulins, cytokines, lymphokines, and chemokines,
including but not limited to IL-2, modified IL-2
(cys125.fwdarw.ser125), GM-CSF, IL-12, .gamma.-interferon, IP-10,
MIP1.beta. and RANTES. The vaccines may be administered as
polypeptides or, alternatively, as naked nucleic acid vaccines
(e.g., DNA), using viral vectors (e.g., retroviral vectors,
adenoviral vectors, adeno-associated viral vectors) or non-viral
vectors (e.g., liposomes, particles coated with nucleic acid or
protein). The vaccines may also comprise a mixture of protein and
nucleic acid, which in turn may be delivered using the same or
different vehicles. The vaccine may be given more than once (e.g.,
a "prime" administration followed by one or more "boosts") to
achieve the desired effects. The same composition can be
administered as the prime and as the one or more boosts.
Alternatively, different compositions can be used for priming and
boosting.
[0130] The vaccines will generally include one or more
"pharmaceutically acceptable excipients or vehicles" such as water,
saline, glycerol, ethanol, etc. Additionally, auxiliary substances,
such as wetting or emulsifying agents, pH buffering substances, and
the like, may be present in such vehicles.
[0131] A carrier is optionally present which is a molecule that
does not itself induce the production of antibodies harmful to the
individual receiving the composition. Suitable carriers are
typically large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycollic acids,
polymeric amino acids, amino acid copolymers, lipid aggregates
(such as oil droplets or liposomes), and inactive virus particles.
Such carriers are well known to those of ordinary skill in the art.
Furthermore, the Env polypeptide may be conjugated to a bacterial
toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.
[0132] Adjuvants may also be used to enhance the effectiveness of
the vaccines. Such adjuvants include, but are not limited to: (1)
aluminum salts (alum), such as aluminum hydroxide, aluminum
phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion
formulations (with or without other specific immunostimulating
agents such as muramyl peptides (see below) or bacterial cell wall
components), such as for example (a) MF59 (International
Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween
80, and 0.5% Span 85 (optionally containing various amounts of
MTP-PE (see below), although not required) formulated into
submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing
10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and
thr-MDP (see below) either microfluidized into a submicron emulsion
or vortexed to generate a larger particle size emulsion, and (c)
Ribi.TM. adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.)
containing 2% Squalene, 0.2% Tween 80, and one or more bacterial
cell wall components from the group consisting of
monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell
wall skeleton (CWS), preferably MPL+CWS (Detox.TM.); (3) saponin
adjuvants, such as Stimulon.TM. (Cambridge Bioscience, Worcester,
Mass.) may be used or particle generated therefrom such as ISCOMs
(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA)
and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as
interleukins (IL-1, IL-2, etc.), macrophage colony stimulating
factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) detoxified
mutants of a bacterial ADP-ribosylating toxin such as a cholera
toxin (CT), a pertussis toxin (PT), or an E. coli heat-labile toxin
(LT), particularly LT-K63 (where lysine is substituted for the
wild-type amino acid at position 63) LT-R72 (where arginine is
substituted for the wild-type amino acid at position 72), CT-S109
(where serine is substituted for the wild-type amino acid at
position 109), and PT-K9/G129 (where lysine is substituted for the
wild-type amino acid at position 9 and glycine substituted at
position 129) (see, e.g., International Publication Nos. WO93/13202
and WO92/19265); and (7) other substances that act as
immunostimulating agents to enhance the effectiveness of the
composition.
[0133] Muramyl peptides include, but are not limited to,
N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1'-2'-dipalmitoyl-s-
n-glycero-3-huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
[0134] Typically, the vaccine compositions are prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection may also be prepared. The preparation also may be
emulsified or encapsulated in liposomes for enhanced adjuvant
effect, as discussed above.
[0135] The vaccines will comprise a therapeutically effective
amount of the modified Env proteins, or complexes of the proteins,
or nucleotide sequences encoding the same, and any other of the
above-mentioned components, as needed. By "therapeutically
effective amount" is meant an amount of a modified Env (e.g.,
gp120) protein which will induce a protective immunological
response in the uninfected, infected or unexposed individual to
which it is administered. Such a response will generally result in
the development in the subject of a secretory, cellular and/or
antibody-mediated immune response to the vaccine. Usually, such a
response includes but is not limited to one or more of the
following effects; the production of antibodies from any of the
immunological classes, such as immunoglobulins A, D, E, G or M; the
proliferation of B and T lymphocytes; the provision of activation,
growth and differentiation signals to immunological cells;
expansion of helper T cell, suppressor T cell, and/or cytotoxic T
cell.
[0136] Preferably, the effective amount is sufficient to bring
about treatment or prevention of disease symptoms. The exact amount
necessary will vary depending on the subject being treated; the age
and general condition of the individual to be treated; the capacity
of the individual's immune system to synthesize antibodies; the
degree of protection desired; the severity of the condition being
treated; the particular Env polypeptide selected and its mode of
administration, among other factors. An appropriate effective
amount can be readily determined by one of skill in the art. A
"therapeutically effective amount" will fall in a relatively broad
range that can be determined through routine trials.
[0137] Once formulated, the nucleic acid vaccines may be
accomplished with or without viral vectors, as described above, by
injection using either a conventional syringe or a gene gun, such
as the Accell.RTM. gene delivery system (PowderJect Technologies,
Inc., Oxford, England). Delivery of DNA into cells of the epidermis
is particularly preferred as this mode of administration provides
access to skin-associated lymphoid cells and provides for a
transient presence of DNA in the recipient. Both nucleic acids
and/or peptides can be injected either subcutaneously, epidermally,
intradermally, intramucosally such as nasally, rectally and
vaginally, intraperitoneally, intravenously, orally or
intramuscularly. Other modes of administration include oral and
pulmonary administration, suppositories, needle-less injection,
transcutaneous and transdermal applications. Dosage treatment may
be a single dose schedule or a multiple dose schedule.
Administration of nucleic acids may also be combined with
administration of peptides or other substances.
[0138] While the invention has been described in conjunction with
the preferred specific embodiments thereof, it is to be understood
that the foregoing description as well as the examples which follow
are intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modifications within the
scope of the invention will be apparent to those skilled in the art
to which the invention pertains.
EXPERIMENTAL
[0139] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0140] Efforts have been made to ensure accuracy with respect to
numbers used (e.g., amounts, temperatures, etc.), but some
experimental error and deviation should, of course, be allowed
for.
Example 1
A.1. Best-Fit and Homology Searches
[0141] The crystal structure of HXB-2 gp120 was downloaded from the
Brookhaven database (COMPLEX (HIV ENVELOPE PROTEIN/CD4/FAB)
15-JUN-98 1GC1 TITLE: HIV-1 GP120 CORE COMPLEXED WITH CD4 AND A
NEUTRALIZING HUMAN ANTIBODY). Beta strands 3, 2, 21, and 20 of
gp120 form a sheet near the CD4 binding site. Strands .beta.-3 and
.beta.-2 are connected by the V1/V2 loop. Strands .beta.-21 and
.beta.-20 are connected by another small loop. The H-bonds at the
interface between strands .beta.-2 and .beta.-21 are the only
connection between domains of the "lower" half of the protein
(joining helix alpha 1 to the CD4 binding site). This beta sheet
and these loops mask some antigens (e.g., antigens which may
generate neutralizing antibodies) that are only exposed during the
CD4 binding.
[0142] Constructs that remove enough of the beta sheet to expose
the antigens in the CD4 binding site, but leave enough of the
protein to allow correct folding were designed. Specifically
targeted were modifications to the small loop and, optional
deletion of the V1/V2 loops. Three different types of constructs
were designed: (1) constructs encoding polypeptides that leave the
number of residues making up the entire 4-strand beta sheet intact,
but replace one or more residues; (2) constructs that encode
polypeptide having at least one residue of at least one beta strand
excised or (3) constructs encoding polypeptides having at least two
residues of at least one beta strand excised. Thus, a total of 6
different turns were needed to rejoin the ends of the strands.
[0143] Initially, residues in the small loop (residues 427-430,
relative to HXB-2) and connected beta strands (.beta.-20 and
.beta.-21) were modified to contain Gly and Pro (common in beta
turns). These sequences were then used as the target to match in
each search. The geometry of the target was matched to known
proteins in the Brookhaven Protein Data Bank. In particular,
5-residue turns (including an overlapping single residue at the
N-terminal, the 2 residue target turn and 2 overlapping residues at
the C-terminal) were searched in the databases. In other words,
these modified loops add a 2 residue turn that should be able to
support a geometry that will maintain the beta-sheet structure of
the wild type protein. The calculations were performed using the
default parameters in the Loop Search feature of the Biopolymer
module of the Sybyl molecular modeling package. In each case, the
25 best fits based on geometry alone were reviewed and, of those,
several selected for homology and fit.
[0144] In addition, it was also determined what modifications could
be made to remove most of the V1/V2 loop (residues 124-198,
relative to HXB-2) yet leave the geometry of the protein intact. As
with the small loop, constructs were also designed which excised
one or more residues from the .beta.-2 strand (residues 119-123 of
HXB-2), the .beta.-3 strand (residues 199-201 of HXB-2) or both
.beta.-2 and .beta.-3. For these constructs, known loops were
searched to match the geometry of a pentamer (including two
remaining residues from the N-terminal side, a 2 residue turn and 1
C-terminal residue). For these searches, Gly-Gly was preferred as
the insert along with at least one C-terminal substitution.
A.2. Small Loop Replacements
[0145] In one aspect, the native sequence was replaced with
residues that expose the CD4 binding site, but leave the overall
geometry of the protein relatively unchanged. For the small loop
replacements, the target to match was:
ASN425-MET426-GLY427-GLY428-GLY431. Results of the search are
summarized in Table 1.
TABLE-US-00001 TABLE 1 Search of Small Loop (Asn425 through Gly431)
Seq % Id Rank Sequence RMSD Homology No. Best fit
LYS-ASP-SER-ASN-ASN 0.16689 62.5 27 3 TYR-GLY-LEU-GLY-LEU 0.220308
62.5 28 4 GLU-ARG-GLU-ASP-GLY 0.241754 62.5 29 7
ARG-LYS-GLY-GLY-ASN 0.24881 100 30 12 TRP-THR-GLY-SER-TYR 0.26417
83.33 31
[0146] Based on these results, constructs encoding Gly-Gly (#7),
Gly-Ser (#12) or Gly-Gly-Asn (#7) were recommended.
[0147] As V1/V2 and one or more residues of .beta.-2 and .beta.-3
are also optionally deleted in the modified polypeptides of the
invention, known loops to match the geometry of the V1/V2 loop were
also searched. The V1/V2 loop the target to match was:
Lys121-Leu-122-Gly123-Gly124-Ser199. Some notable matches are shown
in Table 2:
TABLE-US-00002 TABLE 2 Search of V1/V2 loop (Lys121 through Ser199)
Seq % Id. Rank Sequence RMSD Homology No. Best fit
GLN-VAL-HIS-ASP-GLU 0.154764 68.75 32 2 LYS-GLU-GLY-ASP-LYS 0.15718
81.25 33 9 ARG-SER-GLY-ARG-SER 0.173731 68.75 34 11
THR-LEU-GLY-ASN-SER 0.175554 81.25 35 16 HIS-PHE-GLY-ALA-GLY
0.178772 93.75 36
[0148] Based on these searches, constructs encoding Gly-Asn in
place of V1/V2 were recommended.
A.3. One Additional Residue Excisions
[0149] For a slightly truncated small loop, one more residue was
trimmed from each beta strand to slightly shorten the beta sheet.
The target to match was: ILE424-ASN425-GLY426-GLY427-LYS432.
Results are shown in Table 3:
TABLE-US-00003 TABLE 3 Search of Beta sheet shortened by One
residue (Ile424 through Lys432) Seq % Id Rank Sequence RMSD
Homology No. Best fit: ARG-MET-ALA-PRO-VAL 0.316805 58.33 37 Best
hom: ASP-SER-ASP-GLY-PRO 0.440896 83.33 38
[0150] Although these searches showed more variation and worse fits
than the previous truncation, the Pro-Val or Pro-Leu encoding
constructs were very similar. Accordingly, Ala-Pro encoding
constructs were recommended.
[0151] Sequences encoding gp120 polypeptides having V1/V2 deleted
and an additional residue from .beta.-2 or .beta.-3 excised were
also searched. The V1/V2 loop the target to match was:
VAL120-LYS121-GLY122-GLY123-VAL200. Some notable matches are shown
in Table 4.
TABLE-US-00004 TABLE 4 Search of V1/V2 loop (Val120 through Val200)
Seq % Id Rank Sequence RMSD Homology No Best fit:
THR-VAL-ASP-PRO-TYR 0.400892 58.33333 39 2 SER-THR-ASN-PRO-LEU
0.402575 54.16667 40 3 THR-ARG-SER-PRO-LEU 0.403965 58.33333 41 7
ARG-MET-ALA-PRO-VAL 0.440118 58.33333 42
[0152] The construct encoding Ala-Pro (e.g., #7) was
recommended.
A.4. Further Excisions
[0153] In yet another truncation, an additional residue was trimmed
from the .beta.-20 and .beta.-21 strands to further shorten the
beta sheet. The target to match was
ILE423-ILE424-GLY425-GLY426-ALA433. Notable matches are shown in
Table 5.
TABLE-US-00005 TABLE 5 Search of Beta sheet shortened by Two
Residues (Ile423 through Ala433) Seq % Id Rank Sequence RMSD
Homology No Best fit: THR-TYR-GLU-GLY-VAL 0.130107 79.16666 43 2
GLN-VAL-GLY-ASN-THR 0.138245 79.16666 44 3: THR-VAL-GLY-GLY-ILE
0.153362 100 45
[0154] A construct encoding Gly-Gly (e.g., #3), which has 100%
homology, was recommended.
[0155] Also searched were sequences encoding a deleted V1/V2 region
and at least two residues excised from .beta.-2, .beta.-3 or at
least one residue excised from .beta.-2 and .beta.-3. The target to
match was: CYS119-VAL120-GLY121-GLY122-ILE201. Notable matches are
shown in Table 6.
TABLE-US-00006 TABLE 6 Search of V1/V2 loop (Cys119 through Ile201)
Seq % Id Rank Sequence RMSD Homology No Best fit:
ASP-LEU-PRO-GLY-CYS 0.250501 75 46 4 ASP-VAL-GLY-GLY-LEU 0.290383
100 47
[0156] It was determined that both constructs would be used.
B.1. Constructs Encoding Modified Env Polypeptides
[0157] As described above, the native loops extruding from the
4-.beta. antiparallel-stands were excised and replaced with 1 to 3
residue turns. The loops were replaced so as to leave the entire
.beta.-strands or excised by trimming one or more amino acid from
each side of the connected strands. The ends of the strands were
rejoined with turns that preserve the same backbone geometry (e.g.,
tertiary structure of .beta.-20 and .beta.-21), as determined by
searching the Brookhaven Protein Data Bank.
[0158] Table 7A is a summary of the truncations of the variable
regions 1 and 2 recommended for this study, as determined in
Example 1.A. above.
TABLE-US-00007 TABLE 7A V1/V2 Modifications SEQ ID NO FIG.
-LEU122-GLY-ASN-SER199 7 10 -LYS121-ALA-PRO-VAL200- 6 9
-VAL120-GLY-GLY-ILE201- 4 7 -VAL120-PRO-GLY-ILE201B- 5 8
-VAL120-GLY-ALA-GLY-ALA204- 3 6 -VAL120-GLY-GLY-ALA-THR202- 8 11
-VAL127-GLY-ALA-GLY-ASN195- 25 28
[0159] As previously noted, the polypeptides encoded by the
constructs of the present invention are numbered relative to HXB-2,
but the particular amino acid residue of the polypeptides encoded
by these exemplary constructs is based on SF-162. Thus, for
example, although amino acid residue 195 in HXB-2 is a serine (S),
constructs encoding polypeptides having then wild type SF162
sequence will have an asparagine (N) at this position. Table 7B
shows just three of the variations in amino acid sequence between
strains HXB-2 and SF162. The entire sequences, including
differences in residue and amino acid number, of HXB-2 and SF162
are shown in the alignment of FIG. 2 (SEQ ID NOs:1 and 2).
TABLE-US-00008 TABLE 7B HXB-2 amino SF162 Residue/ acid number
HXB-2 Residue amino acid number 128 Serine (S) Thr (T)/114 195
Serine (S) Asn (N)/188 426 Met (M) Arg (R)/411
[0160] Constructs containing deletions in the .beta.-20 strand,
.beta.-21 stand and small loop were also constructed. Shown in
Table 8 are constructs encoding truncations in these regions. The
constructs in Table 8 are numbered relative to HXB-2 but the
unmodified amino acid sequence is based on SF162. Thus, the
construct encodes an arginine (Arg) as is found in SF162 in the
amino acid position numbered 426 relative to HXB-2 (See, also,
Table 7B). Changes from wildtype (SF162) are shown in bold in Table
8B.
TABLE-US-00009 TABLE 8 Small Loop/.beta.-20 and .beta.-21
(Modified) SEQ ID NO FIG. -TRP427-GLY-GLY431- 9 12
-ARG426-GLY-GLY-GLY431- 10 13 -ARG426-GLY-SER-GLY431B- 11 14
-ARG426-GLY-GLY-ASN-LYS432- 12 15 -ASN425-ALA-PRO-LYS432- 13 16
-ILE424-GLY-GLY-ALA433- 14 17 -ILE423-GLY-GLY-MET434- 15 18
GLN422-GLY-GLY-TYR435- 16 19 -GLN422-ALA-PRO-TYR435B- 17 20
[0161] The deletion constructs shown in Tables 7 and 8 for each one
of the n-strands and combinations of them are constructed. These
deletions will be tested in the Env forms gp120, gp140 and gp160
from different HIV strains like subtype B strains (e.g., SF162,
US4, SF2), subtype E strains (e.g., CM235) and subtype C strains
(e.g., AF110968 or AF110975). Exemplary constructs for SF162 are
shown in the Figures and are summarized in Table 9. As noted above
in FIG. 2 and Table 7B, in the bridging sheet region, the amino
acid sequence of SF162 differs from HXB-2 in that the Met426 of
HXB-2 is an Arg in SF162. In Table 9, V1/V2 refers to deletions in
the V1/V2 region; # bsm refers to a modification in the bridging
sheet small loop.
TABLE-US-00010 TABLE 9 Seq. Modification/ Construct Id. FIG. Amino
acid sequence Val120-Ala204 3 6 V1/V2: Val120-Gly-Ala-Gly- Ala204
Val120-Ile201 4 7 V1/V2: Val120-Gly-Gly- Ile201 Val120-Ile201B 5 8
V1/V2: Val120-Pro-Gly- Ile201 Lys121-Val200 6 9 V1/V2:
Lys121-Ala-Pro- Val200 Leu122-Ser199 7 10 V1/V2: Leu122-Gly-Asn-
Ser199 Val120-Thr202 8 11 V1/V2: Val120-Gly-Gly-Ala- Thr202
Trp427-Gly431 9 12 bsm: Trp427-Gly-Gly431 Arg426-Gly431 10 13 bsm:
Arg426-Gly-Gly-Gly431 Arg426-Gly431B 11 14 bsm:
Arg426-Gly-Ser-Gly431 Arg426-Lys432 12 15 bsm: Arg426-Gly-Gly-Asn-
Lys432 Asn425-Lys432 13 16 bsm: Asn425-Ala-Pro-Lys432 Ile424-Ala433
14 17 bsm: Ile424-Gly-Gly-Ala433 Ile423-Met434 15 18 bsm:
Ile423-Gly-Gly-Met434 Gln422-Tyr435 16 19 bsm:
Gln422-Gly-Gly-Tyr435 Val127-Asn195 25 28 bsm: Val127-Gly-Ala-Gly-
Asn195 Gln422-Tyr435B 17 20 bsm: Gln422-Ala-Pro-Tyr435
Leu122-Ser199; 18 21 V1/V2/bsm: Leu122-Gly-Asn- Arg426-Gly431
Ser199 --- Arg426-Gly-Gly- Gly431 Leu122-Ser199; 19 22 V1/V2/bsm:
Leu122-Gly-Asn- Arg426-Lys432 Ser199 --- Arg426-Gly-Gly- Asn-Lys432
Leu122-Ser199- 20 23 V1/V2/bsm: Leu122-Gly-Asn- Trp427-Gly431
Ser199 --- Trp427-Gly- Gly431 Lys121-Val200- 21 24 V1/V2/bsm:
Lys121-Ala-Pro- Asn425-Lys432 Val200 --- Asn425-Ala-Pro- Lys432
Val120-Ile201- 22 25 V1/V2/bsm: Val120-Gly-Gly- Ile424-Ala433
Ile201 --- Ile424-Gly-Gly- Ala433 Val120-Ile201B- 23 26 V1/V2/bsm:
Val120-Pro-Gly- Ile424-Ala433 Ile201 --- Ile424-Gly-Gly- Ala43
Val120-Thr202; 24 27 V1/V2/bsm: Val120-Gly-Gly- Ile424-Ala433
Ala-Thr202 --- Ile424-Gly- Gly-Ala433 Val127-Asn195; 25 29
V1/V2/bsm: Val127-Gly-Ala- Arg426-Gly431 Gly-Asn195 --- Arg426-Gly-
Gly-Gly431
[0162] Combinations of V1/V2 deletions and bridging sheet small
loop modifications in addition to those specifically shown in Table
9 are also within the scope of the present invention. Various forms
of the different embodiments of the invention, described herein,
may be combined.
[0163] The first screening will be done after transient expression
in COS-7, RD and/or 293 cells. The proteins that are expressed will
be analyzed by immunoblot, ELISA, and for binding to mAbs directed
to the CD4 binding site and other important epitopes on gp120 to
determine integrity of structure. They will also be tested in a CD4
binding assay and, in addition, the binding of neutralizing
antibodies, for example using patient sera or mAb 448D (directed to
Glu370 and Tyr384, a region of the CD4 binding groove that is not
altered by the deletions).
[0164] The immunogenicity of these novel Env glycoproteins will be
tested in rodents and primates. The structures will be administered
as DNA vaccines or adjuvanted protein vaccines or in combined
modalities. The goal of these vaccinations will be to archive
broadly reactive neutralizing antibody responses.
Sequence CWU 1
1
261856PRTHuman Immunodeficiency Virusmodified ENV construct 1Met
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 Asn Met Trp Gln Lys Val Gly Lys
420 425 430Ala Met Tyr Ala Pro Pro Ile Ser Gly Gln Ile Arg Cys Ser
Ser Asn 435 440 445Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Asn
Ser Asn Asn Glu 450 455 460Ser Glu Ile Phe Arg Pro Gly Gly Gly Asp
Met Arg Asp Asn Trp Arg465 470 475 480Ser Glu Leu Tyr Lys Tyr Lys
Val Val Lys Ile Glu Pro Leu Gly Val 485 490 495Ala Pro Thr Lys Ala
Lys Arg Arg Val Val Gln Arg Glu Lys Arg Ala 500 505 510Val Gly Ile
Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser 515 520 525Thr
Met Gly Ala Ala Ser Met Thr Leu Thr Val Gln Ala Arg Gln Leu 530 535
540Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile
Glu545 550 555 560Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
Ile Lys Gln Leu 565 570 575Gln Ala Arg Ile Leu Ala Val Glu Arg Tyr
Leu Lys Asp Gln Gln Leu 580 585 590Leu Gly Ile Trp Gly Cys Ser Gly
Lys Leu Ile Cys Thr Thr Ala Val 595 600 605Pro Trp Asn Ala Ser Trp
Ser Asn Lys Ser Leu Glu Gln Ile Trp Asn 610 615 620His Thr Thr Trp
Met Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser625 630 635 640Leu
Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Lys Asn 645 650
655Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu Trp Asn Trp
660 665 670Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys Leu Phe Ile
Met Ile 675 680 685Val Gly Gly Leu Val Gly Leu Arg Ile Val Phe Ala
Val Leu Ser Ile 690 695 700Val Asn Arg Val Arg Gln Gly Tyr Ser Pro
Leu Ser Phe Gln Thr His705 710 715 720Leu Pro Thr Pro Arg Gly Pro
Asp Arg Pro Glu Gly Ile Glu Glu Glu 725 730 735Gly Gly Glu Arg Asp
Arg Asp Arg Ser Ile Arg Leu Val Asn Gly Ser 740 745 750Leu Ala Leu
Ile Trp Asp Asp Leu Arg Ser Leu Cys Leu Phe Ser Tyr 755 760 765His
Arg Leu Arg Asp Leu Leu Leu Ile Val Thr Arg Ile Val Glu Leu 770 775
780Leu Gly Arg Arg Gly Trp Glu Ala Leu Lys Tyr Trp Trp Asn Leu
Leu785 790 795 800Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Val
Ser Leu Leu Asn 805 810 815Ala Thr Ala Ile Ala Val Ala Glu Gly Thr
Asp Arg Val Ile Glu Val 820 825 830Val Gln Gly Ala Cys Arg Ala Ile
Arg His Ile Pro Arg Arg Ile Arg 835 840 845Gln Gly Leu Glu Arg Ile
Leu Leu 850 8552847PRTHuman Immunodeficiency Virusmodified ENV
construct 2Met Arg Val Lys Gly Ile Arg Lys Asn Tyr Gln His Leu Trp
Arg Gly1 5 10 15Gly Thr Leu Leu Leu Gly Met Leu Met Ile Cys Ser Ala
Val Glu Lys 20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp
Lys Glu Ala Thr 35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala
Tyr Asp 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 Val Leu Glu Asn Val
Thr Glu Asn Phe Asn Met Trp Lys 85 90 95Asn Asn Met Val Glu Gln Met
His Glu Asp Ile Ile Ser Leu Trp Asp 100 105 110Gln Ser Leu Lys Pro
Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu 115 120 125His Cys Thr
Asn Leu Lys Asn Ala Thr Asn Thr Lys Ser Ser Asn Trp 130 135 140Lys
Glu Met Asp Arg Gly Glu Ile Lys Asn Cys Ser Phe Lys Val Thr145 150
155 160Thr Ser Ile Arg Asn Lys Met Gln Lys Glu Tyr Ala Leu Phe Tyr
Lys 165 170 175Leu Asp Val Val Pro Ile Asp Asn Asp Asn Thr Ser Tyr
Lys Leu Ile 180 185 190Asn Cys Asn Thr Ser Val Ile Thr Gln Ala Cys
Pro Lys Val Ser Phe 195 200 205Glu Pro Ile Pro Ile His Tyr Cys Ala
Pro Ala Gly Phe Ala Ile Leu 210 215 220Lys Cys Asn Asp Lys Lys Phe
Asn Gly Ser Gly Pro Cys Thr Asn Val225 230 235 240Ser Thr Val Gln
Cys Thr His Gly Ile Arg Pro Val Val Ser Thr Gln 245 250 255Leu Leu
Leu Asn Gly Ser Leu Ala Glu Glu Gly Val Val Ile Arg Ser 260 265
270Glu Asn Phe Thr Asp Asn Ala Lys Thr Ile Ile Val Gln Leu Lys Glu
275 280 285Ser Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg
Lys Ser 290 295 300Ile Thr Ile Gly Pro Gly Arg Ala Phe Tyr Ala Thr
Gly Asp Ile Ile305 310 315 320Gly Asp Ile Arg Gln Ala His Cys Asn
Ile Ser Gly Glu Lys Trp Asn 325 330 335Asn Thr Leu Lys Gln Ile Val
Thr Lys Leu Gln Ala Gln Phe Gly Asn 340 345 350Lys Thr Ile Val Phe
Lys Gln Ser Ser Gly Gly Asp Pro Glu Ile Val 355 360 365Met His Ser
Phe Asn Cys Gly Gly Glu Phe Phe Tyr Cys Asn Ser Thr 370 375 380Gln
Leu Phe Asn Ser Thr Trp Asn Asn Thr Ile Gly Pro Asn Asn Thr385 390
395 400Asn Gly Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn
Arg 405 410 415Trp Gln Glu Val Gly Lys Ala Met Tyr Ala Pro Pro Ile
Arg Gly Gln 420 425 430Ile Arg Cys Ser Ser Asn Ile Thr Gly Leu Leu
Leu Thr Arg Asp Gly 435 440 445Gly Lys Glu Ile Ser Asn Thr Thr Glu
Ile Phe Arg Pro Gly Gly Gly 450 455 460Asp Met Arg Asp Asn Trp Arg
Ser Glu Leu Tyr Lys Tyr Lys Val Val465 470 475 480Lys Ile Glu Pro
Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val 485 490 495Val Gln
Arg Glu Lys Arg Ala Val Thr Leu Gly Ala Met Phe Leu Gly 500 505
510Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Arg Ser Leu Thr Leu
515 520 525Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln
Gln Asn 530 535 540Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
Leu Gln Leu Thr545 550 555 560Val Trp Gly Ile Lys Gln Leu Gln Ala
Arg Val Leu Ala Val Glu Arg 565 570 575Tyr Leu Lys Asp Gln Gln Leu
Leu Gly Ile Trp Gly Cys Ser Gly Lys 580 585 590Leu Ile Cys Thr Thr
Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Lys 595 600 605Ser Leu Asp
Gln Ile Trp Asn Asn Met Thr Trp Met Glu Trp Glu Arg 610 615 620Glu
Ile Asp Asn Tyr Thr Asn Leu Ile Tyr Thr Leu Ile Glu Glu Ser625 630
635 640Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp
Lys 645 650 655Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Ser Lys Trp
Leu Trp Tyr 660 665 670Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu
Val Gly Leu Arg Ile 675 680 685Val Phe Thr Val Leu Ser Ile Val Asn
Arg Val Arg Gln Gly Tyr Ser 690 695 700Pro Leu Ser Phe Gln Thr Arg
Phe Pro Ala Pro Arg Gly Pro Asp Arg705 710 715 720Pro Glu Gly Ile
Glu Glu Glu Gly Gly Glu Arg Asp Arg Asp Arg Ser 725 730 735Ser Pro
Leu Val His Gly Leu Leu Ala Leu Ile Trp Asp Asp Leu Arg 740 745
750Ser Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Leu Ile Leu Ile
755 760 765Ala Ala Arg Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu
Ala Leu 770 775 780Lys Tyr Trp Gly Asn Leu Leu Gln Tyr Trp Ile Gln
Glu Leu Lys Asn785 790 795 800Ser Ala Val Ser Leu Phe Asp Ala Ile
Ala Ile Ala Val Ala Glu Gly 805 810 815Thr Asp Arg Ile Ile Glu Val
Ala Gln Arg Ile Gly Arg Ala Phe Leu 820 825 830His Ile Pro Arg Arg
Ile Arg Gln Gly Phe Glu Arg Ala Leu Leu 835 840
84532310DNAArtificial Sequencemodified ENV construct 3gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgggcgcc 360ggcgcctgcc
ccaaggtgag cttcgagccc atccccatcc actactgcgc ccccgccggc
420ttcgccatcc tgaagtgcaa cgacaagaag ttcaacggca gcggcccctg
caccaacgtg 480agcaccgtgc agtgcaccca cggcatccgc cccgtggtga
gcacccagct gctgctgaac 540ggcagcctgg ccgaggaggg cgtggtgatc
cgcagcgaga acttcaccga caacgccaag 600accatcatcg tgcagctgaa
ggagagcgtg gagatcaact gcacccgccc caacaacaac 660acccgcaaga
gcatcaccat cggccccggc cgcgccttct acgccaccgg cgacatcatc
720ggcgacatcc gccaggccca ctgcaacatc agcggcgaga agtggaacaa
caccctgaag 780cagatcgtga ccaagctgca ggcccagttc ggcaacaaga
ccatcgtgtt caagcagagc 840agcggcggcg accccgagat cgtgatgcac
agcttcaact gcggcggcga gttcttctac 900tgcaacagca cccagctgtt
caacagcacc tggaacaaca ccatcggccc caacaacacc 960aacggcacca
tcaccctgcc ctgccgcatc aagcagatca tcaaccgctg gcaggaggtg
1020ggcaaggcca tgtacgcccc ccccatccgc ggccagatcc gctgcagcag
caacatcacc 1080ggcctgctgc tgacccgcga cggcggcaag gagatcagca
acaccaccga gatcttccgc 1140cccggcggcg gcgacatgcg cgacaactgg
cgcagcgagc tgtacaagta caaggtggtg 1200aagatcgagc ccctgggcgt
ggcccccacc aaggccaagc gccgcgtggt gcagcgcgag 1260aagcgcgccg
tgaccctggg cgccatgttc ctgggcttcc tgggcgccgc cggcagcacc
1320atgggcgccc gcagcctgac cctgaccgtg caggcccgcc agctgctgag
cggcatcgtg 1380cagcagcaga acaacctgct gcgcgccatc gaggcccagc
agcacctgct gcagctgacc 1440gtgtggggca tcaagcagct gcaggcccgc
gtgctggccg tggagcgcta cctgaaggac 1500cagcagctgc tgggcatctg
gggctgcagc ggcaagctga tctgcaccac cgccgtgccc 1560tggaacgcca
gctggagcaa caagagcctg gaccagatct ggaacaacat gacctggatg
1620gagtgggagc gcgagatcga caactacacc aacctgatct acaccctgat
cgaggagagc 1680cagaaccagc aggagaagaa cgagcaggag ctgctggagc
tggacaagtg ggccagcctg 1740tggaactggt tcgacatcag caagtggctg
tggtacatca agatcttcat catgatcgtg 1800ggcggcctgg tgggcctgcg
catcgtgttc accgtgctga gcatcgtgaa ccgcgtgcgc 1860cagggctaca
gccccctgag cttccagacc cgcttccccg ccccccgcgg ccccgaccgc
1920cccgagggca tcgaggagga gggcggcgag cgcgaccgcg accgcagcag
ccccctggtg 1980cacggcctgc tggccctgat ctgggacgac ctgcgcagcc
tgtgcctgtt cagctaccac 2040cgcctgcgcg acctgatcct gatcgccgcc
cgcatcgtgg agctgctggg ccgccgcggc 2100tgggaggccc tgaagtactg
gggcaacctg ctgcagtact ggatccagga gctgaagaac 2160agcgccgtga
gcctgttcga cgccatcgcc atcgccgtgg ccgagggcac cgaccgcatc
2220atcgaggtgg cccagcgcat cggccgcgcc ttcctgcaca tcccccgccg
catccgccag 2280ggcttcgagc gcgccctgct gtaactcgag
231042316DNAArtificial Sequencemodified ENV construct 4gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgggcggc 360atcacccagg
cctgccccaa ggtgagcttc gagcccatcc ccatccacta ctgcgccccc
420gccggcttcg ccatcctgaa gtgcaacgac aagaagttca acggcagcgg
cccctgcacc 480aacgtgagca ccgtgcagtg cacccacggc atccgccccg
tggtgagcac ccagctgctg 540ctgaacggca gcctggccga ggagggcgtg
gtgatccgca gcgagaactt caccgacaac 600gccaagacca tcatcgtgca
gctgaaggag agcgtggaga tcaactgcac ccgccccaac 660aacaacaccc
gcaagagcat caccatcggc cccggccgcg ccttctacgc caccggcgac
720atcatcggcg acatccgcca ggcccactgc aacatcagcg gcgagaagtg
gaacaacacc 780ctgaagcaga tcgtgaccaa gctgcaggcc cagttcggca
acaagaccat cgtgttcaag 840cagagcagcg gcggcgaccc cgagatcgtg
atgcacagct tcaactgcgg cggcgagttc 900ttctactgca acagcaccca
gctgttcaac agcacctgga acaacaccat cggccccaac 960aacaccaacg
gcaccatcac cctgccctgc cgcatcaagc agatcatcaa ccgctggcag
1020gaggtgggca aggccatgta cgcccccccc atccgcggcc agatccgctg
cagcagcaac 1080atcaccggcc tgctgctgac ccgcgacggc ggcaaggaga
tcagcaacac caccgagatc 1140ttccgccccg gcggcggcga catgcgcgac
aactggcgca gcgagctgta caagtacaag 1200gtggtgaaga tcgagcccct
gggcgtggcc cccaccaagg ccaagcgccg cgtggtgcag 1260cgcgagaagc
gcgccgtgac cctgggcgcc atgttcctgg gcttcctggg cgccgccggc
1320agcaccatgg gcgcccgcag cctgaccctg accgtgcagg cccgccagct
gctgagcggc 1380atcgtgcagc agcagaacaa cctgctgcgc gccatcgagg
cccagcagca cctgctgcag 1440ctgaccgtgt ggggcatcaa gcagctgcag
gcccgcgtgc tggccgtgga gcgctacctg 1500aaggaccagc agctgctggg
catctggggc tgcagcggca agctgatctg caccaccgcc 1560gtgccctgga
acgccagctg gagcaacaag agcctggacc agatctggaa caacatgacc
1620tggatggagt gggagcgcga gatcgacaac tacaccaacc tgatctacac
cctgatcgag 1680gagagccaga accagcagga gaagaacgag caggagctgc
tggagctgga caagtgggcc 1740agcctgtgga actggttcga catcagcaag
tggctgtggt acatcaagat cttcatcatg 1800atcgtgggcg gcctggtggg
cctgcgcatc gtgttcaccg tgctgagcat cgtgaaccgc 1860gtgcgccagg
gctacagccc cctgagcttc cagacccgct tccccgcccc ccgcggcccc
1920gaccgccccg agggcatcga ggaggagggc ggcgagcgcg accgcgaccg
cagcagcccc 1980ctggtgcacg gcctgctggc cctgatctgg gacgacctgc
gcagcctgtg cctgttcagc 2040taccaccgcc tgcgcgacct gatcctgatc
gccgcccgca tcgtggagct gctgggccgc 2100cgcggctggg aggccctgaa
gtactggggc aacctgctgc agtactggat ccaggagctg 2160aagaacagcg
ccgtgagcct gttcgacgcc atcgccatcg ccgtggccga gggcaccgac
2220cgcatcatcg aggtggccca gcgcatcggc cgcgccttcc tgcacatccc
ccgccgcatc 2280cgccagggct tcgagcgcgc cctgctgtaa ctcgag
231652322DNAArtificial Sequencemodified ENV construct 5gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgcccggc 360atcacccagg
cctgccccaa ggtgagcttc gagcccatcc ccatccacta ctgcgccccc
420gccggcttcg ccatcctgaa gtgcaacgac aagaagttca acggcagcgg
cccctgcacc 480aacgtgagca ccgtgcagtg cacccacggc atccgccccg
tggtgagcac ccagctgctg 540ctgaacggca gcctggccga ggagggcgtg
gtgatccgca gcgagaactt caccgacaac 600gccaagacca tcatcgtgca
gctgaaggag agcgtggaga tcaactgcac ccgccccaac 660aacaacaccc
gcaagagcat caccatcggc cccggccgcg ccttctacgc caccggcgac
720atcatcggcg acatccgcca ggcccactgc aacatcagcg gcgagaagtg
gaacaacacc 780ctgaagcaga tcgtgaccaa gctgcaggcc cagttcggca
acaagaccat cgtgttcaag 840cagagcagcg gcggcgaccc cgagatcgtg
atgcacagct tcaactgcgg cggcgagttc 900ttctactgca acagcaccca
gctgttcaac agcacctgga acaacaccat cggccccaac 960aacaccaacg
gcaccatcac cctgccctgc cgcatcaagc agatcatcaa ccgctggcag
1020gaggtgggca aggccatgta cgcccccccc atccgcggcc agatccgctg
cagcagcaac 1080atcaccggcc tgctgctgac ccgcgacggc ggcaaggaga
tcagcaacac caccgagatc 1140ttccgccccg gcggcggcga catgcgcgac
aactggcgca gcgagctgta caagtacaag 1200gtggtgaaga tcgagcccct
gggcgtggcc cccaccaagg ccaagcgccg cgtggtgcag 1260cgcgagaagc
gcgccgtgac cctgggcgcc atgttcctgg gcttcctggg cgccgccggc
1320agcaccatgg gcgcccgcag cctgaccctg accgtgcagg cccgccagct
gctgagcggc 1380atcgtgcagc agcagaacaa cctgctgcgc gccatcgagg
cccagcagca cctgctgcag 1440ctgaccgtgt ggggcatcaa gcagctgcag
gcccgcgtgc tggccgtgga gcgctacctg 1500aaggaccagc agctgctggg
catctggggc tgcagcggca agctgatctg caccaccgcc 1560gtgccctgga
acgccagctg gagcaacaag agcctggacc agatctggaa caacatgacc
1620tggatggagt gggagcgcga gatcgacaac tacaccaacc tgatctacac
cctgatcgag 1680gagagccaga accagcagga gaagaacgag caggagctgc
tggagctgga caagtgggcc 1740agcctgtgga actggttcga catcagcaag
tggctgtggt acatcaagat cttcatcatg 1800atcgtgggcg gcctggtggg
cctgcgcatc gtgttcaccg tgctgagcat cgtgaaccgc 1860gtgcgccagg
gctacagccc cctgagcttc cagacccgct tccccgcccc ccgcggcccc
1920gaccgccccg agggcatcga ggaggagggc ggcgagcgcg accgcgaccg
cagcagcccc 1980ctggtgcacg gcctgctggc cctgatctgg gacgacctgc
gcagcctgtg cctgttcagc 2040taccaccgcc tgcgcgacct gatcctgatc
gccgcccgca tcgtggagct gctgggccgc 2100cgcggctggg aggccctgaa
gtactggggc aacctgctgc agtactggat ccaggagctg 2160aagaacagcg
ccgtgagcct gttcgacgcc atcgccatcg ccgtggccga gggcaccgac
2220cgcatcatcg aggtggccca gcgcatcggc cgcgccttcc tgcacatccc
ccgccgcatc 2280cgccagggct tcgagcgcgc cctgctgtaa ctcgagcgtg ct
232262328DNAArtificial Sequencemodified ENV construct 6gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaaggcc 360cccgtgatca
cccaggcctg ccccaaggtg agcttcgagc ccatccccat ccactactgc
420gcccccgccg gcttcgccat cctgaagtgc aacgacaaga agttcaacgg
cagcggcccc 480tgcaccaacg tgagcaccgt gcagtgcacc cacggcatcc
gccccgtggt gagcacccag 540ctgctgctga acggcagcct ggccgaggag
ggcgtggtga tccgcagcga gaacttcacc 600gacaacgcca agaccatcat
cgtgcagctg aaggagagcg tggagatcaa ctgcacccgc 660cccaacaaca
acacccgcaa gagcatcacc atcggccccg gccgcgcctt ctacgccacc
720ggcgacatca tcggcgacat ccgccaggcc cactgcaaca tcagcggcga
gaagtggaac 780aacaccctga agcagatcgt gaccaagctg caggcccagt
tcggcaacaa gaccatcgtg 840ttcaagcaga gcagcggcgg cgaccccgag
atcgtgatgc acagcttcaa ctgcggcggc 900gagttcttct actgcaacag
cacccagctg ttcaacagca cctggaacaa caccatcggc 960cccaacaaca
ccaacggcac catcaccctg ccctgccgca tcaagcagat catcaaccgc
1020tggcaggagg tgggcaaggc catgtacgcc ccccccatcc gcggccagat
ccgctgcagc 1080agcaacatca ccggcctgct gctgacccgc gacggcggca
aggagatcag caacaccacc 1140gagatcttcc gccccggcgg cggcgacatg
cgcgacaact ggcgcagcga gctgtacaag 1200tacaaggtgg tgaagatcga
gcccctgggc gtggccccca ccaaggccaa gcgccgcgtg 1260gtgcagcgcg
agaagcgcgc cgtgaccctg ggcgccatgt tcctgggctt cctgggcgcc
1320gccggcagca ccatgggcgc ccgcagcctg accctgaccg tgcaggcccg
ccagctgctg 1380agcggcatcg tgcagcagca gaacaacctg ctgcgcgcca
tcgaggccca gcagcacctg 1440ctgcagctga ccgtgtgggg catcaagcag
ctgcaggccc gcgtgctggc cgtggagcgc 1500tacctgaagg accagcagct
gctgggcatc tggggctgca gcggcaagct gatctgcacc 1560accgccgtgc
cctggaacgc cagctggagc aacaagagcc tggaccagat ctggaacaac
1620atgacctgga tggagtggga gcgcgagatc gacaactaca ccaacctgat
ctacaccctg 1680atcgaggaga gccagaacca gcaggagaag aacgagcagg
agctgctgga gctggacaag 1740tgggccagcc tgtggaactg gttcgacatc
agcaagtggc tgtggtacat caagatcttc 1800atcatgatcg tgggcggcct
ggtgggcctg cgcatcgtgt tcaccgtgct gagcatcgtg 1860aaccgcgtgc
gccagggcta cagccccctg agcttccaga cccgcttccc cgccccccgc
1920ggccccgacc gccccgaggg catcgaggag gagggcggcg agcgcgaccg
cgaccgcagc 1980agccccctgg tgcacggcct gctggccctg atctgggacg
acctgcgcag cctgtgcctg 2040ttcagctacc accgcctgcg cgacctgatc
ctgatcgccg cccgcatcgt ggagctgctg 2100ggccgccgcg gctgggaggc
cctgaagtac tggggcaacc tgctgcagta ctggatccag 2160gagctgaaga
acagcgccgt gagcctgttc gacgccatcg ccatcgccgt ggccgagggc
2220accgaccgca tcatcgaggt ggcccagcgc atcggccgcg ccttcctgca
catcccccgc 2280cgcatccgcc agggcttcga gcgcgccctg ctgtaactcg agcgtgct
232872334DNAArtificial Sequencemodified ENV construct 7gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360ggcaacagcg
tgatcaccca ggcctgcccc aaggtgagct tcgagcccat ccccatccac
420tactgcgccc ccgccggctt cgccatcctg aagtgcaacg acaagaagtt
caacggcagc 480ggcccctgca ccaacgtgag caccgtgcag tgcacccacg
gcatccgccc cgtggtgagc 540acccagctgc tgctgaacgg cagcctggcc
gaggagggcg tggtgatccg cagcgagaac 600ttcaccgaca acgccaagac
catcatcgtg cagctgaagg agagcgtgga gatcaactgc 660acccgcccca
acaacaacac ccgcaagagc atcaccatcg gccccggccg cgccttctac
720gccaccggcg acatcatcgg cgacatccgc caggcccact gcaacatcag
cggcgagaag 780tggaacaaca ccctgaagca gatcgtgacc aagctgcagg
cccagttcgg caacaagacc 840atcgtgttca agcagagcag cggcggcgac
cccgagatcg tgatgcacag cttcaactgc 900ggcggcgagt tcttctactg
caacagcacc cagctgttca acagcacctg gaacaacacc 960atcggcccca
acaacaccaa cggcaccatc accctgccct gccgcatcaa gcagatcatc
1020aaccgctggc aggaggtggg caaggccatg tacgcccccc ccatccgcgg
ccagatccgc 1080tgcagcagca acatcaccgg cctgctgctg acccgcgacg
gcggcaagga gatcagcaac 1140accaccgaga tcttccgccc cggcggcggc
gacatgcgcg acaactggcg cagcgagctg 1200tacaagtaca aggtggtgaa
gatcgagccc ctgggcgtgg cccccaccaa ggccaagcgc 1260cgcgtggtgc
agcgcgagaa gcgcgccgtg accctgggcg ccatgttcct gggcttcctg
1320ggcgccgccg gcagcaccat gggcgcccgc agcctgaccc tgaccgtgca
ggcccgccag 1380ctgctgagcg gcatcgtgca gcagcagaac aacctgctgc
gcgccatcga ggcccagcag 1440cacctgctgc agctgaccgt gtggggcatc
aagcagctgc aggcccgcgt gctggccgtg 1500gagcgctacc tgaaggacca
gcagctgctg ggcatctggg gctgcagcgg caagctgatc 1560tgcaccaccg
ccgtgccctg gaacgccagc tggagcaaca agagcctgga ccagatctgg
1620aacaacatga cctggatgga gtgggagcgc gagatcgaca actacaccaa
cctgatctac 1680accctgatcg aggagagcca gaaccagcag gagaagaacg
agcaggagct gctggagctg 1740gacaagtggg ccagcctgtg gaactggttc
gacatcagca agtggctgtg gtacatcaag 1800atcttcatca tgatcgtggg
cggcctggtg ggcctgcgca tcgtgttcac cgtgctgagc 1860atcgtgaacc
gcgtgcgcca gggctacagc cccctgagct tccagacccg cttccccgcc
1920ccccgcggcc ccgaccgccc cgagggcatc gaggaggagg gcggcgagcg
cgaccgcgac 1980cgcagcagcc ccctggtgca cggcctgctg gccctgatct
gggacgacct gcgcagcctg 2040tgcctgttca gctaccaccg cctgcgcgac
ctgatcctga tcgccgcccg catcgtggag 2100ctgctgggcc gccgcggctg
ggaggccctg aagtactggg gcaacctgct gcagtactgg 2160atccaggagc
tgaagaacag cgccgtgagc ctgttcgacg ccatcgccat cgccgtggcc
2220gagggcaccg accgcatcat cgaggtggcc cagcgcatcg gccgcgcctt
cctgcacatc 2280ccccgccgca tccgccaggg cttcgagcgc gccctgctgt
aactcgagcg tgct 233482316DNAArtificial Sequencemodified ENV
construct 8gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct
gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta
ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca
gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac
gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt
gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg
aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgggcggc
360gccacccagg cctgccccaa ggtgagcttc gagcccatcc ccatccacta
ctgcgccccc 420gccggcttcg ccatcctgaa gtgcaacgac aagaagttca
acggcagcgg cccctgcacc 480aacgtgagca ccgtgcagtg cacccacggc
atccgccccg tggtgagcac ccagctgctg 540ctgaacggca gcctggccga
ggagggcgtg gtgatccgca gcgagaactt caccgacaac 600gccaagacca
tcatcgtgca gctgaaggag agcgtggaga tcaactgcac ccgccccaac
660aacaacaccc gcaagagcat caccatcggc cccggccgcg ccttctacgc
caccggcgac 720atcatcggcg acatccgcca ggcccactgc aacatcagcg
gcgagaagtg gaacaacacc 780ctgaagcaga tcgtgaccaa gctgcaggcc
cagttcggca acaagaccat cgtgttcaag 840cagagcagcg gcggcgaccc
cgagatcgtg atgcacagct tcaactgcgg cggcgagttc 900ttctactgca
acagcaccca gctgttcaac agcacctgga acaacaccat cggccccaac
960aacaccaacg gcaccatcac cctgccctgc cgcatcaagc agatcatcaa
ccgctggcag 1020gaggtgggca aggccatgta cgcccccccc atccgcggcc
agatccgctg cagcagcaac 1080atcaccggcc tgctgctgac ccgcgacggc
ggcaaggaga tcagcaacac caccgagatc 1140ttccgccccg gcggcggcga
catgcgcgac aactggcgca gcgagctgta caagtacaag 1200gtggtgaaga
tcgagcccct gggcgtggcc cccaccaagg ccaagcgccg cgtggtgcag
1260cgcgagaagc gcgccgtgac cctgggcgcc atgttcctgg gcttcctggg
cgccgccggc 1320agcaccatgg gcgcccgcag cctgaccctg accgtgcagg
cccgccagct gctgagcggc 1380atcgtgcagc agcagaacaa cctgctgcgc
gccatcgagg cccagcagca cctgctgcag 1440ctgaccgtgt ggggcatcaa
gcagctgcag gcccgcgtgc tggccgtgga gcgctacctg 1500aaggaccagc
agctgctggg catctggggc tgcagcggca agctgatctg caccaccgcc
1560gtgccctgga acgccagctg gagcaacaag agcctggacc agatctggaa
caacatgacc 1620tggatggagt gggagcgcga gatcgacaac tacaccaacc
tgatctacac cctgatcgag 1680gagagccaga accagcagga gaagaacgag
caggagctgc tggagctgga caagtgggcc 1740agcctgtgga actggttcga
catcagcaag tggctgtggt acatcaagat cttcatcatg 1800atcgtgggcg
gcctggtggg cctgcgcatc gtgttcaccg tgctgagcat cgtgaaccgc
1860gtgcgccagg gctacagccc cctgagcttc cagacccgct tccccgcccc
ccgcggcccc 1920gaccgccccg agggcatcga ggaggagggc ggcgagcgcg
accgcgaccg cagcagcccc 1980ctggtgcacg gcctgctggc cctgatctgg
gacgacctgc gcagcctgtg cctgttcagc 2040taccaccgcc tgcgcgacct
gatcctgatc gccgcccgca tcgtggagct gctgggccgc 2100cgcggctggg
aggccctgaa gtactggggc aacctgctgc agtactggat ccaggagctg
2160aagaacagcg ccgtgagcct gttcgacgcc atcgccatcg ccgtggccga
gggcaccgac 2220cgcatcatcg aggtggccca gcgcatcggc cgcgccttcc
tgcacatccc ccgccgcatc 2280cgccagggct tcgagcgcgc cctgctgtaa ctcgag
231692541DNAArtificial Sequencemodified ENV construct 9gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc
420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa
ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct
acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg
atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt
cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga
agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc
720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct
gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact
tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag
atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg
ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc
aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag
1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa
gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg
gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg
aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg
ccgcatcaag cagatcatca accgctgggg cggcaaggcc 1260atgtacgccc
cccccatccg cggccagatc cgctgcagca gcaacatcac cggcctgctg
1320ctgacccgcg acggcggcaa ggagatcagc aacaccaccg agatcttccg
ccccggcggc 1380ggcgacatgc gcgacaactg gcgcagcgag ctgtacaagt
acaaggtggt gaagatcgag 1440cccctgggcg tggcccccac caaggccaag
cgccgcgtgg tgcagcgcga gaagcgcgcc 1500gtgaccctgg gcgccatgtt
cctgggcttc ctgggcgccg ccggcagcac catgggcgcc 1560cgcagcctga
ccctgaccgt gcaggcccgc cagctgctga gcggcatcgt gcagcagcag
1620aacaacctgc tgcgcgccat cgaggcccag cagcacctgc tgcagctgac
cgtgtggggc 1680atcaagcagc tgcaggcccg cgtgctggcc gtggagcgct
acctgaagga ccagcagctg 1740ctgggcatct ggggctgcag cggcaagctg
atctgcacca ccgccgtgcc ctggaacgcc 1800agctggagca acaagagcct
ggaccagatc tggaacaaca tgacctggat ggagtgggag 1860cgcgagatcg
acaactacac caacctgatc tacaccctga tcgaggagag ccagaaccag
1920caggagaaga acgagcagga gctgctggag ctggacaagt gggccagcct
gtggaactgg 1980ttcgacatca gcaagtggct gtggtacatc aagatcttca
tcatgatcgt gggcggcctg 2040gtgggcctgc gcatcgtgtt caccgtgctg
agcatcgtga accgcgtgcg ccagggctac 2100agccccctga gcttccagac
ccgcttcccc gccccccgcg gccccgaccg ccccgagggc 2160atcgaggagg
agggcggcga gcgcgaccgc gaccgcagca gccccctggt gcacggcctg
2220ctggccctga tctgggacga cctgcgcagc ctgtgcctgt tcagctacca
ccgcctgcgc 2280gacctgatcc tgatcgccgc ccgcatcgtg gagctgctgg
gccgccgcgg ctgggaggcc 2340ctgaagtact ggggcaacct gctgcagtac
tggatccagg agctgaagaa cagcgccgtg 2400agcctgttcg acgccatcgc
catcgccgtg gccgagggca ccgaccgcat catcgaggtg 2460gcccagcgca
tcggccgcgc cttcctgcac atcccccgcc gcatccgcca gggcttcgag
2520cgcgccctgc tgtaactcga g 2541102541DNAArtificial
Sequencemodified ENV construct 10gaattcgcca ccatggatgc aatgaagaga
gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag
aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac
caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc
acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag
240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa
catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc
tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc
aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga
ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca
acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc
540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt
gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact
actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc
aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg
catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg
aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc
840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa
caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg
ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc
ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc
ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc
ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc
1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa
caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca
accgcggcgg cggcaaggcc 1260atgtacgccc cccccatccg cggccagatc
cgctgcagca gcaacatcac cggcctgctg 1320ctgacccgcg acggcggcaa
ggagatcagc aacaccaccg agatcttccg ccccggcggc 1380ggcgacatgc
gcgacaactg gcgcagcgag ctgtacaagt acaaggtggt gaagatcgag
1440cccctgggcg tggcccccac caaggccaag cgccgcgtgg tgcagcgcga
gaagcgcgcc 1500gtgaccctgg gcgccatgtt cctgggcttc ctgggcgccg
ccggcagcac catgggcgcc 1560cgcagcctga ccctgaccgt gcaggcccgc
cagctgctga gcggcatcgt gcagcagcag 1620aacaacctgc tgcgcgccat
cgaggcccag cagcacctgc tgcagctgac cgtgtggggc 1680atcaagcagc
tgcaggcccg cgtgctggcc gtggagcgct acctgaagga ccagcagctg
1740ctgggcatct ggggctgcag cggcaagctg atctgcacca ccgccgtgcc
ctggaacgcc 1800agctggagca acaagagcct ggaccagatc tggaacaaca
tgacctggat ggagtgggag 1860cgcgagatcg acaactacac caacctgatc
tacaccctga tcgaggagag ccagaaccag 1920caggagaaga acgagcagga
gctgctggag ctggacaagt gggccagcct gtggaactgg 1980ttcgacatca
gcaagtggct gtggtacatc aagatcttca tcatgatcgt gggcggcctg
2040gtgggcctgc gcatcgtgtt caccgtgctg agcatcgtga accgcgtgcg
ccagggctac 2100agccccctga gcttccagac ccgcttcccc gccccccgcg
gccccgaccg ccccgagggc 2160atcgaggagg agggcggcga gcgcgaccgc
gaccgcagca gccccctggt gcacggcctg 2220ctggccctga tctgggacga
cctgcgcagc ctgtgcctgt tcagctacca ccgcctgcgc 2280gacctgatcc
tgatcgccgc ccgcatcgtg gagctgctgg gccgccgcgg ctgggaggcc
2340ctgaagtact ggggcaacct gctgcagtac tggatccagg agctgaagaa
cagcgccgtg 2400agcctgttcg acgccatcgc catcgccgtg gccgagggca
ccgaccgcat catcgaggtg 2460gcccagcgca tcggccgcgc cttcctgcac
atcccccgcc gcatccgcca gggcttcgag 2520cgcgccctgc tgtaactcga g
2541112541DNAArtificial Sequencemodified ENV construct 11gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc
420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa
ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct
acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg
atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt
cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga
agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc
720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct
gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact
tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag
atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg
ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc
aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag
1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa
gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg
gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg
aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg
ccgcatcaag cagatcatca accgcggcag cggcaaggcc 1260atgtacgccc
cccccatccg cggccagatc cgctgcagca gcaacatcac cggcctgctg
1320ctgacccgcg acggcggcaa ggagatcagc aacaccaccg agatcttccg
ccccggcggc 1380ggcgacatgc gcgacaactg gcgcagcgag ctgtacaagt
acaaggtggt gaagatcgag 1440cccctgggcg tggcccccac caaggccaag
cgccgcgtgg tgcagcgcga gaagcgcgcc 1500gtgaccctgg gcgccatgtt
cctgggcttc ctgggcgccg ccggcagcac catgggcgcc 1560cgcagcctga
ccctgaccgt gcaggcccgc cagctgctga gcggcatcgt gcagcagcag
1620aacaacctgc tgcgcgccat cgaggcccag cagcacctgc tgcagctgac
cgtgtggggc 1680atcaagcagc tgcaggcccg cgtgctggcc gtggagcgct
acctgaagga ccagcagctg 1740ctgggcatct ggggctgcag cggcaagctg
atctgcacca ccgccgtgcc ctggaacgcc 1800agctggagca acaagagcct
ggaccagatc tggaacaaca tgacctggat ggagtgggag 1860cgcgagatcg
acaactacac caacctgatc tacaccctga tcgaggagag ccagaaccag
1920caggagaaga acgagcagga gctgctggag ctggacaagt gggccagcct
gtggaactgg 1980ttcgacatca gcaagtggct gtggtacatc aagatcttca
tcatgatcgt gggcggcctg 2040gtgggcctgc gcatcgtgtt caccgtgctg
agcatcgtga accgcgtgcg ccagggctac 2100agccccctga gcttccagac
ccgcttcccc gccccccgcg gccccgaccg ccccgagggc 2160atcgaggagg
agggcggcga gcgcgaccgc gaccgcagca gccccctggt gcacggcctg
2220ctggccctga tctgggacga cctgcgcagc ctgtgcctgt tcagctacca
ccgcctgcgc 2280gacctgatcc tgatcgccgc ccgcatcgtg gagctgctgg
gccgccgcgg ctgggaggcc 2340ctgaagtact ggggcaacct gctgcagtac
tggatccagg agctgaagaa cagcgccgtg 2400agcctgttcg acgccatcgc
catcgccgtg gccgagggca ccgaccgcat catcgaggtg 2460gcccagcgca
tcggccgcgc cttcctgcac atcccccgcc gcatccgcca gggcttcgag
2520cgcgccctgc tgtaactcga g 2541122541DNAArtificial
Sequencemodified ENV construct 12gaattcgcca ccatggatgc aatgaagaga
gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag
aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac
caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc
acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag
240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa
catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc
tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc
aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga
ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca
acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc
540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt
gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact
actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc
aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg
catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg
aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc
840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa
caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg
ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc
ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc
ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc
ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc
1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa
caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagatcatca
accgcggcgg caacaaggcc 1260atgtacgccc cccccatccg cggccagatc
cgctgcagca gcaacatcac cggcctgctg 1320ctgacccgcg acggcggcaa
ggagatcagc aacaccaccg agatcttccg ccccggcggc 1380ggcgacatgc
gcgacaactg gcgcagcgag ctgtacaagt acaaggtggt gaagatcgag
1440cccctgggcg tggcccccac caaggccaag cgccgcgtgg tgcagcgcga
gaagcgcgcc 1500gtgaccctgg gcgccatgtt cctgggcttc ctgggcgccg
ccggcagcac catgggcgcc 1560cgcagcctga ccctgaccgt gcaggcccgc
cagctgctga gcggcatcgt gcagcagcag 1620aacaacctgc tgcgcgccat
cgaggcccag cagcacctgc tgcagctgac cgtgtggggc 1680atcaagcagc
tgcaggcccg cgtgctggcc gtggagcgct acctgaagga ccagcagctg
1740ctgggcatct ggggctgcag cggcaagctg atctgcacca ccgccgtgcc
ctggaacgcc 1800agctggagca acaagagcct ggaccagatc tggaacaaca
tgacctggat ggagtgggag 1860cgcgagatcg acaactacac caacctgatc
tacaccctga tcgaggagag ccagaaccag 1920caggagaaga acgagcagga
gctgctggag ctggacaagt gggccagcct gtggaactgg 1980ttcgacatca
gcaagtggct gtggtacatc aagatcttca tcatgatcgt gggcggcctg
2040gtgggcctgc gcatcgtgtt caccgtgctg agcatcgtga accgcgtgcg
ccagggctac 2100agccccctga gcttccagac ccgcttcccc gccccccgcg
gccccgaccg ccccgagggc 2160atcgaggagg agggcggcga gcgcgaccgc
gaccgcagca gccccctggt gcacggcctg 2220ctggccctga tctgggacga
cctgcgcagc ctgtgcctgt tcagctacca ccgcctgcgc 2280gacctgatcc
tgatcgccgc ccgcatcgtg gagctgctgg gccgccgcgg ctgggaggcc
2340ctgaagtact ggggcaacct gctgcagtac tggatccagg agctgaagaa
cagcgccgtg 2400agcctgttcg acgccatcgc catcgccgtg gccgagggca
ccgaccgcat catcgaggtg 2460gcccagcgca tcggccgcgc cttcctgcac
atcccccgcc gcatccgcca gggcttcgag 2520cgcgccctgc tgtaactcga g
2541132535DNAArtificial Sequencemodified ENV construct 13gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc
420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa
ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct
acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg
atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt
cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga
agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc
720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct
gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact
tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag
atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg
ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc
aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag
1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa
gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg
gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg
aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg
ccgcatcaag cagatcatca acgcccccaa ggccatgtac 1260gcccccccca
tccgcggcca gatccgctgc agcagcaaca tcaccggcct gctgctgacc
1320cgcgacggcg gcaaggagat cagcaacacc accgagatct tccgccccgg
cggcggcgac 1380atgcgcgaca actggcgcag cgagctgtac aagtacaagg
tggtgaagat cgagcccctg 1440ggcgtggccc ccaccaaggc caagcgccgc
gtggtgcagc gcgagaagcg cgccgtgacc 1500ctgggcgcca tgttcctggg
cttcctgggc gccgccggca gcaccatggg cgcccgcagc 1560ctgaccctga
ccgtgcaggc ccgccagctg ctgagcggca tcgtgcagca gcagaacaac
1620ctgctgcgcg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg
gggcatcaag 1680cagctgcagg cccgcgtgct ggccgtggag cgctacctga
aggaccagca gctgctgggc 1740atctggggct gcagcggcaa gctgatctgc
accaccgccg tgccctggaa cgccagctgg 1800agcaacaaga gcctggacca
gatctggaac aacatgacct ggatggagtg ggagcgcgag 1860atcgacaact
acaccaacct gatctacacc ctgatcgagg agagccagaa ccagcaggag
1920aagaacgagc aggagctgct ggagctggac aagtgggcca gcctgtggaa
ctggttcgac 1980atcagcaagt ggctgtggta catcaagatc ttcatcatga
tcgtgggcgg cctggtgggc 2040ctgcgcatcg tgttcaccgt gctgagcatc
gtgaaccgcg tgcgccaggg ctacagcccc 2100ctgagcttcc agacccgctt
ccccgccccc cgcggccccg accgccccga gggcatcgag 2160gaggagggcg
gcgagcgcga ccgcgaccgc agcagccccc tggtgcacgg cctgctggcc
2220ctgatctggg acgacctgcg cagcctgtgc ctgttcagct accaccgcct
gcgcgacctg 2280atcctgatcg ccgcccgcat cgtggagctg ctgggccgcc
gcggctggga ggccctgaag 2340tactggggca acctgctgca gtactggatc
caggagctga agaacagcgc cgtgagcctg 2400ttcgacgcca tcgccatcgc
cgtggccgag ggcaccgacc gcatcatcga ggtggcccag 2460cgcatcggcc
gcgccttcct gcacatcccc cgccgcatcc gccagggctt cgagcgcgcc
2520ctgctgtaac tcgag 2535142529DNAArtificial Sequencemodified ENV
construct 14gaattcgcca ccatggatgc aatgaagaga gggctctgct gtgtgctgct
gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg tgaccgtgta
ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg ttctgcgcca
gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac
gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt
gaccgagaac ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg
aggacatcat cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg
360acccccctgt gcgtgaccct gcactgcacc aacctgaaga acgccaccaa
caccaagagc 420agcaactgga aggagatgga ccgcggcgag atcaagaact
gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca gaaggagtac
gccctgttct acaagctgga cgtggtgccc 540atcgacaacg acaacaccag
ctacaagctg atcaactgca acaccagcgt gatcacccag 600gcctgcccca
aggtgagctt cgagcccatc cccatccact actgcgcccc cgccggcttc
660gccatcctga agtgcaacga caagaagttc aacggcagcg gcccctgcac
caacgtgagc 720accgtgcagt gcacccacgg catccgcccc gtggtgagca
cccagctgct gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc
agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga
gagcgtggag atcaactgca cccgccccaa caacaacacc 900cgcaagagca
tcaccatcgg ccccggccgc gccttctacg ccaccggcga catcatcggc
960gacatccgcc aggcccactg caacatcagc ggcgagaagt ggaacaacac
cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc aacaagacca
tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc
ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa
cagcacctgg aacaacacca tcggccccaa caacaccaac 1200ggcaccatca
ccctgccctg ccgcatcaag cagatcatcg gcggcgccat gtacgccccc
1260cccatccgcg gccagatccg ctgcagcagc aacatcaccg gcctgctgct
gacccgcgac 1320ggcggcaagg agatcagcaa caccaccgag atcttccgcc
ccggcggcgg cgacatgcgc 1380gacaactggc gcagcgagct gtacaagtac
aaggtggtga agatcgagcc cctgggcgtg 1440gcccccacca aggccaagcg
ccgcgtggtg cagcgcgaga agcgcgccgt gaccctgggc 1500gccatgttcc
tgggcttcct gggcgccgcc ggcagcacca tgggcgcccg cagcctgacc
1560ctgaccgtgc aggcccgcca gctgctgagc ggcatcgtgc agcagcagaa
caacctgctg 1620cgcgccatcg aggcccagca gcacctgctg cagctgaccg
tgtggggcat caagcagctg 1680caggcccgcg tgctggccgt ggagcgctac
ctgaaggacc agcagctgct gggcatctgg 1740ggctgcagcg gcaagctgat
ctgcaccacc gccgtgccct ggaacgccag ctggagcaac 1800aagagcctgg
accagatctg gaacaacatg acctggatgg agtgggagcg cgagatcgac
1860aactacacca acctgatcta caccctgatc gaggagagcc agaaccagca
ggagaagaac 1920gagcaggagc tgctggagct ggacaagtgg gccagcctgt
ggaactggtt cgacatcagc 1980aagtggctgt ggtacatcaa gatcttcatc
atgatcgtgg gcggcctggt gggcctgcgc 2040atcgtgttca ccgtgctgag
catcgtgaac cgcgtgcgcc agggctacag ccccctgagc 2100ttccagaccc
gcttccccgc cccccgcggc cccgaccgcc ccgagggcat cgaggaggag
2160ggcggcgagc gcgaccgcga ccgcagcagc cccctggtgc acggcctgct
ggccctgatc 2220tgggacgacc tgcgcagcct gtgcctgttc agctaccacc
gcctgcgcga cctgatcctg 2280atcgccgccc gcatcgtgga gctgctgggc
cgccgcggct gggaggccct gaagtactgg 2340ggcaacctgc tgcagtactg
gatccaggag ctgaagaaca gcgccgtgag cctgttcgac 2400gccatcgcca
tcgccgtggc cgagggcacc gaccgcatca tcgaggtggc ccagcgcatc
2460ggccgcgcct tcctgcacat cccccgccgc atccgccagg gcttcgagcg
cgccctgctg 2520taactcgag 2529152523DNAArtificial Sequencemodified
ENV construct 15gaattcgcca ccatggatgc aatgaagaga gggctctgct
gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag aagctgtggg
tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac caccaccctg
ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc acaacgtgtg
ggccacccac gcctgcgtgc ccaccgaccc caacccccag 240gagatcgtgc
tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa catggtggag
300cagatgcacg aggacatcat cagcctgtgg gaccagagcc tgaagccctg
cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc aacctgaaga
acgccaccaa caccaagagc 420agcaactgga aggagatgga ccgcggcgag
atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca acaagatgca
gaaggagtac gccctgttct acaagctgga cgtggtgccc 540atcgacaacg
acaacaccag ctacaagctg atcaactgca acaccagcgt gatcacccag
600gcctgcccca aggtgagctt cgagcccatc cccatccact actgcgcccc
cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcagcg
gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg catccgcccc
gtggtgagca cccagctgct gctgaacggc 780agcctggccg aggagggcgt
ggtgatccgc agcgagaact tcaccgacaa cgccaagacc 840atcatcgtgc
agctgaagga gagcgtggag atcaactgca cccgccccaa caacaacacc
900cgcaagagca tcaccatcgg ccccggccgc gccttctacg ccaccggcga
catcatcggc 960gacatccgcc aggcccactg caacatcagc ggcgagaagt
ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc ccagttcggc
aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc ccgagatcgt
gatgcacagc ttcaactgcg gcggcgagtt cttctactgc 1140aacagcaccc
agctgttcaa cagcacctgg aacaacacca tcggccccaa caacaccaac
1200ggcaccatca ccctgccctg ccgcatcaag cagatcggcg gcatgtacgc
cccccccatc 1260cgcggccaga tccgctgcag cagcaacatc accggcctgc
tgctgacccg cgacggcggc 1320aaggagatca gcaacaccac cgagatcttc
cgccccggcg gcggcgacat gcgcgacaac 1380tggcgcagcg agctgtacaa
gtacaaggtg gtgaagatcg agcccctggg cgtggccccc 1440accaaggcca
agcgccgcgt ggtgcagcgc gagaagcgcg ccgtgaccct gggcgccatg
1500ttcctgggct tcctgggcgc cgccggcagc accatgggcg cccgcagcct
gaccctgacc 1560gtgcaggccc gccagctgct gagcggcatc gtgcagcagc
agaacaacct gctgcgcgcc 1620atcgaggccc agcagcacct gctgcagctg
accgtgtggg gcatcaagca gctgcaggcc 1680cgcgtgctgg ccgtggagcg
ctacctgaag gaccagcagc tgctgggcat ctggggctgc 1740agcggcaagc
tgatctgcac caccgccgtg ccctggaacg ccagctggag caacaagagc
1800ctggaccaga tctggaacaa catgacctgg atggagtggg agcgcgagat
cgacaactac 1860accaacctga tctacaccct gatcgaggag agccagaacc
agcaggagaa gaacgagcag 1920gagctgctgg agctggacaa gtgggccagc
ctgtggaact ggttcgacat cagcaagtgg 1980ctgtggtaca tcaagatctt
catcatgatc gtgggcggcc tggtgggcct gcgcatcgtg 2040ttcaccgtgc
tgagcatcgt gaaccgcgtg cgccagggct acagccccct gagcttccag
2100acccgcttcc ccgccccccg cggccccgac cgccccgagg gcatcgagga
ggagggcggc 2160gagcgcgacc gcgaccgcag cagccccctg gtgcacggcc
tgctggccct gatctgggac 2220gacctgcgca gcctgtgcct gttcagctac
caccgcctgc gcgacctgat cctgatcgcc 2280gcccgcatcg tggagctgct
gggccgccgc ggctgggagg ccctgaagta ctggggcaac 2340ctgctgcagt
actggatcca ggagctgaag aacagcgccg tgagcctgtt cgacgccatc
2400gccatcgccg tggccgaggg caccgaccgc atcatcgagg tggcccagcg
catcggccgc 2460gccttcctgc acatcccccg ccgcatccgc cagggcttcg
agcgcgccct gctgtaactc 2520gag 2523162517DNAArtificial
Sequencemodified ENV construct 16gaattcgcca ccatggatgc aatgaagaga
gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag
aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac
caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc
acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag
240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa
catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc
tgaagccctg cgtgaagctg 360acccccctgt gcgtgaccct gcactgcacc
aacctgaaga acgccaccaa caccaagagc 420agcaactgga aggagatgga
ccgcggcgag atcaagaact gcagcttcaa ggtgaccacc 480agcatccgca
acaagatgca gaaggagtac gccctgttct acaagctgga cgtggtgccc
540atcgacaacg acaacaccag ctacaagctg atcaactgca acaccagcgt
gatcacccag 600gcctgcccca aggtgagctt cgagcccatc cccatccact
actgcgcccc cgccggcttc 660gccatcctga agtgcaacga caagaagttc
aacggcagcg gcccctgcac caacgtgagc 720accgtgcagt gcacccacgg
catccgcccc gtggtgagca cccagctgct gctgaacggc 780agcctggccg
aggagggcgt ggtgatccgc agcgagaact tcaccgacaa cgccaagacc
840atcatcgtgc agctgaagga gagcgtggag atcaactgca cccgccccaa
caacaacacc 900cgcaagagca tcaccatcgg ccccggccgc gccttctacg
ccaccggcga catcatcggc 960gacatccgcc aggcccactg caacatcagc
ggcgagaagt ggaacaacac cctgaagcag 1020atcgtgacca agctgcaggc
ccagttcggc aacaagacca tcgtgttcaa gcagagcagc 1080ggcggcgacc
ccgagatcgt gatgcacagc ttcaactgcg gcggcgagtt cttctactgc
1140aacagcaccc agctgttcaa cagcacctgg aacaacacca tcggccccaa
caacaccaac 1200ggcaccatca ccctgccctg ccgcatcaag cagggcggct
acgccccccc catccgcggc 1260cagatccgct gcagcagcaa catcaccggc
ctgctgctga cccgcgacgg cggcaaggag 1320atcagcaaca ccaccgagat
cttccgcccc ggcggcggcg acatgcgcga caactggcgc 1380agcgagctgt
acaagtacaa ggtggtgaag atcgagcccc tgggcgtggc ccccaccaag
1440gccaagcgcc gcgtggtgca gcgcgagaag cgcgccgtga ccctgggcgc
catgttcctg 1500ggcttcctgg gcgccgccgg cagcaccatg ggcgcccgca
gcctgaccct gaccgtgcag 1560gcccgccagc tgctgagcgg catcgtgcag
cagcagaaca acctgctgcg cgccatcgag 1620gcccagcagc acctgctgca
gctgaccgtg tggggcatca agcagctgca ggcccgcgtg 1680ctggccgtgg
agcgctacct gaaggaccag cagctgctgg gcatctgggg ctgcagcggc
1740aagctgatct gcaccaccgc cgtgccctgg aacgccagct ggagcaacaa
gagcctggac 1800cagatctgga
acaacatgac ctggatggag tgggagcgcg agatcgacaa ctacaccaac
1860ctgatctaca ccctgatcga ggagagccag aaccagcagg agaagaacga
gcaggagctg 1920ctggagctgg acaagtgggc cagcctgtgg aactggttcg
acatcagcaa gtggctgtgg 1980tacatcaaga tcttcatcat gatcgtgggc
ggcctggtgg gcctgcgcat cgtgttcacc 2040gtgctgagca tcgtgaaccg
cgtgcgccag ggctacagcc ccctgagctt ccagacccgc 2100ttccccgccc
cccgcggccc cgaccgcccc gagggcatcg aggaggaggg cggcgagcgc
2160gaccgcgacc gcagcagccc cctggtgcac ggcctgctgg ccctgatctg
ggacgacctg 2220cgcagcctgt gcctgttcag ctaccaccgc ctgcgcgacc
tgatcctgat cgccgcccgc 2280atcgtggagc tgctgggccg ccgcggctgg
gaggccctga agtactgggg caacctgctg 2340cagtactgga tccaggagct
gaagaacagc gccgtgagcc tgttcgacgc catcgccatc 2400gccgtggccg
agggcaccga ccgcatcatc gaggtggccc agcgcatcgg ccgcgccttc
2460ctgcacatcc cccgccgcat ccgccagggc ttcgagcgcg ccctgctgta actcgag
2517172517DNAArtificial Sequencemodified ENV construct 17gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgaccct gcactgcacc aacctgaaga acgccaccaa caccaagagc
420agcaactgga aggagatgga ccgcggcgag atcaagaact gcagcttcaa
ggtgaccacc 480agcatccgca acaagatgca gaaggagtac gccctgttct
acaagctgga cgtggtgccc 540atcgacaacg acaacaccag ctacaagctg
atcaactgca acaccagcgt gatcacccag 600gcctgcccca aggtgagctt
cgagcccatc cccatccact actgcgcccc cgccggcttc 660gccatcctga
agtgcaacga caagaagttc aacggcagcg gcccctgcac caacgtgagc
720accgtgcagt gcacccacgg catccgcccc gtggtgagca cccagctgct
gctgaacggc 780agcctggccg aggagggcgt ggtgatccgc agcgagaact
tcaccgacaa cgccaagacc 840atcatcgtgc agctgaagga gagcgtggag
atcaactgca cccgccccaa caacaacacc 900cgcaagagca tcaccatcgg
ccccggccgc gccttctacg ccaccggcga catcatcggc 960gacatccgcc
aggcccactg caacatcagc ggcgagaagt ggaacaacac cctgaagcag
1020atcgtgacca agctgcaggc ccagttcggc aacaagacca tcgtgttcaa
gcagagcagc 1080ggcggcgacc ccgagatcgt gatgcacagc ttcaactgcg
gcggcgagtt cttctactgc 1140aacagcaccc agctgttcaa cagcacctgg
aacaacacca tcggccccaa caacaccaac 1200ggcaccatca ccctgccctg
ccgcatcaag caggccccct acgccccccc catccgcggc 1260cagatccgct
gcagcagcaa catcaccggc ctgctgctga cccgcgacgg cggcaaggag
1320atcagcaaca ccaccgagat cttccgcccc ggcggcggcg acatgcgcga
caactggcgc 1380agcgagctgt acaagtacaa ggtggtgaag atcgagcccc
tgggcgtggc ccccaccaag 1440gccaagcgcc gcgtggtgca gcgcgagaag
cgcgccgtga ccctgggcgc catgttcctg 1500ggcttcctgg gcgccgccgg
cagcaccatg ggcgcccgca gcctgaccct gaccgtgcag 1560gcccgccagc
tgctgagcgg catcgtgcag cagcagaaca acctgctgcg cgccatcgag
1620gcccagcagc acctgctgca gctgaccgtg tggggcatca agcagctgca
ggcccgcgtg 1680ctggccgtgg agcgctacct gaaggaccag cagctgctgg
gcatctgggg ctgcagcggc 1740aagctgatct gcaccaccgc cgtgccctgg
aacgccagct ggagcaacaa gagcctggac 1800cagatctgga acaacatgac
ctggatggag tgggagcgcg agatcgacaa ctacaccaac 1860ctgatctaca
ccctgatcga ggagagccag aaccagcagg agaagaacga gcaggagctg
1920ctggagctgg acaagtgggc cagcctgtgg aactggttcg acatcagcaa
gtggctgtgg 1980tacatcaaga tcttcatcat gatcgtgggc ggcctggtgg
gcctgcgcat cgtgttcacc 2040gtgctgagca tcgtgaaccg cgtgcgccag
ggctacagcc ccctgagctt ccagacccgc 2100ttccccgccc cccgcggccc
cgaccgcccc gagggcatcg aggaggaggg cggcgagcgc 2160gaccgcgacc
gcagcagccc cctggtgcac ggcctgctgg ccctgatctg ggacgacctg
2220cgcagcctgt gcctgttcag ctaccaccgc ctgcgcgacc tgatcctgat
cgccgcccgc 2280atcgtggagc tgctgggccg ccgcggctgg gaggccctga
agtactgggg caacctgctg 2340cagtactgga tccaggagct gaagaacagc
gccgtgagcc tgttcgacgc catcgccatc 2400gccgtggccg agggcaccga
ccgcatcatc gaggtggccc agcgcatcgg ccgcgccttc 2460ctgcacatcc
cccgccgcat ccgccagggc ttcgagcgcg ccctgctgta actcgag
2517182322DNAArtificial Sequencemodified ENV construct 18gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360ggcaacagcg
tgatcaccca ggcctgcccc aaggtgagct tcgagcccat ccccatccac
420tactgcgccc ccgccggctt cgccatcctg aagtgcaacg acaagaagtt
caacggcagc 480ggcccctgca ccaacgtgag caccgtgcag tgcacccacg
gcatccgccc cgtggtgagc 540acccagctgc tgctgaacgg cagcctggcc
gaggagggcg tggtgatccg cagcgagaac 600ttcaccgaca acgccaagac
catcatcgtg cagctgaagg agagcgtgga gatcaactgc 660acccgcccca
acaacaacac ccgcaagagc atcaccatcg gccccggccg cgccttctac
720gccaccggcg acatcatcgg cgacatccgc caggcccact gcaacatcag
cggcgagaag 780tggaacaaca ccctgaagca gatcgtgacc aagctgcagg
cccagttcgg caacaagacc 840atcgtgttca agcagagcag cggcggcgac
cccgagatcg tgatgcacag cttcaactgc 900ggcggcgagt tcttctactg
caacagcacc cagctgttca acagcacctg gaacaacacc 960atcggcccca
acaacaccaa cggcaccatc accctgccct gccgcatcaa gcagatcatc
1020aaccgcggcg gcggcaaggc catgtacgcc ccccccatcc gcggccagat
ccgctgcagc 1080agcaacatca ccggcctgct gctgacccgc gacggcggca
aggagatcag caacaccacc 1140gagatcttcc gccccggcgg cggcgacatg
cgcgacaact ggcgcagcga gctgtacaag 1200tacaaggtgg tgaagatcga
gcccctgggc gtggccccca ccaaggccaa gcgccgcgtg 1260gtgcagcgcg
agaagcgcgc cgtgaccctg ggcgccatgt tcctgggctt cctgggcgcc
1320gccggcagca ccatgggcgc ccgcagcctg accctgaccg tgcaggcccg
ccagctgctg 1380agcggcatcg tgcagcagca gaacaacctg ctgcgcgcca
tcgaggccca gcagcacctg 1440ctgcagctga ccgtgtgggg catcaagcag
ctgcaggccc gcgtgctggc cgtggagcgc 1500tacctgaagg accagcagct
gctgggcatc tggggctgca gcggcaagct gatctgcacc 1560accgccgtgc
cctggaacgc cagctggagc aacaagagcc tggaccagat ctggaacaac
1620atgacctgga tggagtggga gcgcgagatc gacaactaca ccaacctgat
ctacaccctg 1680atcgaggaga gccagaacca gcaggagaag aacgagcagg
agctgctgga gctggacaag 1740tgggccagcc tgtggaactg gttcgacatc
agcaagtggc tgtggtacat caagatcttc 1800atcatgatcg tgggcggcct
ggtgggcctg cgcatcgtgt tcaccgtgct gagcatcgtg 1860aaccgcgtgc
gccagggcta cagccccctg agcttccaga cccgcttccc cgccccccgc
1920ggccccgacc gccccgaggg catcgaggag gagggcggcg agcgcgaccg
cgaccgcagc 1980agccccctgg tgcacggcct gctggccctg atctgggacg
acctgcgcag cctgtgcctg 2040ttcagctacc accgcctgcg cgacctgatc
ctgatcgccg cccgcatcgt ggagctgctg 2100ggccgccgcg gctgggaggc
cctgaagtac tggggcaacc tgctgcagta ctggatccag 2160gagctgaaga
acagcgccgt gagcctgttc gacgccatcg ccatcgccgt ggccgagggc
2220accgaccgca tcatcgaggt ggcccagcgc atcggccgcg ccttcctgca
catcccccgc 2280cgcatccgcc agggcttcga gcgcgccctg ctgtaactcg ag
2322192322DNAArtificial Sequencemodified ENV construct 19gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360ggcaacagcg
tgatcaccca ggcctgcccc aaggtgagct tcgagcccat ccccatccac
420tactgcgccc ccgccggctt cgccatcctg aagtgcaacg acaagaagtt
caacggcagc 480ggcccctgca ccaacgtgag caccgtgcag tgcacccacg
gcatccgccc cgtggtgagc 540acccagctgc tgctgaacgg cagcctggcc
gaggagggcg tggtgatccg cagcgagaac 600ttcaccgaca acgccaagac
catcatcgtg cagctgaagg agagcgtgga gatcaactgc 660acccgcccca
acaacaacac ccgcaagagc atcaccatcg gccccggccg cgccttctac
720gccaccggcg acatcatcgg cgacatccgc caggcccact gcaacatcag
cggcgagaag 780tggaacaaca ccctgaagca gatcgtgacc aagctgcagg
cccagttcgg caacaagacc 840atcgtgttca agcagagcag cggcggcgac
cccgagatcg tgatgcacag cttcaactgc 900ggcggcgagt tcttctactg
caacagcacc cagctgttca acagcacctg gaacaacacc 960atcggcccca
acaacaccaa cggcaccatc accctgccct gccgcatcaa gcagatcatc
1020aaccgcggcg gcaacaaggc catgtacgcc ccccccatcc gcggccagat
ccgctgcagc 1080agcaacatca ccggcctgct gctgacccgc gacggcggca
aggagatcag caacaccacc 1140gagatcttcc gccccggcgg cggcgacatg
cgcgacaact ggcgcagcga gctgtacaag 1200tacaaggtgg tgaagatcga
gcccctgggc gtggccccca ccaaggccaa gcgccgcgtg 1260gtgcagcgcg
agaagcgcgc cgtgaccctg ggcgccatgt tcctgggctt cctgggcgcc
1320gccggcagca ccatgggcgc ccgcagcctg accctgaccg tgcaggcccg
ccagctgctg 1380agcggcatcg tgcagcagca gaacaacctg ctgcgcgcca
tcgaggccca gcagcacctg 1440ctgcagctga ccgtgtgggg catcaagcag
ctgcaggccc gcgtgctggc cgtggagcgc 1500tacctgaagg accagcagct
gctgggcatc tggggctgca gcggcaagct gatctgcacc 1560accgccgtgc
cctggaacgc cagctggagc aacaagagcc tggaccagat ctggaacaac
1620atgacctgga tggagtggga gcgcgagatc gacaactaca ccaacctgat
ctacaccctg 1680atcgaggaga gccagaacca gcaggagaag aacgagcagg
agctgctgga gctggacaag 1740tgggccagcc tgtggaactg gttcgacatc
agcaagtggc tgtggtacat caagatcttc 1800atcatgatcg tgggcggcct
ggtgggcctg cgcatcgtgt tcaccgtgct gagcatcgtg 1860aaccgcgtgc
gccagggcta cagccccctg agcttccaga cccgcttccc cgccccccgc
1920ggccccgacc gccccgaggg catcgaggag gagggcggcg agcgcgaccg
cgaccgcagc 1980agccccctgg tgcacggcct gctggccctg atctgggacg
acctgcgcag cctgtgcctg 2040ttcagctacc accgcctgcg cgacctgatc
ctgatcgccg cccgcatcgt ggagctgctg 2100ggccgccgcg gctgggaggc
cctgaagtac tggggcaacc tgctgcagta ctggatccag 2160gagctgaaga
acagcgccgt gagcctgttc gacgccatcg ccatcgccgt ggccgagggc
2220accgaccgca tcatcgaggt ggcccagcgc atcggccgcg ccttcctgca
catcccccgc 2280cgcatccgcc agggcttcga gcgcgccctg ctgtaactcg ag
2322202322DNAArtificial Sequencemodified ENV construct 20gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360ggcaacagcg
tgatcaccca ggcctgcccc aaggtgagct tcgagcccat ccccatccac
420tactgcgccc ccgccggctt cgccatcctg aagtgcaacg acaagaagtt
caacggcagc 480ggcccctgca ccaacgtgag caccgtgcag tgcacccacg
gcatccgccc cgtggtgagc 540acccagctgc tgctgaacgg cagcctggcc
gaggagggcg tggtgatccg cagcgagaac 600ttcaccgaca acgccaagac
catcatcgtg cagctgaagg agagcgtgga gatcaactgc 660acccgcccca
acaacaacac ccgcaagagc atcaccatcg gccccggccg cgccttctac
720gccaccggcg acatcatcgg cgacatccgc caggcccact gcaacatcag
cggcgagaag 780tggaacaaca ccctgaagca gatcgtgacc aagctgcagg
cccagttcgg caacaagacc 840atcgtgttca agcagagcag cggcggcgac
cccgagatcg tgatgcacag cttcaactgc 900ggcggcgagt tcttctactg
caacagcacc cagctgttca acagcacctg gaacaacacc 960atcggcccca
acaacaccaa cggcaccatc accctgccct gccgcatcaa gcagatcatc
1020aaccgctggg gcggcaaggc catgtacgcc ccccccatcc gcggccagat
ccgctgcagc 1080agcaacatca ccggcctgct gctgacccgc gacggcggca
aggagatcag caacaccacc 1140gagatcttcc gccccggcgg cggcgacatg
cgcgacaact ggcgcagcga gctgtacaag 1200tacaaggtgg tgaagatcga
gcccctgggc gtggccccca ccaaggccaa gcgccgcgtg 1260gtgcagcgcg
agaagcgcgc cgtgaccctg ggcgccatgt tcctgggctt cctgggcgcc
1320gccggcagca ccatgggcgc ccgcagcctg accctgaccg tgcaggcccg
ccagctgctg 1380agcggcatcg tgcagcagca gaacaacctg ctgcgcgcca
tcgaggccca gcagcacctg 1440ctgcagctga ccgtgtgggg catcaagcag
ctgcaggccc gcgtgctggc cgtggagcgc 1500tacctgaagg accagcagct
gctgggcatc tggggctgca gcggcaagct gatctgcacc 1560accgccgtgc
cctggaacgc cagctggagc aacaagagcc tggaccagat ctggaacaac
1620atgacctgga tggagtggga gcgcgagatc gacaactaca ccaacctgat
ctacaccctg 1680atcgaggaga gccagaacca gcaggagaag aacgagcagg
agctgctgga gctggacaag 1740tgggccagcc tgtggaactg gttcgacatc
agcaagtggc tgtggtacat caagatcttc 1800atcatgatcg tgggcggcct
ggtgggcctg cgcatcgtgt tcaccgtgct gagcatcgtg 1860aaccgcgtgc
gccagggcta cagccccctg agcttccaga cccgcttccc cgccccccgc
1920ggccccgacc gccccgaggg catcgaggag gagggcggcg agcgcgaccg
cgaccgcagc 1980agccccctgg tgcacggcct gctggccctg atctgggacg
acctgcgcag cctgtgcctg 2040ttcagctacc accgcctgcg cgacctgatc
ctgatcgccg cccgcatcgt ggagctgctg 2100ggccgccgcg gctgggaggc
cctgaagtac tggggcaacc tgctgcagta ctggatccag 2160gagctgaaga
acagcgccgt gagcctgttc gacgccatcg ccatcgccgt ggccgagggc
2220accgaccgca tcatcgaggt ggcccagcgc atcggccgcg ccttcctgca
catcccccgc 2280cgcatccgcc agggcttcga gcgcgccctg ctgtaactcg ag
2322212310DNAArtificial Sequencemodified ENV construct 21gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaaggcc 360cccgtgatca
cccaggcctg ccccaaggtg agcttcgagc ccatccccat ccactactgc
420gcccccgccg gcttcgccat cctgaagtgc aacgacaaga agttcaacgg
cagcggcccc 480tgcaccaacg tgagcaccgt gcagtgcacc cacggcatcc
gccccgtggt gagcacccag 540ctgctgctga acggcagcct ggccgaggag
ggcgtggtga tccgcagcga gaacttcacc 600gacaacgcca agaccatcat
cgtgcagctg aaggagagcg tggagatcaa ctgcacccgc 660cccaacaaca
acacccgcaa gagcatcacc atcggccccg gccgcgcctt ctacgccacc
720ggcgacatca tcggcgacat ccgccaggcc cactgcaaca tcagcggcga
gaagtggaac 780aacaccctga agcagatcgt gaccaagctg caggcccagt
tcggcaacaa gaccatcgtg 840ttcaagcaga gcagcggcgg cgaccccgag
atcgtgatgc acagcttcaa ctgcggcggc 900gagttcttct actgcaacag
cacccagctg ttcaacagca cctggaacaa caccatcggc 960cccaacaaca
ccaacggcac catcaccctg ccctgccgca tcaagcagat catcaacgcc
1020cccaaggcca tgtacgcccc ccccatccgc ggccagatcc gctgcagcag
caacatcacc 1080ggcctgctgc tgacccgcga cggcggcaag gagatcagca
acaccaccga gatcttccgc 1140cccggcggcg gcgacatgcg cgacaactgg
cgcagcgagc tgtacaagta caaggtggtg 1200aagatcgagc ccctgggcgt
ggcccccacc aaggccaagc gccgcgtggt gcagcgcgag 1260aagcgcgccg
tgaccctggg cgccatgttc ctgggcttcc tgggcgccgc cggcagcacc
1320atgggcgccc gcagcctgac cctgaccgtg caggcccgcc agctgctgag
cggcatcgtg 1380cagcagcaga acaacctgct gcgcgccatc gaggcccagc
agcacctgct gcagctgacc 1440gtgtggggca tcaagcagct gcaggcccgc
gtgctggccg tggagcgcta cctgaaggac 1500cagcagctgc tgggcatctg
gggctgcagc ggcaagctga tctgcaccac cgccgtgccc 1560tggaacgcca
gctggagcaa caagagcctg gaccagatct ggaacaacat gacctggatg
1620gagtgggagc gcgagatcga caactacacc aacctgatct acaccctgat
cgaggagagc 1680cagaaccagc aggagaagaa cgagcaggag ctgctggagc
tggacaagtg ggccagcctg 1740tggaactggt tcgacatcag caagtggctg
tggtacatca agatcttcat catgatcgtg 1800ggcggcctgg tgggcctgcg
catcgtgttc accgtgctga gcatcgtgaa ccgcgtgcgc 1860cagggctaca
gccccctgag cttccagacc cgcttccccg ccccccgcgg ccccgaccgc
1920cccgagggca tcgaggagga gggcggcgag cgcgaccgcg accgcagcag
ccccctggtg 1980cacggcctgc tggccctgat ctgggacgac ctgcgcagcc
tgtgcctgtt cagctaccac 2040cgcctgcgcg acctgatcct gatcgccgcc
cgcatcgtgg agctgctggg ccgccgcggc 2100tgggaggccc tgaagtactg
gggcaacctg ctgcagtact ggatccagga gctgaagaac 2160agcgccgtga
gcctgttcga cgccatcgcc atcgccgtgg ccgagggcac cgaccgcatc
2220atcgaggtgg cccagcgcat cggccgcgcc ttcctgcaca tcccccgccg
catccgccag 2280ggcttcgagc gcgccctgct gtaactcgag
2310222298DNAArtificial Sequencemodified ENV construct 22gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgggcggc 360atcacccagg
cctgccccaa ggtgagcttc gagcccatcc ccatccacta ctgcgccccc
420gccggcttcg ccatcctgaa gtgcaacgac aagaagttca acggcagcgg
cccctgcacc 480aacgtgagca ccgtgcagtg cacccacggc atccgccccg
tggtgagcac ccagctgctg 540ctgaacggca gcctggccga ggagggcgtg
gtgatccgca gcgagaactt caccgacaac 600gccaagacca tcatcgtgca
gctgaaggag agcgtggaga tcaactgcac ccgccccaac 660aacaacaccc
gcaagagcat caccatcggc cccggccgcg ccttctacgc caccggcgac
720atcatcggcg acatccgcca ggcccactgc aacatcagcg gcgagaagtg
gaacaacacc 780ctgaagcaga tcgtgaccaa gctgcaggcc cagttcggca
acaagaccat cgtgttcaag 840cagagcagcg gcggcgaccc cgagatcgtg
atgcacagct tcaactgcgg cggcgagttc 900ttctactgca acagcaccca
gctgttcaac agcacctgga acaacaccat cggccccaac 960aacaccaacg
gcaccatcac cctgccctgc cgcatcaagc agatcatcgg cggcgccatg
1020tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg
cctgctgctg 1080acccgcgacg gcggcaagga gatcagcaac accaccgaga
tcttccgccc cggcggcggc 1140gacatgcgcg acaactggcg cagcgagctg
tacaagtaca aggtggtgaa gatcgagccc 1200ctgggcgtgg cccccaccaa
ggccaagcgc cgcgtggtgc agcgcgagaa gcgcgccgtg 1260accctgggcg
ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc
1320agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca
gcagcagaac 1380aacctgctgc gcgccatcga ggcccagcag cacctgctgc
agctgaccgt gtggggcatc 1440aagcagctgc aggcccgcgt gctggccgtg
gagcgctacc tgaaggacca gcagctgctg 1500ggcatctggg gctgcagcgg
caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1560tggagcaaca
agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc
1620gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca
gaaccagcag 1680gagaagaacg agcaggagct gctggagctg gacaagtggg
ccagcctgtg gaactggttc 1740gacatcagca agtggctgtg gtacatcaag
atcttcatca tgatcgtggg cggcctggtg 1800ggcctgcgca tcgtgttcac
cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc 1860cccctgagct
tccagacccg cttccccgcc ccccgcggcc ccgaccgccc cgagggcatc
1920gaggaggagg gcggcgagcg cgaccgcgac cgcagcagcc ccctggtgca
cggcctgctg 1980gccctgatct gggacgacct gcgcagcctg tgcctgttca
gctaccaccg cctgcgcgac 2040ctgatcctga tcgccgcccg catcgtggag
ctgctgggcc gccgcggctg ggaggccctg 2100aagtactggg
gcaacctgct gcagtactgg atccaggagc tgaagaacag cgccgtgagc
2160ctgttcgacg ccatcgccat cgccgtggcc gagggcaccg accgcatcat
cgaggtggcc 2220cagcgcatcg gccgcgcctt cctgcacatc ccccgccgca
tccgccaggg cttcgagcgc 2280gccctgctgt aactcgag
2298232298DNAArtificial Sequencemodified ENV construct 23gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgcccggc 360atcacccagg
cctgccccaa ggtgagcttc gagcccatcc ccatccacta ctgcgccccc
420gccggcttcg ccatcctgaa gtgcaacgac aagaagttca acggcagcgg
cccctgcacc 480aacgtgagca ccgtgcagtg cacccacggc atccgccccg
tggtgagcac ccagctgctg 540ctgaacggca gcctggccga ggagggcgtg
gtgatccgca gcgagaactt caccgacaac 600gccaagacca tcatcgtgca
gctgaaggag agcgtggaga tcaactgcac ccgccccaac 660aacaacaccc
gcaagagcat caccatcggc cccggccgcg ccttctacgc caccggcgac
720atcatcggcg acatccgcca ggcccactgc aacatcagcg gcgagaagtg
gaacaacacc 780ctgaagcaga tcgtgaccaa gctgcaggcc cagttcggca
acaagaccat cgtgttcaag 840cagagcagcg gcggcgaccc cgagatcgtg
atgcacagct tcaactgcgg cggcgagttc 900ttctactgca acagcaccca
gctgttcaac agcacctgga acaacaccat cggccccaac 960aacaccaacg
gcaccatcac cctgccctgc cgcatcaagc agatcatcgg cggcgccatg
1020tacgcccccc ccatccgcgg ccagatccgc tgcagcagca acatcaccgg
cctgctgctg 1080acccgcgacg gcggcaagga gatcagcaac accaccgaga
tcttccgccc cggcggcggc 1140gacatgcgcg acaactggcg cagcgagctg
tacaagtaca aggtggtgaa gatcgagccc 1200ctgggcgtgg cccccaccaa
ggccaagcgc cgcgtggtgc agcgcgagaa gcgcgccgtg 1260accctgggcg
ccatgttcct gggcttcctg ggcgccgccg gcagcaccat gggcgcccgc
1320agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg gcatcgtgca
gcagcagaac 1380aacctgctgc gcgccatcga ggcccagcag cacctgctgc
agctgaccgt gtggggcatc 1440aagcagctgc aggcccgcgt gctggccgtg
gagcgctacc tgaaggacca gcagctgctg 1500ggcatctggg gctgcagcgg
caagctgatc tgcaccaccg ccgtgccctg gaacgccagc 1560tggagcaaca
agagcctgga ccagatctgg aacaacatga cctggatgga gtgggagcgc
1620gagatcgaca actacaccaa cctgatctac accctgatcg aggagagcca
gaaccagcag 1680gagaagaacg agcaggagct gctggagctg gacaagtggg
ccagcctgtg gaactggttc 1740gacatcagca agtggctgtg gtacatcaag
atcttcatca tgatcgtggg cggcctggtg 1800ggcctgcgca tcgtgttcac
cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc 1860cccctgagct
tccagacccg cttccccgcc ccccgcggcc ccgaccgccc cgagggcatc
1920gaggaggagg gcggcgagcg cgaccgcgac cgcagcagcc ccctggtgca
cggcctgctg 1980gccctgatct gggacgacct gcgcagcctg tgcctgttca
gctaccaccg cctgcgcgac 2040ctgatcctga tcgccgcccg catcgtggag
ctgctgggcc gccgcggctg ggaggccctg 2100aagtactggg gcaacctgct
gcagtactgg atccaggagc tgaagaacag cgccgtgagc 2160ctgttcgacg
ccatcgccat cgccgtggcc gagggcaccg accgcatcat cgaggtggcc
2220cagcgcatcg gccgcgcctt cctgcacatc ccccgccgca tccgccaggg
cttcgagcgc 2280gccctgctgt aactcgag 2298242298DNAArtificial
Sequencemodified ENV construct 24gaattcgcca ccatggatgc aatgaagaga
gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg tttcgcccag cgccgtggag
aagctgtggg tgaccgtgta ctacggcgtg 120cccgtgtgga aggaggccac
caccaccctg ttctgcgcca gcgacgccaa ggcctacgac 180accgaggtgc
acaacgtgtg ggccacccac gcctgcgtgc ccaccgaccc caacccccag
240gagatcgtgc tggagaacgt gaccgagaac ttcaacatgt ggaagaacaa
catggtggag 300cagatgcacg aggacatcat cagcctgtgg gaccagagcc
tgaagccctg cgtgggcggc 360gccacccagg cctgccccaa ggtgagcttc
gagcccatcc ccatccacta ctgcgccccc 420gccggcttcg ccatcctgaa
gtgcaacgac aagaagttca acggcagcgg cccctgcacc 480aacgtgagca
ccgtgcagtg cacccacggc atccgccccg tggtgagcac ccagctgctg
540ctgaacggca gcctggccga ggagggcgtg gtgatccgca gcgagaactt
caccgacaac 600gccaagacca tcatcgtgca gctgaaggag agcgtggaga
tcaactgcac ccgccccaac 660aacaacaccc gcaagagcat caccatcggc
cccggccgcg ccttctacgc caccggcgac 720atcatcggcg acatccgcca
ggcccactgc aacatcagcg gcgagaagtg gaacaacacc 780ctgaagcaga
tcgtgaccaa gctgcaggcc cagttcggca acaagaccat cgtgttcaag
840cagagcagcg gcggcgaccc cgagatcgtg atgcacagct tcaactgcgg
cggcgagttc 900ttctactgca acagcaccca gctgttcaac agcacctgga
acaacaccat cggccccaac 960aacaccaacg gcaccatcac cctgccctgc
cgcatcaagc agatcatcgg cggcgccatg 1020tacgcccccc ccatccgcgg
ccagatccgc tgcagcagca acatcaccgg cctgctgctg 1080acccgcgacg
gcggcaagga gatcagcaac accaccgaga tcttccgccc cggcggcggc
1140gacatgcgcg acaactggcg cagcgagctg tacaagtaca aggtggtgaa
gatcgagccc 1200ctgggcgtgg cccccaccaa ggccaagcgc cgcgtggtgc
agcgcgagaa gcgcgccgtg 1260accctgggcg ccatgttcct gggcttcctg
ggcgccgccg gcagcaccat gggcgcccgc 1320agcctgaccc tgaccgtgca
ggcccgccag ctgctgagcg gcatcgtgca gcagcagaac 1380aacctgctgc
gcgccatcga ggcccagcag cacctgctgc agctgaccgt gtggggcatc
1440aagcagctgc aggcccgcgt gctggccgtg gagcgctacc tgaaggacca
gcagctgctg 1500ggcatctggg gctgcagcgg caagctgatc tgcaccaccg
ccgtgccctg gaacgccagc 1560tggagcaaca agagcctgga ccagatctgg
aacaacatga cctggatgga gtgggagcgc 1620gagatcgaca actacaccaa
cctgatctac accctgatcg aggagagcca gaaccagcag 1680gagaagaacg
agcaggagct gctggagctg gacaagtggg ccagcctgtg gaactggttc
1740gacatcagca agtggctgtg gtacatcaag atcttcatca tgatcgtggg
cggcctggtg 1800ggcctgcgca tcgtgttcac cgtgctgagc atcgtgaacc
gcgtgcgcca gggctacagc 1860cccctgagct tccagacccg cttccccgcc
ccccgcggcc ccgaccgccc cgagggcatc 1920gaggaggagg gcggcgagcg
cgaccgcgac cgcagcagcc ccctggtgca cggcctgctg 1980gccctgatct
gggacgacct gcgcagcctg tgcctgttca gctaccaccg cctgcgcgac
2040ctgatcctga tcgccgcccg catcgtggag ctgctgggcc gccgcggctg
ggaggccctg 2100aagtactggg gcaacctgct gcagtactgg atccaggagc
tgaagaacag cgccgtgagc 2160ctgttcgacg ccatcgccat cgccgtggcc
gagggcaccg accgcatcat cgaggtggcc 2220cagcgcatcg gccgcgcctt
cctgcacatc ccccgccgca tccgccaggg cttcgagcgc 2280gccctgctgt aactcgag
2298252358DNAArtificial Sequencemodified ENV construct 25gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgggggc agggaactgc aacaccagcg tgatcaccca ggcctgcccc
420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt
cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca
ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc
acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg
cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg
agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc
720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg
cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca
ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc
atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag
cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca
acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc
1020accctgccct gccgcatcaa gcagatcatc aaccgctggc aggaggtggg
caaggccatg 1080tacgcccccc ccatccgcgg ccagatccgc tgcagcagca
acatcaccgg cctgctgctg 1140acccgcgacg gcggcaagga gatcagcaac
accaccgaga tcttccgccc cggcggcggc 1200gacatgcgcg acaactggcg
cagcgagctg tacaagtaca aggtggtgaa gatcgagccc 1260ctgggcgtgg
cccccaccaa ggccaagcgc cgcgtggtgc agcgcgagaa gcgcgccgtg
1320accctgggcg ccatgttcct gggcttcctg ggcgccgccg gcagcaccat
gggcgcccgc 1380agcctgaccc tgaccgtgca ggcccgccag ctgctgagcg
gcatcgtgca gcagcagaac 1440aacctgctgc gcgccatcga ggcccagcag
cacctgctgc agctgaccgt gtggggcatc 1500aagcagctgc aggcccgcgt
gctggccgtg gagcgctacc tgaaggacca gcagctgctg 1560ggcatctggg
gctgcagcgg caagctgatc tgcaccaccg ccgtgccctg gaacgccagc
1620tggagcaaca agagcctgga ccagatctgg aacaacatga cctggatgga
gtgggagcgc 1680gagatcgaca actacaccaa cctgatctac accctgatcg
aggagagcca gaaccagcag 1740gagaagaacg agcaggagct gctggagctg
gacaagtggg ccagcctgtg gaactggttc 1800gacatcagca agtggctgtg
gtacatcaag atcttcatca tgatcgtggg cggcctggtg 1860ggcctgcgca
tcgtgttcac cgtgctgagc atcgtgaacc gcgtgcgcca gggctacagc
1920cccctgagct tccagacccg cttccccgcc ccccgcggcc ccgaccgccc
cgagggcatc 1980gaggaggagg gcggcgagcg cgaccgcgac cgcagcagcc
ccctggtgca cggcctgctg 2040gccctgatct gggacgacct gcgcagcctg
tgcctgttca gctaccaccg cctgcgcgac 2100ctgatcctga tcgccgcccg
catcgtggag ctgctgggcc gccgcggctg ggaggccctg 2160aagtactggg
gcaacctgct gcagtactgg atccaggagc tgaagaacag cgccgtgagc
2220ctgttcgacg ccatcgccat cgccgtggcc gagggcaccg accgcatcat
cgaggtggcc 2280cagcgcatcg gccgcgcctt cctgcacatc ccccgccgca
tccgccaggg cttcgagcgc 2340gccctgctgt aactcgag
2358262352DNAArtificial SequenceModified ENV construct 26gaattcgcca
ccatggatgc aatgaagaga gggctctgct gtgtgctgct gctgtgtgga 60gcagtcttcg
tttcgcccag cgccgtggag aagctgtggg tgaccgtgta ctacggcgtg
120cccgtgtgga aggaggccac caccaccctg ttctgcgcca gcgacgccaa
ggcctacgac 180accgaggtgc acaacgtgtg ggccacccac gcctgcgtgc
ccaccgaccc caacccccag 240gagatcgtgc tggagaacgt gaccgagaac
ttcaacatgt ggaagaacaa catggtggag 300cagatgcacg aggacatcat
cagcctgtgg gaccagagcc tgaagccctg cgtgaagctg 360acccccctgt
gcgtgggggc agggaactgc aacaccagcg tgatcaccca ggcctgcccc
420aaggtgagct tcgagcccat ccccatccac tactgcgccc ccgccggctt
cgccatcctg 480aagtgcaacg acaagaagtt caacggcagc ggcccctgca
ccaacgtgag caccgtgcag 540tgcacccacg gcatccgccc cgtggtgagc
acccagctgc tgctgaacgg cagcctggcc 600gaggagggcg tggtgatccg
cagcgagaac ttcaccgaca acgccaagac catcatcgtg 660cagctgaagg
agagcgtgga gatcaactgc acccgcccca acaacaacac ccgcaagagc
720atcaccatcg gccccggccg cgccttctac gccaccggcg acatcatcgg
cgacatccgc 780caggcccact gcaacatcag cggcgagaag tggaacaaca
ccctgaagca gatcgtgacc 840aagctgcagg cccagttcgg caacaagacc
atcgtgttca agcagagcag cggcggcgac 900cccgagatcg tgatgcacag
cttcaactgc ggcggcgagt tcttctactg caacagcacc 960cagctgttca
acagcacctg gaacaacacc atcggcccca acaacaccaa cggcaccatc
1020accctgccct gccgcatcaa gcagatcatc aaccgcggcg gcggcaaggc
catgtacgcc 1080ccccccatcc gcggccagat ccgctgcagc agcaacatca
ccggcctgct gctgacccgc 1140gacggcggca aggagatcag caacaccacc
gagatcttcc gccccggggg cggcgacatg 1200cgcgacaact ggcgcagcga
gctgtacaag tacaaggtgg tgaagatcga gcccctgggc 1260gtggccccca
ccaaggccaa gcgccgcgtg gtgcagcgcg agaagcgcgc cgtgaccctg
1320ggcgccatgt tcctgggctt cctgggcgcc gccggcagca ccatgggcgc
ccgcagcctg 1380accctgaccg tgcaggcccg ccagctgctg agcggcatcg
tgcagcagca gaacaacctg 1440ctgcgcgcca tcgaggccca gcagcacctg
ctgcagctga ccgtgtgggg catcaagcag 1500ctgcaggccc gcgtgctggc
cgtggagcgc tacctgaagg accagcagct gctgggcatc 1560tggggctgca
gcggcaagct gatctgcacc accgccgtgc cctggaacgc cagctggagc
1620aacaagagcc tggaccagat ctggaacaac atgacctgga tggagtggga
gcgcgagatc 1680gacaactaca ccaacctgat ctacaccctg atcgaggaga
gccagaacca gcaggagaag 1740aacgagcagg agctgctgga gctggacaag
tgggccagcc tgtggaactg gttcgacatc 1800agcaagtggc tgtggtacat
caagatcttc atcatgatcg tgggcggcct ggtgggcctg 1860cgcatcgtgt
tcaccgtgct gagcatcgtg aaccgcgtgc gccagggcta cagccccctg
1920agcttccaga cccgcttccc cgccccccgc ggccccgacc gccccgaggg
catcgaggag 1980gagggcggcg agcgcgaccg cgaccgcagc agccccctgg
tgcacggcct gctggccctg 2040atctgggacg acctgcgcag cctgtgcctg
ttcagctacc accgcctgcg cgacctgatc 2100ctgatcgccg cccgcatcgt
ggagctgctg ggccgccgcg gctgggaggc cctgaagtac 2160tggggcaacc
tgctgcagta ctggatccag gagctgaaga acagcgccgt gagcctgttc
2220gacgccatcg ccatcgccgt ggccgagggc accgaccgca tcatcgaggt
ggcccagcgc 2280atcggccgcg ccttcctgca catcccccgc cgcatccgcc
agggcttcga gcgcgccctg 2340ctgtaactcg ag 2352
* * * * *
References