U.S. patent application number 11/918219 was filed with the patent office on 2010-02-04 for method of inducing neutralizing antibodies to human immunodeficiency virus.
Invention is credited to S. Munir Alam, Barton F. Haynes, Hua-Xin Liao.
Application Number | 20100028415 11/918219 |
Document ID | / |
Family ID | 37087677 |
Filed Date | 2010-02-04 |
United States Patent
Application |
20100028415 |
Kind Code |
A1 |
Haynes; Barton F. ; et
al. |
February 4, 2010 |
Method of Inducing Neutralizing Antibodies to Human
Immunodeficiency Virus
Abstract
The present invention relates, in general, to human
immunodeficiency virus (HIV), and, in particular, to a method of
inducing neutralizing antibodies to HIV and to compounds and
compositions suitable for use in such a method.
Inventors: |
Haynes; Barton F.; (Durham,
NC) ; Alam; S. Munir; (Durham, NC) ; Liao;
Hua-Xin; (Durham, NC) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
901 NORTH GLEBE ROAD, 11TH FLOOR
ARLINGTON
VA
22203
US
|
Family ID: |
37087677 |
Appl. No.: |
11/918219 |
Filed: |
April 12, 2006 |
PCT Filed: |
April 12, 2006 |
PCT NO: |
PCT/US2006/013684 |
371 Date: |
December 22, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60670243 |
Apr 12, 2005 |
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60675091 |
Apr 27, 2005 |
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60697997 |
Jul 12, 2005 |
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60757478 |
Jan 10, 2006 |
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Current U.S.
Class: |
424/450 ;
424/184.1; 435/71.1 |
Current CPC
Class: |
A61K 2039/6018 20130101;
C12N 2740/16134 20130101; C12N 7/00 20130101; C07K 16/18 20130101;
A61K 39/0005 20130101; A61K 39/385 20130101; C12N 2740/16011
20130101; C12N 2740/16122 20130101; A61K 39/21 20130101; A61K
2039/55561 20130101; A61K 2039/55566 20130101; Y02A 50/30 20180101;
C07K 14/005 20130101; C12N 2740/16111 20130101; C07K 2317/76
20130101; A61K 39/12 20130101; A61K 9/127 20130101; A61K 2039/55555
20130101; A61P 31/18 20180101; A61K 2039/55505 20130101; Y02A 50/41
20180101; A61P 37/04 20180101; C07K 16/1063 20130101; A61K 2039/57
20130101; C07K 2319/00 20130101 |
Class at
Publication: |
424/450 ;
424/184.1; 435/71.1 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 39/00 20060101 A61K039/00; A61P 31/18 20060101
A61P031/18; C12P 21/00 20060101 C12P021/00 |
Claims
1. A method of inducing the production in a patient of anti-human
immunodeficiency virus (HIV) antibodies comprising administering to
a patient in need thereof an amount of at least one autoantigen
cross-reactive with HIV envelope sufficient to effect said
induction.
2. The method according to claim 1 wherein said autoantigen is
cardiolipin, SS-A/RO, double stranded (ds) DNA, centromere B
protein or RiBo nucleoprotein (RNP), or fragment thereof that
induces production of said antibodies.
3. The method according to claim 2 wherein said autoantigen is
cardiolipin or fragment thereof that induces production of said
antibodies.
4. The method according to claim 1 wherein said antibodies bind a
gp41 membrane proximal external region (MPER) epitope.
5. The method according to claim 4 wherein said MPER epitope
comprises the sequence NWFDIT (SEQ ID NO:22) or ELDKWA (SEQ ID
NO:23).
6. The method according to claim 1 wherein said method further
comprises administering to said patient an HIV envelope protein,
polypeptide or peptide comprising an epitope cross-reactive with
said autoantigen.
7. The method according to claim 6 wherein a DNA sequence encoding
said HIV protein, polypeptide or peptide is administered under
conditions such that said DNA sequence is expressed and said HIV
protein, polypeptide or peptide is thereby produced in said
patient.
8. The method according to claim 1 further comprising administering
to said patient an adjuvant that breaks tolerance to said
autoantigen.
9. The method according to claim 8 wherein said adjuvant comprises
a TRL9 agonist.
10. The method according to claim 9 wherein said TRL9 agonist
comprises a CpG oligonucleotide.
11. A composition comprising an autoantigen that is cross-reactive
with HIV envelope and an agent that breaks tolerance to said
autoantigen.
12. The composition according to claim 11 wherein said autoantigen
is cardiolipin, SS-A/RO, dsDNA, centromere B protein or RiBo RNP,
or fragment thereof comprising an epitope cross-reactive with HIV
envelope.
13. The method according to claim 11 wherein said agent is a TRL9
agonist.
14. The method according to claim 13 wherein said TRL9 agonist
comprises a CpG oligonucleotide.
15. A method of producing autoantibodies that are cross-reactive
with HIV envelope comprising isolating B cells from a patient with
primary autoimmune disease or from a non-HIV infected patient with
an infectious disease selected from the group consisting of
syphilis, leishmaniasis and leprosy, and creating therefrom an
immortal cell line that produces-said autoantibodies.
16. The method according to claim 15 wherein said patient is a
primary autoimmune patient that is HIV infected or that has
received an envelope-based HIV vaccine.
17. The method according to claim 15 wherein said B cells are fused
with myeloma cells to form hybridomas that produce said
autoantibodies.
18. The method according to claim 15 wherein said patient is a
systemic lupus erythematosus (SLE) patient, an anti-phospholipid
antibody syndrome patient or a non-HIV infected patient with an
infectious disease selected from the group consisting of syphilis,
leishmaniasis and leprosy.
19. The method according to claim 18 wherein said patient is a
systemic lupus erythematosus (SLE) patient that is HIV infected or
that has received an envelope-based HIV vaccine, or said patient is
a non-HIV infected patient with an infectious disease selected from
the group consisting of syphilis, leishmaniasis and leprosy that
has received an envelope-based HIV vaccine.
20. A method of inducing the production in a patient of anti-HIV
antibodies comprising administering to a patient in need thereof an
amount of at least one autoantigen cross-reactive with an HIV
virion sufficient to effect said induction.
21. The method according to claim 20 wherein said autoantigen is a
phospholipid or a derivative thereof.
22. The method according to claim 21 wherein said phospholipid is
cardiolipin, phosphatidylserine, phosphatidylethanolamine,
phosphatidylcholine, phosphotidylinositol or sphingomyelin, or
derivative thereof.
23. The method according to claim 22 wherein said phospholipid is
dioleoyl phosphatidylethanolamine (DOPE) (hexagonal phase).
24. The method according to claim 20 wherein said autoantigen is
cross-reactive with HIV envelope.
25. The method according to claim 20 wherein said autoantigen is
centromere F protein, or fragment thereof comprising an epitope
cross-reactive with the HIV virion.
26. The method according to claim 25 wherein said autoantigen is
cross-reactive with HIV envelope.
27. A composition comprising an autoantigen that is cross-reactive
with an HIV virion and an agent that breaks tolerance to said
autoantigen.
28. The composition according to claim 27 wherein said autoantigen
is a phospholipid or a derivative thereof.
29. The composition according to claim 28 wherein said phospholipid
is cardiolipin, phosphatidylserine, phosphatidylethanolamine,
phosphatidylcholine, phosphotidylinositol or sphingomyelin, or
derivative thereof.
30. The composition according to claim 29 wherein said phospholipid
is dioleoyl phosphatidylethanolamine (DOPE) (hexagonal phase).
31. The composition according to claim 27 wherein said autoantigen
is cross-reactive with HIV envelope.
32. The method according to claim 1 or 20 wherein said patient is
not infected with HIV.
33. A method of inducing the production in a patient of anti-HIV
antibodies comprising administering to a patient in need thereof an
amount of at least one liposome-peptide conjugate in an amount
sufficient to effect said induction, wherein said peptide comprises
a membrane external proximal region (MPER) epitope.
34. The method according to claim 33 wherein said peptide comprises
the sequence ELDKWAS (SEQ ID NO:12) or WFNITNW (SEQ ID NO:21).
35. The method according to claim 33 wherein said liposome-peptide
conjugate comprises a hydrophobic linker.
36. An immunogen comprising an MPER epitope embedded in a
liposome.
37. The immunogen according to claim 36 bound to a detectable
label.
Description
[0001] This application claims priority from U.S. Prov. Appln. No.
60/670,243, filed Apr. 12, 2005, U.S. Prov. Appln. No. 60/675,091,
filed Apr. 27, 2005, U.S. Prov. Appln. No. 60/697,997, filed Jul.
12, 2005, and U.S. Prov. Appln. No. 60/757,478, filed Jan. 10,
2006, the entire contents of which are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates, in general, to human
immunodeficiency virus (HIV), and, in particular, to a method of
inducing neutralizing antibodies to HIV and to compounds and
compositions suitable for use in such a method.
BACKGROUND
[0003] The first antibodies that are made in acute HIV-1 infection
are against the CD4 binding site (Moore et al, J. Virol. 68(8) 5142
(1994)), the CCR5 co-receptor binding site (Choe et al, Cell
114(2):161-170 (2003)), and the V3 loop (Moore et al, J. Acquir.
Immun. Def. Syn. 7(4):332 (1994)). However, these antibodies do not
control HIV-1 and are easily escaped (Burton et al, Nature Immun.
5:233-236 (2004), Wei et al, Nature 422(6929):307-312 (2003)).
Neutralizing antibodies against autologous virus develop fifty to
sixty days after infection, but antibodies capable of neutralizing
heterologous HIV-1 strains do not arise until after the first year
of infection (Richman et al, Proc. Natl. Acad. Sci. USA
100(7):4144-4149 (2003), Wei et al, Nature 422(6929):307-312
(2003)).
[0004] The four epitopes on HIV-1 envelope to which rare broadly
reactive neutralizing antibodies bind are the CD4 binding site
(CD4BS) (mab (monoclonal antibody) IgG1b12) (Zwick et al, J. Virol.
77(10):5863-5876 (2003)), the membrane proximal external region
(MPER) epitopes defined by human mabs 2F5 and 4E10 (Armbruster et
al, J. Antimicrob. Chemother. 54:915-920 (2004), Stiegler and
Katinger, J. Antimicrob. Chemother. 51:757-759 (2003), Zwick et al,
Journal of Virology 79:1252-1261 (2005), Purtscher et al, AIDS
10:587 (1996)) (FIG. 1), and the mannan glycan epitope defined by
human mab 2G12 (Scanlan et al, Adv. Exper. Med. Biol. 535:205-218
(2003)). These four rare human mabs are all unusual: two are IgG3
(2F5 and 4E10), one has a unique Ig dimer structure (2G12), one has
a very hydrophobic CDR3 (2F5) (Ofek et al, J. Virol. 198:10724
(2004)), and, in all four, the CDR3 is unusually long (Burton et
al, Nature Immunol. 5(3):233-236 (2004), Kunert et al, AIDS Res.
Hum. Retroviruses 20(7):755-762 (2004), Zwick et al, J. Virol.
78(6):3155-3161 (2004), Cardoso et al, Immunity 22:163-172 (2005)).
Of these, 2F5- and 4E10-like human mabs are quite rare. Acute HIV-1
patients do not make antibodies against the MPER or 2G12 epitopes
(Robinson, unpublished (2005), Shaw, unpublished (2005), MPER can
be defined as amino acids 652 to 683 of HIV envelope (Cardoso et
al, Immunity 22:163-173 (2005) (e.g.,
QQEKNEQELLELDKWASLWNWFDITNWLWYIK). CD4 binding site (BS) antibodies
are commonly made early in HIV-1 infection, but these antibodies
generally do not have the broad spectrum of neutralization shown by
mab IgG1b12 (Burton et al, Nat. Immunol. 5(3):233-236 (2004)).
[0005] A number of epitopes of the HIV-1 envelope have been shown
to cross-react with host tissues (Pinto et al., AIDS Res. Hum.
Retrov. 10:823-828 (1994), Douvas et al, AIDS Res. Hum. Retrov.
10:253-262 (1994), Douvas et al, AIDS Res. Hum. Retrov.
12:1509-1517 (1996)), and autoimmune patients have been shown to
make antibodies that cross-react with HIV proteins (Pinto et al,
AIDS Res. Hum. Retrov. 10:823-828 (1994), Douvas et al, AIDS Res.
Hum. Retrov. 10:253-262 (1994), Douvas et al, AIDS Res. Hum.
Retrov. 12:1509-1517 (1996), Barthel et al, Semin. Arthr. Rheum.
23:1-7 (1993)). Similarly, induction of immune responses to
self-epitopes has been suggested to be a cause of the autoimmune
abnormalities and T cell depletion in AIDS (Douvas et al, AIDS Res.
Hum. Retrov. 12:1509-1517 (1996), Ziegler et al, Clin. Immunol.
Immunopath. 41:305-313 (1986)).
[0006] High affinity peptide ligands for the 2F5 mab have been made
that induce high levels of antibody against the peptide but do not
broadly neutralize HIV-1 primary isolates (McGaughey et al,
Biochemistry 42(11):3214-3223 (2003), Zhang et al, J. Virol.
78(15):8342-8348 (2004), rev. in Zwick et al, J. Virol.
79:1252-1261 (2005)). These results have been interpreted to mean
that the peptide ligands for 2F5 are not in the appropriate
conformation for induction of anti-MPER antibodies (Burton et al,
Nature Immunology 5(3):233-236 (2004), Zwick et al, J. Virol.
79:1252-1261 (2005)). A series of highly constrained HIV-1 Env
immunogens have been made with the IgG1b12, 2G12, 2F5 and 4E10
epitopes stably expressed, and it has been demonstrated that these
immunogens do not induce broadly reactive neutralizing antibodies
in guinea pigs or rabbits, and, specifically, do not make
neutralizing antibodies to the MPER epitopes (Liao et al., J.
Virol. 78(10):5270-5278 (2004); Haynes, unpublished (2005)). These
results have raised the question as to whether broadly reactive
neutralizing antibodies to HIV-1 envelope are not made in normal
animals and humans because they cannot be made.
[0007] Because long, hydrophobic CDR3 regions are typical of
natural polyreactive autoantibodies (Meffre et al, J. Clin. Invest.
108:879-886 (2001), Ramsland et al, Exp. Clin. Immun. 18:176-198
(2001)), and HIV-1-infected patient B lymphocytes are polyclonally
driven to make cardiolipin antibodies (Weiss et al, Clin. Immunol.
Immunopathol. 77:69-74 (1995), Grunewald et al, Clin. Exp. Immunol.
15:464-71 (1999)), studies were undertaken to assay these and other
anti-HIV-1 mabs for cardiolipin and other autoantigen reactivities.
The present invention results, at least in part, from the
realization that two broadly reactive HIV-1 envelope gp 41 human
mabs, 2F5 and 4E10, are polyspecific autoantibodies reactive with
cardiolipin.
SUMMARY OF THE INVENTION
[0008] The present invention relates generally to human HIV. More
specifically, the invention relates to a method of inducing
neutralizing antibodies to HIV and to compounds and compositions
suitable for use in such a method. In a specific embodiment, the
present invention provides immunogens that present MPER epitopes in
their native membrane bound environment, and immunization methods
using such immunogens that break tolerance.
[0009] Objects and advantages of the present invention will be
clear from the description that follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1. Broadly neutralizing antibodies (2F5, 4E10) bind to
epitopes that lie proximal to the host membrane. Both 2F5 and 4E1
mAbs are IgG3, have long CDR3s, and bind to epitopes that lie
within HIV-1 gp41 (aa 660-683) membrane proximal external region
(MPER).
[0011] FIGS. 2A-2D. Reactivity of 2F5, 4E10, IgG1b12 Mabs with
human Hep-2 epithelial cells. FIG. 2A shows Mab 2F5 reacting with
Hep-2 cells in a diffuse cytoplasmic and nuclear pattern, FIG. 2B
shows Mab 4E10 reacting with HEp-2 cells in a pattern similar to
2F5. FIG. 2C shows Mab IgG1b 12 reacting with Hep-2 cells in a
diffuse cytoplasmic pattern, with nucleoli reactive in the nucleus.
FIG. 2C insert shows higher magnification of cells showing the
nucleolar reactivity of IgG1b12 (arrows). FIG. 2D shows negative
reactivity of Mab 1.9F on Hep-2 cells. Antibody amounts per slide
assayed in FIGS. 2A-2D were 3.75 .mu.g per slide of Mab. Mab 2F5
was positive on HEp-2 cells at 0.125 .mu.g per slide (5 .mu.g/ml).
Mab 4E10 was positive on HEp-2 at 0.125 .mu.g per slide (5
.mu.g/ml), and IgG1b12 was positive at 1.25 .mu.g per slide (50
.mu.g/ml). All Figs. X200; FIG. 2C insert X400. Images shown are
from an experiment representative of three performed.
[0012] FIGS. 3A-3D. Assay of Mabs 2F5 and 4E10 against lipids and
specificity of binding. FIG. 3A shows ELISA reactivity of MAbs 4E10
(solid bars) and 2F5 (open bars) to cardiolipin (CL),
phosphatidylserine (PS), phosphatidylcholine (PC),
phosphatidylethanolamine (PE), and sphingomyelin (SM). Whereas both
4E10 and 2F5 reacted with cardiolipin, only 4E10 reacted with the
other lipids tested. Reactivity of control human anti-CCR5 binding
site MAb 1.7b was negative (data not shown). Reactivity of MAbs
against empty coated plate was similarly negative (not shown). To
show specificity of binding of MAb 2F5 to cardiolipin, 150-300
.mu.g/ml of 2F5 and 1000 .mu.g/ml of anti-2F5 idiotype murine MAb
3H6, which blocks the neutralization of HIV-1 by MAb 2F5 (Kunert et
al, AIDS 16:667 (2002)), were used. The 2F5 anti-idiotype
significantly blocked the binding of MAb 2F5 to cardiolipin by a
mean of 70% in 3 separate experiments (p<0.03) (FIG. 3B). In a
separate ELISA, MAb 2F5 bound to cardiolipin in half-maximal (EC50)
response of 660 nM (not shown). FIG. 3C shows the dose response
curve of 4E10 MAb binding to cardiolipin. The half-maximal (EC50)
response of 4E10 binding (80 nM) was calculated from a four
parametric, sigmoidal curve fitting analysis. Binding data was
acquired from an ELISA of 4E10 MAb binding (0.5 nM-1000 nM) to
cardiolipin coated on ELISA plate (1.35 .mu.g/well). FIG. 3D shows
soluble HIV-1 Env gp140 oligomers (CON-S) expressing the 4E10
epitope inhibits binding of 4E10 MAb to cardiolipin. The IC50 of
inhibition of 4E10 binding to cardiolipin was calculated to be 145
nM. The inhibition assay was carried out by using varying
concentrations of gp140 (19.25-1230 nM) mixed with 10 .mu.g/ml of
4E10 MAb, which were then added to wells containing 1.35 .mu.g of
cardiolipin. MAb 3H6 (1 mg/ml) (but not control MAb) also blocked
the binding of MAb 2F5 to SSA/Ro, centromere B, and histones (not
shown). All data in FIGS. 3A-3D are representative of at least two
experiments performed.
[0013] FIGS. 4A and 4B. Amino acid (FIG. 4A) and nucleic acid (FIG.
4B) sequences of CON-S Env gp160. A CFI form of the protein of FIG.
4A was used in Example 2. (Gp140CFI refers to an HIV-1 envelope
design with the cleavage site (C), fusion site (F), and gp41
immunodominant region (I) deleted in addition to the deletion of
the transmembrane and cytoplasmic domains.)
[0014] FIG. 5. Structures of phosphospholipids used in immunization
regimens and resulting neutralization titers.
[0015] FIGS. 6A and 6B. Peptide sequences used in the generation of
peptide-liposome conjugates. The nominal epitopes of mAbs 2F5 and
4E10 binding epitopes include sequences ELDKWAS and WFNITNW,
respectively, and are underlined. The V3 sequences were derived
from gp120 of HIV-1 MN strain and were used as a control construct.
Scrambled sequences are used controls.
[0016] FIG. 7. Schematic presentation of various designs of MPER
gp41 constructs. The functional regions are indicated above the
schematic constructs. Amino acid sequences are indicated below each
of schematic constructs. Initiation and maturation signal sequences
are highlighted in blue; immunodominant regions are highlighted in
bold; MPER regions are highlighted in brown and GTH1 domains are
highlighted in red and transmembrane domains are underlined.
His-tags were added to the C-terminal ends of the constructs for
easy purification and are highlighted in green.
[0017] FIG. 8. Binding of mAb 4E10 to peptide-liposome conjugates.
BIAcore binding curves show specific and markedly higher binding of
mAb 4E10 to GTH1-4E10 liposomes. Low levels of binding with fast
kinetics to GTH1-2F5 liposomes were also detected.
[0018] FIG. 9. Binding of 2F5 mAb to peptide-liposomes. MAb 2F5
bound specifically to GTH1-2F5 liposomes and showed no binding to
GTH1-4E10 liposomes.
[0019] FIG. 10. A32 mAb binding to peptide-liposomes. A control
anti-gp120 Mab, A32, showed no binding to any of the liposome
conjugates. 17b, a CD4-inducible mAb, also showed no binding to the
above liposome conjugates (data not shown).
[0020] FIG. 11. Generation of fluorescein conjugated
peptide-liposomes. Peptide-liposomes were conjugated with a
fluorescein tag by incorporating fluorescein-POPE in the lipid
composition. Binding assays show that the specificity of mAb 4E10
binding is retained in fluorescein conjugated liposomes.
Fluorescein-conjugated GTH1-2F5 liposomes gave similar results.
[0021] FIG. 12. Reactivity of immunized guinea pig sera with 4E10
peptide. ELISA binding assay show strong positive reactivity of
sera to 4E10 peptide from two guinea pigs immunized with GTH1-4E10
liposomes. All pre-bleed sera gave background binding while a low
level of binding was observed in a serum from an animal immunized
with 4E10 peptide. Both the positive sera from the peptide-liposome
immunized animals also showed neutralizing activity (Table 2). One
serum (1102) showed neutralization of MN and SS1196 strains with
antibody titers at 1:209 and 1:32 respectively. The second serum
(1103) was only effective against the MN virus (1:60).
DETAILED DESCRIPTION OF THE INVENTION
[0022] The present invention results, at least in part, from
studies demonstrating that certain broadly neutralizing HIV-1
antibodies are autoantibodies. A large number of HIV.sup.+ patients
transiently make low levels of such antibodies, however, the
studies described herein indicate that gp41 epitopes do not induce
these antibody specificities but, rather, that cross-reactive
autoantigens, including cardiolipin, are the priming antigen.
[0023] The present invention provides a method of inducing
antibodies that neutralize HIV. The method comprises administering
to a patient in need thereof an amount of at least one heterologous
(e.g., non-human) or homologous (e.g., human) cross-reactive
autoantigen sufficient to effect the induction. Cross-reactive
autoantigens suitable for use in the instant invention include
cardiolipin, SS-A/RO, dsDNA from bacteria or mammalian cells,
centromere B protein and RiBo nucleoprotein (RNP).
[0024] Suitable autoantigens also include phospholipids in addition
to cardiolipin, such as phosphatidylserine,
phosphatidylethanolamine, phosphatidylcholine,
phosphotidylinositol, sphingomyelin, and derivatives thereof, e.g.,
1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS),
1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), and dioleoyl
phosphatidylethanolamine (DOPE). Use of hexagonal II phases of
phospholipids can be advantageous and phospholipids that readily
form hexagonally packed cylinders of the hexagonal II tubular phase
(e.g., under physiological conditions) are preferred, as are
phospholipids that can be stabilized in the hexagonal II phase.
(See Rauch et al, Proc. Natl. Acad. Sci. USA 87:4112-4114 (1990);
Aguilar et al, J. Biol. Chem. 274: 25193-25196 (1999)).
[0025] Fragments of such autoantigens comprising the cross-reactive
epitopes can also be used.
[0026] The autoantigen, or fragment thereof, can be used, for
example, in prime boost regimens that can be readily optimized by
one skilled in the art (DNA sequences encoding proteinaceous
components of such regimens can be administered under conditions
such that the proteinaceous component is produced in vivo). By way
of example, cross-reactive autoantigen can be used as a first
vaccine prime to boost natural auto-antibodies (e.g.,
anti-cardiolipin 4E10- and 2F5-like antibodies). Either autoantigen
(e.g., cardiolipin (or fragment thereof)), or an HIV-envelope
protein/polypeptide/peptide comprising a cross-reactive epitope(s),
such as the 2F5 and/or 4E10 epitopes (which epitopes can include at
least the sequences ELDKWA and NWFDIT, respectively), can be used
as the boost. (See sequences disclosed in PCT/US04/30397.) (It will
be appreciated that HIV-envelope is not an autoantigen.)
[0027] The mode of administration of the autoantigen and/or
HIV-protein/polypeptide/peptide, or encoding sequence, can vary
with the immunogen, the patient and the effect sought, similarly,
the dose administered. Optimum dosage regimens can be readily
determined by one skilled in the art. Typically, administration is
subcutaneous, intramuscular, intravenous, intranasal or oral.
[0028] The immunogenic agents can be administered in combination
with an adjuvant. While a variety of adjuvants can be used,
preferred adjuvants include CpG oligonucleotides and other agents
(e.g., TRL9 agonists) that can break tolerance to autoantigens
without inducing autoimmune disease (Tran et al, Clin. Immunol.
109:278-287 (2003), US Appln Nos. 20030181406, 20040006242,
20040006032, 20040092472, 20040067905, 20040053880, 20040152649,
20040171086, 20040198680, 200500059619).
[0029] The invention includes compositions suitable for use in the
instant method, including compositions comprising the autoantigen,
and/or HIV protein/polypeptide/peptide comprising one or more
cross-reactive epitopes (e.g., 4E10 and/or 2F5 epitopes), or 4E10
or 2F5 epitope mimics, and a carrier. When a DNA prime or boost can
be used, suitable formulations include a DNA prime and a
recombinant adenovirus boost and a DNA prime and a recombinant
mycobacteria boost, where the DNA or the vectors encode, for
example, either HIV envelope or a protein autoantigen, such as
SS-A/Ro. Other combinations of these vectors can be used as primes
or boosts, either with or without HIV protein/polypeptide/peptide
and/or autoantigen. The composition can be present, for example, in
a form suitable for injection or nasal administration.
Advantageously, the composition is sterile. The composition can be
present in dosage unit form.
[0030] The present invention also relates to a passive
immunotherapy approach wherein B cells from patients with a primary
autoimmune disease, such as systemic lupus erythematosis (SLE) or
anti-phospholipid antibody syndrome or patients with infectious
diseases such as syphilis, leishmaniasis, and leprosy, are used in
the production of cross-reactive antibodies (including monoclonal
antibodies other than 4E10 and 2F5). That is, the invention
includes the use of B cells from SLE patients, as well as other
patients with disordered immunoregulation (that is, patients with a
primary autoimmune disease, or a non-HIV infection such as those
noted above, that produce autoantibodies cross-reactive with HIV
envelope), in the production of immortal cell lines that provide a
source of antibodies that cross-react with HIV envelope (such as
2F5-like and 4E10-like antibodies) (see Stiegler et al, AIDS Res.
Hum. Retroviruses 17:1757-1765 (2001), Armbruster et al, J.
Antimicrob. Chemother. 54:915-920 (2004), U.S. Pat. No. 5,831,034).
Advantageously, the B cells are from an SLE patient (or patient
with another primary autoimmune disease) that is HIV infected or
that has received an envelope-based HIV vaccine (while not wishing
to be bound by theory, HIV infection or vaccination may serve to
"boost" primed B1 cells (e.g., cardiolipin-primed B1 cells) to
produce 2F5- and/or 4E10-like antibodies and escape deletion (which
would occur in a normal subject)--the "boost" may trigger somatic
hypermutation so that the resulting Ig genes encode antibodies that
fit 2F5 and or 4E10-like epitopes--or that fit other gp 160
epitopes that induce broadly neutralizing antibodies but are
deleted in normal subjects). The production of immortal cell lines
from B cells can be effected using any of a variety of art
recognized techniques, including, but not limited to, fusing such B
cells with myeloma cells to produce hybridomas.
[0031] Once selected, sequences encoding such cross-reactive
antibodies (or binding fragments thereof) can be cloned and
amplified (see, for example, Huse et al, Science 246:1275-1281
(1989), and phage-display technology as described in WO 91/17271,
WO 92/01047, U.S. Pat. Nos. 5,877,218, 5,871,907, 5,858,657,
5,837,242, 5,733,743 and 5,565,332). Soluble antibodies for therapy
can then be designed and produced using art recognized techniques
(Stiegler et al, AIDS Res. Hum. Retroviruses 17:1757-1765 (2001),
Armbruster et al, J. Antimicrob. Chemother. 54:915-920 (2004)).
[0032] In accordance with this approach, the antibody (or binding
fragment thereof) can be administered in doses ranging from about
10 to 100 mg/dose, preferably 25 mg/dose. The dosage and frequency
can vary with the antibody (or binding fragment thereof), the
patient and the effect sought (see Armbruster et al, J. Antimicrob.
Chemother. 54:915-920 (2004)). The antibodies described above can
be used prophylactically or therapeutically.
[0033] The antibodies (or binding fragments thereof), or DNA
encoding the antibodies or binding fragments, can be formulated
with a carrier (e.g., pharmaceutically acceptable carrier) and can
be administered by, for example, parenteral, intravenous,
subcutaneous, intramuscular or intranasal routes.
[0034] Finally, animal species such as camels (Ramsland et al, Exp.
Clin. Immunogenet. 18:176-198 (2001), Litman et al, Annu. Rev.
Immunol. 7:109-147 (1999)), cows (Ramsland et al, Exp. Clin.
Immunogenet. 18:176-198 (2001), Litman et al, Annu. Rev. Immunol.
7:109-147 (1999)) and sharks (Ramsland et al, Exp. Clin.
Immunogenet. 18:176-198 (2001), Litman et al, Annu. Rev. Immunol.
7:109-147 (1999), Hohman et al, Proc. Natl. Acad. Sci. USA.
90:9882-9886 (1993)) have very long CDR3 lengths, and their
antibodies show polyreactivitiy. These engineered CDR3s that show
polyreactivity to HIV envelope can be utilized for making potent
therapeutic antibodies (e.g, monoclonal antibodies, including, for
example, chimeric and humanized antibodies, and antigen binding
fragments thereof) to HIV and to many infectious agents.
[0035] In a specific embodiment, the present invention further
relates to synthetic liposome-peptide conjugates and to methods of
using same as immunogens for the generation of broadly neutralizing
antibodies against HIV-1. This embodiment of the invention provides
compositions and methods for embedding into synthetic liposomes
nominal epitope peptides of broadly neutralizing antibodies that
bind to the MPER of HIV-1 gp41. Also provided are immunization
strategies and protocols for the generation of anti-HIV-1
neutralizing antibodies and for the detection of antigen specific B
cell responses.
[0036] In accordance with this embodiment of the invention, peptide
sequences that include a nominal epitope of a broadly neutralizing
anti-HIV antibody and a hydrophobic linker, such as GTH1 (see FIG.
6 for sequence), are embedded into synthetic liposomes. In a
preferred aspect, the nominal epitope is that of mAbs 2F5 (ELDKWAS)
or 4E10 (WFNITNW), which, as noted above, lie in the MPER of HIV-1
envelope gp41. The epitope can be present in the peptide such that
antibodies specific therefor have relatively unconstrained access
or, alternatively, the epitope can be present in the peptide in
relation to the hydrophobic linker so as to mimic the native
orientation of the MPER region. Specific examples of peptide
sequences suitable for use in the invention are set forth in FIG.
6. In addition, the MPER gp41 region can be expressed as
recombinant proteins in recombinant vaccinia virus, in human cell
expression systems, and formulated with amphipathic alpha helices
at the N or C termini of the gp41 component for ease in association
with liposomes (FIG. 7).
[0037] Liposomes suitable for use in the invention include, but are
not limited to, those comprising POPC, POPE, DMPA (or sphingomyelin
(SM)) and cholesterol (Ch). While optimum ratios can be determined
by one skilled in the art, examples include POPC:POPE:SM:Ch or
POPC:POPE:DMPA:Ch at ratios of 45:25:20:10. Alternative
formulations of liposomes that can be used include DMPC
(1,2-dimyristoyl-sn-glycero-3-phoshphocholine), cholesterol (Ch)
and DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol)
formulated at a molar ratio of 9:7.5:1 (Wassef et al, ImmunoMethods
4:217-222 (1994); Alving et al., G. Gregoriadis (ed.), Liposome
technology 2.sup.nd ed., vol. III CRC Press, Inc., Boca Raton, Fla.
(1993); Richards et al, Infect. Immun. 66(6):285902865 (1998)).
[0038] The optimum ratio of peptide to total lipid can vary, for
example, with the peptide and the liposome. For the peptides of
Example 3, a ratio 1:420 was advantageous.
[0039] The liposome-peptide conjugates can be prepared using
standard techniques (see too Example 3 that follows).
[0040] The peptide-liposome immunogens of the invention can be
formulated with, and/or administered with, adjuvants such as lipid
A, oCpGs, TRL4 agonists or TLR 7 agonists that facilitate robust
antibody responses (Rao et al, Immunobiol. Cell Biol. 82(5):523
(2004)). Other adjuvants that can be used include alum and Q521
(which do not break existing B cell tolerance). Preferred
formulations comprise an adjuvant that is designed to break forms
of B cell tolerance, such as oCpGs in an oil emulsion such as
Emulsigen (an oil in water emulsion) (Tran et al, Clin. Immunol.
109(3):278-287 (2003)). Additional suitable adjuvants include those
described in 11/302,505, filed Dec. 14, 2005, including the TRL
agonists disclosed therein.
[0041] The peptide-liposome immunogens can be administered, for
example, IV, intranasally, subcutaneously, intraperitoneally,
intravaginally, or intrarectally. The route of administration can
vary, for example, with the patient, the conjugate and/or the
effect sought, likewise the dosing regimen. The peptide-liposome
immunogens are preferred for use prophylactically, however, their
administration to infected individuals may reduce viral load.
[0042] As described in Example 3 that follows, the peptide-liposome
conjugates can be used as reagents for the detection of
MPER-specific B cell responses. For example, the peptide-liposome
constructs can be conjugated with a detectable label, e.g., a
fluorescent label, such as fluorescein. The fluorescein-conjugated
liposomes can be used in flow cytometric assays as a reagent for
the detection of anti-MPER specific B cell responses in hosts
immunized with HIV-1 Env proteins that present exposed MPER region.
These reagents can be used to study peripheral blood B cells to
determine the effectiveness of immunization for anti-MPER antibody
induction by measuring the number of circulating memory B cells
after immunization.
[0043] It will be appreciated from a reading of the foregoing that
if HIV has evolved to escape the host immune response by making the
immune system blind to it, other infectious agents may have evolved
similarly. That is, this may represent a general mechanism of
escape. That being the case, approaches comparable to those
described herein can be expected to be useful in the treatment of
such other agents well.
[0044] Certain aspects of the invention are described in greater
detail in the non-limiting Examples that follow (see also Maksyutov
et al, J. Clin. Virol. December; 31 Suppl 1:S26-38 (2004), US
Appln. 20040161429, and Haynes et al, Science 308:1906 (2005)).
Example 1
[0045] Design of an HIV-1 immunogen that can induce broadly
reactive neutralizing antibodies is a major goal of HIV-1 vaccine
development. While rare human mabs exist that broadly neutralize
HIV-1, HIV-1 envelope immunogens do not induce these antibody
specificities. In this study, it was demonstrated that the two most
broadly reactive HIV-1 envelope gp41 human mabs, 2F5 and 4E10, are
polyspecific, autoantibodies reactive with cardiolipin. Thus,
current HIV-1 vaccines may not induce antibodies against membrane
proximal gp41 epitopes because of gp41 membrane proximal epitopes
mimicry of autoantigens.
Experimental Details
[0046] Monoclonal Antibodies. Mabs 2F5, 2G12, and 4E10 were
produced as described (Steigler et al, AID Res. Human Retroviruses
17:1757 (2001), Purtscher et al, AIDS 10:587 (1996), Trkola et al,
J. Virol. 70:1100 (1996)). IgG1b12 (Burton et al, Science
266:1024-1027 (1994)) was the generous gift of Dennis Burton,
Scripps Institute, La Jolla, Calif. Mab 447-52D (Zolla-Pazner et
al, AIDS Res. Human Retrovirol. 20:1254 (2004)) was obtained from
the AIDS Reagent Repository, NIAID, NIH. The remainder of the mabs
in Table 1 were produced from HIV-1 infected subjects and used as
described (Robinson et al., AIDS Res. Human Retrovirol. 6:567
(1990), Binley et al, J. Virol. 78:13232 (2004)).
[0047] Autoantibody Assays. An anti-cardiolipin ELISA was used as
described (DeRoe et al, J. Obstet. Gynecol. Neonatal Nurs. 5:207
(1985), Harris et al, Clin. Exp. Immunol. 68:215 (1987)). A similar
ELISA was adapted for assay for mab reactivity to
phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine,
and sphingomyelin (all purchased from Sigma, St. Louis, Mo.). The
Luminex AtheNA Multi-Lyte ANA Test (Wampole Laboratories,
Princeton, N.J.) was used for mab reactivity to SS-A/Ro, SS-B/La,
Sm, ribonucleoprotein (RNP), Scl-70, Jo-1, double stranded (ds)
DNA, centromere B, and histone. Mab concentrations assayed were 150
.mu.g, 50 .mu.g, 15 .mu.g, and 5 .mu.g/ml. Ten .mu.l of each
concentration (0.15 .mu.g, 0.05 .mu.g, 0.015 .mu.g, and 0.005
.mu.g, respectively, per assay) were incubated with the Luminex
fluorescence beads and the test performed per manufacturer's
specifications. Values in Table 1 are results of assays with 0.15
.mu.g added per test. In addition, an ELISA for SS-A/Ro
(ImmunoVision, Springdale, Ark.) and dsDNA (Inova Diagnostics, San
Diego, Calif.) was also used to confirm these autoantigen
specificities. Reactivity to human epithelial Hep-2 cells was
determined using indirect immunofluoresence on Hep-2 slides using
Evans Blue as a counterstain and FITC-conjugated goat anti-human
IgG (Zeus Scientific, Raritan N.J.). Slides were photographed on a
Nikon Optiphot fluorescence microscope. Rheumatoid factor was
performed by nephelometry (Dade Behring, Inc (Newark, Del.). Lupus
anticoagulant assay was performed by activated partial
thromboplastin (aPTT) and dilute Russell viper venom testing, as
described (Moll and Ortel, Ann. Int. Med. 127:177 (1997)). Forty
.mu.l of 1 mg/ml of 2F5, 4E10 and control mabs were added to pooled
normal plasma (final mab concentration, 200 .mu.g/ml) for lupus
anticoagulant assay. Anti-.beta.2 glycoprotein-1 assay was an ELISA
(Inova Diagnostics, Inc.). Serum antibodies to dsDNA, SS-A/Ro,
SS-B/La, Sm, RNP and histone occur in patients with SLE; serum
antibodies to centromere B and scl-70 (topoisomerase I) are found
in systemic sclerosis; and antibodies to Jo-1 are found in
association with polymyositis (Rose and MacKay, The Autoimmune
Diseases, Third Ed. Academic Press, Sand Diego, Calif. (1998)).
Results
[0048] The reactivity of mabs 2F5 and 4E10, two additional rare
broadly reactive neutralizing mabs (2G12 and IgG1b12), and
thirty-one common anti-HIV-1 Env human mabs, with cardiolipin
(Robinson et al, AIDS Res. Human Retrovirol. 6:567 (1990)) was
determined (Table 1). Both 2F5 and 4E10 reacted with cardiolipin,
whereas all 33 of the other mabs were negative. Mab 2F5 also
reacted with SS-A/Ro, histones and centromere B autoantigen, while
mab 4E10 reacted with the systemic lupus erythematosus (SLE)
autoantigen, SS-A/Ro. Both 2F5 and 4E10 reacted with Hep-2 human
epithelial cells in a diffuse cytoplasmic and nuclear pattern
(Robinson et al, AIDS Res. Human Retrovirol. 6:567 (1990)) (FIG.
2). Thus, both 2F5 and 4E10 are characterized by polyspecific
autoreactivity.
TABLE-US-00001 TABLE 1 Hep-2 Cell Mab Type and Antibody Name
Cardiolipin Reactivity Ro(SSA) dsDNA Centromere B Histones Membrane
Proximal External 47 +Cytoplasmic 290 - 1,776 1,011 Region (2F5)
nuclear Membrane Proximal External 15,434 +Cytoplasmic 221 - - -
Region (4E10) nuclear CD4 Binding Site (IgG1b12) - +Cytoplasmic -
513 479 185 nucleolar CD4 Binding Site (F1.5E, 25G) - - - - - -
Adjacent CD4 Binding Site - - - - 1,131 - (A32) Adjacent CD4
Binding Site - - - 768 1,422 539 (1.4G) Adjacent CD4 Binding Site -
- - - - - (1.4C, 4.6H, 4.11C) Third variable loop (CO11, - - - - -
- F2A3, F3.9F, LA21, 447-52D) gp41 immunodominant region - - - - -
- (7B2, KU32) gp41 immunodominant region - +intermediate - - 314 -
(2.2B) - filament C1-C4 gp120 (8.2A, 2.3B) - - - - - - C1-C4 gp120
(EH21, C11) - - - - - - Glycan-dependent (2G12) - - - - - - CCR5
binding site (1.7B, 2.1C, - - - - - - LF17, E51 1.9F, LA15, 4.8E,
LA28, 1.9E, E047, 2.5E, ED10) Positive control serum 34
+homogeneous 1365 228 624 34 nuclear Negative controls <16 -
<120 <120 <120 <120 All Mabs were negative in assays
for reactivity with La (SSB), Sm, Sci-70 and Jo-1, except for Ku32
mab that reacted with Sm. Ro (SSA), dsDNA, centromere B, histone
and cardiolipin antibody values are in relative units based on a
standard curve. - = negative
[0049] Of the two other rare neutralizing mabs, one mab, 2G12, was
not autoreactive, while another mab against the CD4 binding site,
IgG1b12 (Stiegler et al, AIDS Res. Hum. Retroviruses 17:1757
(2001)), reacted with ribonucleoprotein, dsDNA, and centromere B as
well as with Hep-2 cells in a cytoplamic and nucleolar pattern
(Table 1 and FIG. 2). Of the 31 more common anti-HIV-1 mabs
studied, only two mabs with specificity for binding near the CD4
binding site (A32, 1.4G) and a mab to a non-neutralizing gp41
epitope (2.2 B) showed evidence of polyreactivity (Table 1).
[0050] To determine if 2F5 and 4E10 were similar to prothrombotic
anti-cardiolipin antibodies found in SLE-associated
anti-phospholipid syndrome (Burton et al, Science 266:1024-1027
(1994)), both mabs were tested for lupus anticoagulant activity,
and for the ability to bind to prothombin (PT), beta-2
glycoprotein-1, phosphatidylserine (PS), phosphatidylcholine (PC),
phosphatidylethanolamine (PE), and sphingomyelin (SM) (Robinson et
al, AIDS Res. Human Retrovirol. 6:567 (1990)). Whereas 2F5 was
negative for these reactivities, 4E10 had lupus anticoagulant
reactivity, and reacted strongly with PS, PC, PE, weakly with SM
and PT, and negatively with .beta.2 glycoprotein-1. (See FIG.
3.)
[0051] Anti-cardiolipin antibodies can be found in patients with
disordered immunoregulation due to autoimmune disease or infection
(Burton et al, Science 266:1024-1027 (1994)). Anti-cardiolipin
autoantibodies are induced by syphilis, leprosy, leishmaniasis,
Epstein Barr virus, and HIV-1 (Burton et al, Science 266:1024-1027
(1994)). Unlike anti-cardiolipin antibodies found in SLE,
"infectious" anti-cardiolipin antibodies are rarely prothrombotic,
and are transient. Thus, 4E10 is similar to anti-cardiolipin
antibodies in autoimmune disease, and 2F5 is similar to
anti-cardiolipin antibodies in infectious diseases.
[0052] Autoreactive B cell clones with long CDR3 lengths are
normally deleted or made tolerant to self antigens ((Zolla-Pazner
et al, AIDS Res. Human Retrovirol. 20:1254 (2004)). Thus, HIV-1 may
have evolved to escape membrane proximal antibody responses by
having conserved neutralizing epitopes as mimics of autoantibody
epitopes. These data suggest that current HIV-1 vaccines do not
routinely induce robust membrane proximal anti-envelope
neutralizing antibodies because antibodies targeting these epitopes
are derived from autoreactive B cell clones that are normally
deleted or made tolerant upon antigenic stimulation by HIV-1 Env.
These observations may also explain the rare occurrence of HIV-1 in
SLE patients who may be unable to delete such clones (Fox et al,
Arth. Rhum. 40:1168 (1997)).
Example 2
[0053] The ability of autoantigens of the invention to induce the
production of neutralizing antibodies was studied using, as
autoantigen, cardiolipin (lamellar and hexagonal phases),
1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (POPS)
(lamellar and hexagonal phases),
1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) (lamellar
phase) and dioleoyl phosphatidylethanolamine (DOPE) (hexagonal
phase). Guinea pigs (4 per group) were immunized with phospholipid
(cardiolipin lamellar phase, cardiolipin hexagonal phase, POPS
lamellar phase, POPS hexagonal phase, POPE lamellar phase or DOPE
hexagonal phase) in 10 .mu.g of oCpGs, four times, with each
immunization being two weeks apart. Following the four phospholipid
immunizations, a final immunization was made IP with 10 .mu.g of
oCpGs with 100 .mu.g of group M consensus Env, CON-S gp140CFI
oligomer (that is, the CFI form of the protein shown in FIG.
4A).
[0054] Neutralization assays were performed using an Env pseudotype
neutralization assay in TMZ cells (Wei et al, Nature 422:307-312
(2003), Derdeyn et al, J Virol 74:8358-8367 (2000), Wei et al,
Antimicrob Agents Chemother 46:1896-1905 (2002), Platt et al, J
Virol 72:2855-2864 (1998), Mascola et al, J. Virol. 79:10103-10107
(2005)), as described below:
Cell Culture
[0055] TZM-bl an adherent cell line and is maintained in T-75
culture flasks. Complete growth medium (GM) consists of D-MEM
supplemented with 10% fetal bovine serum (FBS, heat-inactivated)
and gentamicin (50 .mu.g/ml). Cell monolayers are disrupted and
removed by treatment with trypsin/EDTA:
Trypsin-EDTA Treatment for Disruption of TZM-bl Cell
Monolayers:
[0056] Cell monolayers maintained in T-75 culture flasks are
disrupted and removed by treatment with trypsin/EDTA at confluency
when splitting cells for routine maintenance and when preparing
cells for assay. 1. Decant the culture medium and remove residual
serum by rinsing monolayers with 6 ml of sterile PBS. 2. Slowly add
2.5 ml of an 0.25% Trypin-EDTA solution to cover the cell
monolayer. Incubate at room temp for 30-45 seconds. Decant the
trypsin solution and incubate at 37.degree. C. for 4 minutes. Do
not agitate the cells by hitting or shaking the flask while waiting
for the cells to detach. 3. Add 10 ml of GM and suspend the cells
by gentle pipet action. Count cells. 4. Seed new T-75 culture
flasks with approximately 10.sup.6 cells in 15 ml of GM. Cultures
are incubated at 37.degree. C. in a 5% CO.sub.2/95% air
environment. Cells should be split approximately every 3 days.
Virus Stocks
[0057] Stocks of uncloned viruses may be produced in either PBMC or
T cell lines. Pseudoviruses may be produced by transfection in an
appropriate cell type, such as 293T cells. All virus stocks should
be made cell free by low speed centrifugation and filtration
(0.45-micron) and stored at -80.degree. C. in GM containing 20%
FBS.
TCID50 Determination
[0058] It is necessary to determine the TCID50 of each virus stock
in a single-cycle infection assay (2-day incubation) in TZM-bl
cells prior to performing neutralization assays. A cut-off value of
2.5-times background RLU is used when quantifying positive
infection in TCID50 assays.
[0059] Too much virus in the neutralization assay can result in
strong virus-induced cytopathic effects that interfere with
accurate measurements. Most virus stocks must be diluted at least
10-fold to avoid cell-killing. A standard inoculum of 200 TCID50
was chosen for the neutralization assay to minimize virus-induced
cytopathic effects while maintaining an ability to measure a 2-log
reduction in virus infectivity. It should be noted that different
strains vary significantly in their cytopathicity. Virus-induced
cytopathic effects may be monitored by visual inspection of
syncytium formation under light microscopy. Cytopathic effects may
also be observed as reductions in luminescence at high virus doses
in the TCID50 assay.
Neutralizing Antibody Assay Protocol
[0060] NOTE 1: All incubations are performed in a humidified
37.degree. C., 5% CO.sub.2 incubator unless otherwise specified.
NOTE 2: Assays with replication-competent viruses are performed in
DEAE-GM containing 1 .mu.M indinavir. 1. Using the format of a
96-well flat-bottom culture plate, place 150 .mu.l of GM in all
wells of column 1 (cell control). Place 100 .mu.l in all wells of
columns 2-11 (column 2 will be the virus control). Place an
additional 40 .mu.l in all wells of columns 3-12, row H (to receive
test samples). 2. Add 11 .mu.l of test sample to each well in
columns 3 & 4, row H. Add 11 .mu.l of a second test sample to
each well in columns 5 & 6, row H. Add 11 .mu.l of a third test
sample to each well in columns 7 & 8, row H. Add 11 .mu.l of a
fourth test sample to each well in columns 9 & 10, row H. Add
11 .mu.l of a fifth test sample to each well in columns 11 &
12, row H. Mix the samples in row H and transfer 50 .mu.l to row G.
Repeat the transfer and dilution of samples through row A (these
are serial 3-fold dilutions). After final transfer and mixing is
complete, discard 50 .mu.lfrom the wells in columns 3-12, row A
into a waste container of disinfectant. 3. Thaw the required number
of vials of virus by placing in an ambient temperature water bath.
When completely thawed, dilute the virus in GM to achieve a
concentration of 4,000 TCID.sub.50/ml.
[0061] Cell-free stocks of virus should be prepared in advance and
cryopreserved in working aliquots of approximately 1 ml.
4. Dispense 50 .mu.l of cell-free virus (200 TCID.sub.50) to all
wells in columns 2-12, rows A through H. Mix by pipet action after
each transfer. Rinse pipet tips in a reagent reservoir containing
40 ml sterile PBS between each transfer to avoid carry-over. 5.
Cover plates and incubate for 1 hour. 6. Prepare a suspension of
TZM-bl cells (trypsinize approximately 10-15 minutes prior to use)
at a density of 1.times.10.sup.5 cells/ml in GM containing DEAE
dextran (37.5 .mu.g/ml). Dispense 100 .mu.l of cell suspension
(10,000 cells per well) to each well in columns 1-12, rows A though
H. Rinse pipet tips in a reagent reservoir filled with sterile PBS
between each transfer to avoid carry-over. The final concentration
of DEAE dextran is 15 .mu.g/ml. 7. Cover plates and incubate for 48
hours. 8. Remove 150 .mu.l of culture medium from each well,
leaving approximately 100 .mu.l. Dispense 100 .mu.l of Bright
Glo.TM. Reagent to each well. Incubate at room temperature for 2
minutes to allow complete cell lysis. Mix by pipet action (at least
two strokes) and transfer 150 .mu.l to a corresponding 96-well
black plate. Read the plate immediately in a luminometer. 9.
Percent neutralization is determined by calculating the difference
in average RLU between test wells (cells+serum sample+virus) and
cell control wells (cells only, column 1), dividing this result by
the difference in average RLU between virus control (cell+virus,
column 2) and cell control wells (column 1), subtracting from 1 and
multiplying by 100. Neutralizing antibody titers are expressed as
the reciprocal of the serum dilution required to reduce RLU by
50%.
[0062] As shown in FIG. 5, animals receiving DOPE (hexagonal phase)
had a neutralization titer of 170.
Example 3
Immunogen Design
[0063] Peptide sequences that include the nominal epitopes of mAbs
2F5 and 4E10, respectively, linked to a hydrophobic linker (GTH1)
were synthesized and embedded into synthetic liposomes (FIG. 6).
The first generation of immunogens was designed with the 2F5 and
4E10 epitope sequences at the distal end of the lipid bilayer (FIG.
6A). These constructs provided unconstrained access of mAbs to
their respective epitopes. The second generation constructs have
been designed to mimic the native orientation of the MPER region
with the 2F5 and 4E10 mAb epitope sequences linked proximal to the
hydrophobic linker (FIGS. 6A, 6B).
[0064] The composition of the synthetic liposomes comprised the
following phospholipids, POPC
(1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine), POPE
(1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphoethanolamine), DMPA
(1,2-Dimyristoyl-sn-Glycero-3-Phosphate), and Cholesterol dissolved
in chloroform (purchased from Avanti Polar Lipids (Alabaster,
Ala.)).
[0065] Synthetic liposomes were prepared by dispensing appropriate
molar amounts of phospholipids (POPC:POPE:DMPA:Ch=45:25:20:10) in
chloroform resistant tubes. The phospholipids were mixed by
vortexing and the mixture was dried in the fume hood under a gentle
stream of nitrogen. Any residual chloroform was removed by storing
the lipids under a high vacuum (15 h). Aqueous suspensions of
phospholipids were prepared by adding PBS or TBS buffer, pH 7.4,
and incubating at 37.degree. C. for 10-30 minutes, with
intermittent, vigorous vortexing to resuspend the phospholipids.
The milky, uniform suspension of phospholipids was then sonicated
in a bath sonicator (Misonix Sonicator 3000, Misonix Inc.,
Farmingdale, N.Y.). The sonicator was programmed to run 3
consecutive cycles of 45 seconds of total sonication per cycle.
Each cycle included 5 seconds of sonication pulse (70 watts power
output) followed by a pulse off period of 12 seconds. At the end of
sonication, the suspension of lamellar liposomes was stored at
4.degree. C.
[0066] HIV-1 MPER peptides GTH1-2F5 and GTH1-4E10 (FIG. 6) were
dissolved in 70% chloroform, 30% methanol, Chloroform solutions of
lipids were added to the peptide solution, in the molar ratios of
45:25:20:10 (POPC:POPE:DMPA:Cholesterol). Each peptide was added to
a ratio of peptide:total phospholipids of 1:420. The mixture was
vortexed, then dried and resuspended as described above.
[0067] Binding assays to test specificity of mAb binding to each
peptide-lipid conjugate were performed following capture of the
liposomes on a BAcore L1 sensor chip, which allows immobilization
of lipid bilayer via a hydrophobic linker. 2F5, 4E10 and control
mAbs (A32 or 17b) were injected over each of the sensor surfaces
with either synthetic liposomes, or peptide-lipid conjugates and
the binding monitored on a BIAcore 3000 instrument (FIGS.
8-11).
Immunization Strategy
[0068] The immunization strategy incorporated a regimen that allows
temporary breaks in tolerance. The protocol involves the use of
oCpGs, the TLR9 ligand that has been used to break tolerance for
the production of anti-dsDNA antibodies in mice (Tran et al, Clin.
Immunol. 109(3):278-287 (2003)). The peptide-liposome conjugates
were mixed (1:1) with the adjuvant, Emulsigen plus oCpG. The
Emulsigen mixed adjuvant (2.times.) was prepared by mixing 375
.mu.L of Emulsigen, 250 .mu.L of oCpG and 625 .mu.L of saline. Each
guinea pig was immunized on a 21-day interval with 250 .mu.g of
either peptide alone or peptide-liposome conjugates with equivalent
amount of peptide. Serum samples were harvested as pre-bleed prior
to first immunization and at each subsequent immunizations. Serum
samples were analyzed by ELISA assay (FIG. 12) for binding to
peptide epitopes and for viral neutralization assay (Table 2). Data
in FIG. 12, show strong reactivity to 4E10 peptide of sera from two
guinea pigs immunized with GTH1-4E10 liposomes, while only low
level of reactivity was observed in a serum from 4E10 peptide
immunized animal. Both the positive sera also neutralized HIV-1 MN
strain (Table 2).
TABLE-US-00002 TABLE 2 Induction of neutralizing antibodies in
guinea pigs immunized with 4E10 peptide-liposomes HIV-1
Strain/antibody titer Animal No. MN SS1196 1102 Bleed 4 209 32 1103
Bleed 4 60 <20
Application of Peptide-Liposome Conjugates in the Detection of
Antigen Specific B Cell Responses.
[0069] The above peptide-liposome conjugates have been utilized as
a reagent for the detection of MPER specific B cell responses. The
peptide-liposome constructs (2F5 and 4E10) were conjugated with
fluorescein by incorporating fluorescein-POPE in the lipid
composition. The flourescein-POPE was mixed with unconjugated POPE
at a ratio of 45:55 and then mixed with the rest of the lipids in
the molar ratio as described above. In BIAcore binding assays, both
fluorescein conjugated 2F5 and 4E10-peptide-liposomes retained
their specificity in binding to their respective mAbs (FIG.
11).
[0070] All documents and other information sources cited above are
hereby incorporated in their entirety by reference.
Sequence CWU 1
1
27132PRTArtificial SequenceDescription of Artificial SequenceHIV
1Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala1 5
10 15Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile
Lys 20 25 30257PRTArtificial SequenceDescription of Artificial
SequenceHIV 2Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr1 5 10 15Ser Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly
Lys Ala Met Tyr 20 25 30Ala Cys Thr Arg Pro Asn Tyr Asn Lys Arg Lys
Arg Ile His Ile Gly 35 40 45Pro Gly Arg Ala Phe Tyr Thr Thr Lys 50
55349PRTArtificial SequenceDescription of Artificial SequenceHIV
3Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala1 5
10 15Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile
Lys 20 25 30Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg
Met Tyr 35 40 45Ser445PRTArtificial SequenceDescription of
Artificial SequenceHIV 4Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn1 5 10 15Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr
Ile Lys Tyr Lys Arg Trp 20 25 30Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr Ser 35 40 45549PRTArtificial SequenceDescription of
Artificial SequenceHIV 5Glu Ala Trp Leu Trp Asp Leu Leu Ile Trp Asn
Leu Gln Phe Glu Trp1 5 10 15Lys Asn Asn Trp Thr Glu Gln Asn Gln Leu
Glu Lys Ser Tyr Ile Lys 20 25 30Tyr Lys Arg Trp Ile Ile Leu Gly Leu
Asn Lys Ile Val Arg Met Tyr 35 40 45Ser636PRTArtificial
SequenceDescription of Artificial SequenceHIV 6Ser Leu Trp Asn Trp
Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys1 5 10 15Gly Gly Gly Tyr
Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val 20 25 30Arg Met Tyr
Ser 35736PRTArtificial SequenceDescription of Artificial
SequenceHIV 7Lys Asn Ile Trp Leu Ser Asn Tyr Phe Trp Leu Ile Asn
Trp Trp Thr1 5 10 15Gly Gly Gly Tyr Lys Arg Trp Ile Ile Leu Gly Leu
Asn Lys Ile Val 20 25 30Arg Met Tyr Ser 35840PRTArtificial
SequenceDescription of Artificial SequenceHIV 8Gln Gln Glu Lys Asn
Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala1 5 10 15Ser Leu Trp Asn
Gly Gly Gly Tyr Lys Arg Trp Ile Ile Leu Gly Leu 20 25 30Asn Lys Ile
Val Arg Met Tyr Ser 35 40940PRTArtificial SequenceDescription of
Artificial SequenceHIV 9Asn Lys Glu Gln Asp Gln Ala Glu Glu Ser Leu
Gln Leu Trp Glu Lys1 5 10 15Leu Asn Trp Leu Gly Gly Gly Tyr Lys Arg
Trp Ile Ile Leu Gly Leu 20 25 30Asn Lys Ile Val Arg Met Tyr Ser 35
401033PRTArtificial SequenceDescription of Artificial SequenceHIV
10Ser Leu Trp Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile Lys1
5 10 15Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val Arg Met
Tyr 20 25 30Ser1133PRTArtificial SequenceDescription of Artificial
SequenceHIV 11Lys Asn Ile Trp Leu Ser Asn Tyr Phe Trp Leu Ile Asn
Trp Trp Thr1 5 10 15Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile
Val Arg Met Tyr 20 25 30Ser127PRTArtificial SequenceDescription of
Artificial SequenceHIV 12Glu Leu Asp Lys Trp Ala Ser1
51337PRTArtificial SequenceDescription of Artificial SequenceHIV
13Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala1
5 10 15Ser Leu Trp Asn Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys
Ile 20 25 30Val Arg Met Tyr Ser 351437PRTArtificial
SequenceDescription of Artificial SequenceHIV 14Asn Lys Glu Gln Asp
Gln Ala Glu Glu Ser Leu Gln Leu Trp Glu Lys1 5 10 15Leu Asn Trp Leu
Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile 20 25 30Val Arg Met
Tyr Ser 3515117PRTArtificial SequenceDescription of Artificial
SequenceHIV 15Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Leu Gly 20 25 30Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr
Thr Ala Val Pro Trp 35 40 45Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu
Tyr Thr Ser Leu Ile His 50 55 60Ser Leu Ile Glu Glu Ser Gln Asn Gln
Gln Glu Lys Asn Glu Gln Glu65 70 75 80Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn Trp Phe Tyr Lys 85 90 95Arg Trp Ile Ile Leu Gly
Leu Asn Lys Ile Val Arg Met Tyr Ser His 100 105 110His His His His
His 1151689PRTArtificial SequenceDescription of Artificial
SequenceHIV 16Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Tyr Thr 20 25 30Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln Glu Lys 35 40 45Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu Trp Asn 50 55 60Trp Phe Tyr Lys Arg Trp Ile Ile Leu
Gly Leu Asn Lys Ile Val Arg65 70 75 80Met Tyr Ser His His His His
His His 8517117PRTArtificial SequenceDescription of Artificial
SequenceHIV 17Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Tyr Lys 20 25 30Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr Ser Leu 35 40 45Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile
Cys Thr Thr Ala Val Pro 50 55 60Trp Asn Ala Ser Trp Ser Asn Lys Ser
Leu Glu Tyr Thr Ser Leu Ile65 70 75 80His Ser Leu Ile Glu Glu Ser
Gln Asn Gln Gln Glu Lys Asn Glu Gln 85 90 95Glu Leu Leu Glu Leu Asp
Lys Trp Ala Ser Leu Trp Asn Trp Phe His 100 105 110His His His His
His 1151889PRTArtificial SequenceDescription of Artificial
SequenceHIV 18Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Tyr Lys 20 25 30Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr Ser Tyr 35 40 45Thr Ser Leu Ile His Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu 50 55 60Lys Asn Glu Gln Glu Leu Leu Glu Leu
Asp Lys Trp Ala Ser Leu Trp65 70 75 80Asn Trp Phe His His His His
His His 8519132PRTArtificial SequenceDescription of Artificial
SequenceHIV 19Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Leu Gly 20 25 30Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr
Thr Ala Val Pro Trp 35 40 45Asn Ala Ser Trp Ser Asn Lys Ser Leu Glu
Tyr Thr Ser Leu Ile His 50 55 60Ser Leu Ile Glu Glu Ser Gln Asn Gln
Gln Glu Lys Asn Glu Gln Glu65 70 75 80Leu Leu Glu Leu Asp Lys Trp
Ala Ser Leu Trp Asn Trp Phe Asn Ile 85 90 95Thr Asn Trp Leu Trp Tyr
Ile Lys Leu Phe Ile Met Ile Val Gly Gly 100 105 110Leu Val Gly Leu
Arg Ile Val Phe Ala Val Leu Ser Val Val His His 115 120 125His His
His His 13020104PRTArtificial SequenceDescription of Artificial
SequenceHIV 20Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu
Trp Arg Trp1 5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser
Ala Ala Tyr Thr 20 25 30Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln
Asn Gln Gln Glu Lys 35 40 45Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys
Trp Ala Ser Leu Trp Asn 50 55 60Trp Phe Asn Ile Thr Asn Trp Leu Trp
Tyr Ile Lys Leu Phe Ile Met65 70 75 80Ile Val Gly Gly Leu Val Gly
Leu Arg Ile Val Phe Ala Val Leu Ser 85 90 95Val Val His His His His
His His 100217PRTArtificial SequenceDescription of Artificial
SequenceHIV 21Trp Phe Asn Ile Thr Asn Trp1 5226PRTArtificial
SequenceDescription of Artificial SequenceHIV 22Asn Trp Phe Asp Ile
Thr1 5236PRTArtificial SequenceDescription of Artificial
SequenceHIV 23Glu Leu Asp Lys Trp Ala1 524852PRTArtificial
SequenceDescription of Artificial SequenceHIV consensus sequence
24Met Arg Val Arg Gly Ile Gln Arg Asn Cys Gln His Leu Trp Arg Trp1
5 10 15Gly Thr Leu Ile Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu
Asn 20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu
Ala Asn 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 125Asn Cys Thr Asn Val
Asn Val Thr Asn Thr Thr Asn Asn Thr Glu Glu 130 135 140Lys Gly Glu
Ile Lys Asn Cys Ser Phe Asn Ile Thr Thr Glu Ile Arg145 150 155
160Asp Lys Lys Gln Lys Val Tyr Ala Leu Phe Tyr Arg Leu Asp Val Val
165 170 175Pro Ile Asp Asp Asn Asn Asn Asn Ser Ser Asn Tyr Arg Leu
Ile Asn 180 185 190Cys Asn Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys
Val Ser Phe Glu 195 200 205Pro Ile Pro Ile His Tyr Cys Ala Pro Ala
Gly Phe Ala Ile Leu Lys 210 215 220Cys Asn Asp Lys Lys Phe Asn Gly
Thr Gly Pro Cys Lys Asn Val Ser225 230 235 240Thr Val Gln Cys Thr
His Gly Ile Lys Pro Val Val Ser Thr Gln Leu 245 250 255Leu Leu Asn
Gly Ser Leu Ala Glu Glu Glu Ile Ile Ile Arg Ser Glu 260 265 270Asn
Ile Thr Asn Asn Ala Lys Thr Ile Ile Val Gln Leu Asn Glu Ser 275 280
285Val Glu Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile
290 295 300Arg Ile Gly Pro Gly Gln Ala Phe Tyr Ala Thr Gly Asp Ile
Ile Gly305 310 315 320Asp Ile Arg Gln Ala His Cys Asn Ile Ser Gly
Thr Lys Trp Asn Lys 325 330 335Thr Leu Gln Gln Val Ala Lys Lys Leu
Arg Glu His Phe Asn Asn Lys 340 345 350Thr Ile Ile Phe Lys Pro Ser
Ser Gly Gly Asp Leu Glu Ile Thr Thr 355 360 365His Ser Phe Asn Cys
Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Gly 370 375 380Leu Phe Asn
Ser Thr Trp Ile Gly Asn Gly Thr Lys Asn Asn Asn Asn385 390 395
400Thr Asn Asp Thr Ile Thr Leu Pro Cys Arg Ile Lys Gln Ile Ile Asn
405 410 415Met Trp Gln Gly Val Gly Gln Ala Met Tyr Ala Pro Pro Ile
Glu Gly 420 425 430Lys Ile Thr Cys Lys Ser Asn Ile Thr Gly Leu Leu
Leu Thr Arg Asp 435 440 445Gly Gly Asn Asn Asn Thr Asn Glu Thr Glu
Ile Phe Arg Pro Gly Gly 450 455 460Gly Asp Met Arg Asp Asn Trp Arg
Ser Glu Leu Tyr Lys Tyr Lys Val465 470 475 480Val Lys Ile Glu Pro
Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg 485 490 495Val Val Glu
Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu 500 505 510Gly
Phe Leu Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr 515 520
525Leu Thr Val Gln Ala Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln
530 535 540Ser Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu
Gln Leu545 550 555 560Thr Val Trp Gly Ile Lys Gln Leu Gln Ala Arg
Val Leu Ala Val Glu 565 570 575Arg Tyr Leu Lys Asp Gln Gln Leu Leu
Gly Ile Trp Gly Cys Ser Gly 580 585 590Lys Leu Ile Cys Thr Thr Thr
Val Pro Trp Asn Ser Ser Trp Ser Asn 595 600 605Lys Ser Gln Asp Glu
Ile Trp Asp Asn Met Thr Trp Met Glu Trp Glu 610 615 620Arg Glu Ile
Asn Asn Tyr Thr Asp Ile Ile Tyr Ser Leu Ile Glu Glu625 630 635
640Ser Gln Asn Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Ala Leu Asp
645 650 655Lys Trp Ala Ser Leu Trp Asn Trp Phe Asp Ile Thr Asn Trp
Leu Trp 660 665 670Tyr Ile Lys Ile Phe Ile Met Ile Val Gly Gly Leu
Ile Gly Leu Arg 675 680 685Ile Val Phe Ala Val Leu Ser Ile Val Asn
Arg Val Arg Gln Gly Tyr 690 695 700Ser Pro Leu Ser Phe Gln Thr Leu
Ile Pro Asn Pro Arg Gly Pro Asp705 710 715 720Arg Pro Glu Gly Ile
Glu Glu Glu Gly Gly Glu Gln Asp Arg Asp Arg 725 730 735Ser Ile Arg
Leu Val Asn Gly Phe Leu Ala Leu Ala Trp Asp Asp Leu 740 745 750Arg
Ser Leu Cys Leu Phe Ser Tyr His Arg Leu Arg Asp Phe Ile Leu 755 760
765Ile Ala Ala Arg Thr Val Glu Leu Leu Gly Arg Lys Gly Leu Arg Arg
770 775 780Gly Trp Glu Ala Leu Lys Tyr Leu Trp Asn Leu Leu Gln Tyr
Trp Gly785 790 795 800Gln Glu Leu Lys Asn Ser Ala Ile Ser Leu Leu
Asp Thr Thr Ala Ile 805 810 815Ala Val Ala Glu Gly Thr Asp Arg Val
Ile Glu Val Val Gln Arg Ala 820 825 830Cys Arg Ala Ile Leu Asn Ile
Pro Arg Arg Ile Arg Gln Gly Leu Glu 835 840 845Arg Ala Leu Leu
850252559DNAArtificial SequenceDescription of Artificial
SequenceProbe 25atgcgcgtgc gcggcatcca gcgcaactgc cagcacctgt
ggcgctgggg caccctgatc 60ctgggcatgc tgatgatctg ctccgccgcc gagaacctgt
gggtgaccgt gtactacggc 120gtgcccgtgt ggaaggaggc caacaccacc
ctgttctgcg cctccgacgc caaggcctac 180gacaccgagg tgcacaacgt
gtgggccacc cacgcctgcg tgcccaccga ccccaacccc 240caggagatcg
tgctggagaa cgtgaccgag aacttcaaca tgtggaagaa caacatggtg
300gagcagatgc acgaggacat catctccctg tgggaccagt ccctgaagcc
ctgcgtgaag 360ctgacccccc tgtgcgtgac cctgaactgc accaacgtga
acgtgaccaa caccaccaac 420aacaccgagg agaagggcga gatcaagaac
tgctccttca acatcaccac cgagatccgc 480gacaagaagc agaaggtgta
cgccctgttc taccgcctgg acgtggtgcc catcgacgac 540aacaacaaca
actcctccaa ctaccgcctg atcaactgca acacctccgc catcacccag
600gcctgcccca aggtgtcctt cgagcccatc cccatccact actgcgcccc
cgccggcttc 660gccatcctga agtgcaacga caagaagttc aacggcaccg
gcccctgcaa gaacgtgtcc 720accgtgcagt gcacccacgg catcaagccc
gtggtgtcca cccagctgct gctgaacggc 780tccctggccg aggaggagat
catcatccgc tccgagaaca tcaccaacaa cgccaagacc 840atcatcgtgc
agctgaacga gtccgtggag atcaactgca cccgccccaa caacaacacc
900cgcaagtcca tccgcatcgg ccccggccag gccttctacg ccaccggcga
catcatcggc 960gacatccgcc aggcccactg caacatctcc ggcaccaagt
ggaacaagac cctgcagcag 1020gtggccaaga agctgcgcga gcacttcaac
aacaagacca tcatcttcaa gccctcctcc 1080ggcggcgacc tggagatcac
cacccactcc ttcaactgcc gcggcgagtt cttctactgc 1140aacacctccg
gcctgttcaa ctccacctgg atcggcaacg gcaccaagaa caacaacaac
1200accaacgaca ccatcaccct gccctgccgc atcaagcaga tcatcaacat
gtggcagggc 1260gtgggccagg ccatgtacgc cccccccatc gagggcaaga
tcacctgcaa gtccaacatc 1320accggcctgc tgctgacccg cgacggcggc
aacaacaaca ccaacgagac cgagatcttc 1380cgccccggcg gcggcgacat
gcgcgacaac tggcgctccg agctgtacaa gtacaaggtg 1440gtgaagatcg
agcccctggg cgtggccccc accaaggcca agcgccgcgt ggtggagcgc
1500gagaagcgcg ccgtgggcat cggcgccgtg ttcctgggct tcctgggcgc
cgccggctcc 1560accatgggcg ccgcctccat caccctgacc gtgcaggccc
gccagctgct gtccggcatc 1620gtgcagcagc agtccaacct gctgcgcgcc
atcgaggccc agcagcacct gctgcagctg 1680accgtgtggg gcatcaagca
gctgcaggcc cgcgtgctgg ccgtggagcg ctacctgaag 1740gaccagcagc
tgctgggcat ctggggctgc tccggcaagc tgatctgcac caccaccgtg
1800ccctggaact cctcctggtc caacaagtcc caggacgaga tctgggacaa
catgacctgg 1860atggagtggg agcgcgagat caacaactac accgacatca
tctactccct gatcgaggag 1920tcccagaacc agcaggagaa gaacgagcag
gagctgctgg ccctggacaa gtgggcctcc 1980ctgtggaact ggttcgacat
caccaactgg ctgtggtaca tcaagatctt catcatgatc 2040gtgggcggcc
tgatcggcct gcgcatcgtg ttcgccgtgc tgtccatcgt gaaccgcgtg
2100cgccagggct actcccccct gtccttccag accctgatcc ccaacccccg
cggccccgac 2160cgccccgagg gcatcgagga ggagggcggc gagcaggacc
gcgaccgctc catccgcctg 2220gtgaacggct tcctggccct ggcctgggac
gacctgcgct ccctgtgcct gttctcctac 2280caccgcctgc gcgacttcat
cctgatcgcc gcccgcaccg tggagctgct gggccgcaag 2340ggcctgcgcc
gcggctggga ggccctgaag tacctgtgga acctgctgca gtactggggc
2400caggagctga agaactccgc catctccctg ctggacacca ccgccatcgc
cgtggccgag 2460ggcaccgacc gcgtgatcga ggtggtgcag cgcgcctgcc
gcgccatcct gaacatcccc 2520cgccgcatcc gccagggcct ggagcgcgcc
ctgctgtaa 25592637PRTArtificial SequenceDescription of Artificial
SequenceHIV 26Tyr Lys Arg Trp Ile Ile Leu Gly Leu Asn Lys Ile Val
Arg Met Tyr1 5 10 15Ser Gln Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu
Leu Asp Lys Trp 20 25 30Ala Ser Leu Trp Asn 352733PRTArtificial
SequenceDescription of Artificial SequenceHIV 27Tyr Lys Arg Trp Ile
Ile Leu Gly Leu Asn Lys Ile Val Arg Met Tyr1 5 10 15Ser Ser Leu Trp
Asn Trp Phe Asn Ile Thr Asn Trp Leu Trp Tyr Ile 20 25 30Lys
* * * * *