U.S. patent application number 13/363313 was filed with the patent office on 2012-10-11 for antigen-antibody complexes as hiv-1 vaccines.
Invention is credited to Wayne C. Koff, Sanjay Phogat.
Application Number | 20120258124 13/363313 |
Document ID | / |
Family ID | 40623925 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258124 |
Kind Code |
A1 |
Koff; Wayne C. ; et
al. |
October 11, 2012 |
ANTIGEN-ANTIBODY COMPLEXES AS HIV-1 VACCINES
Abstract
The present relation relates to antigen-antibody complexes for
use as prophylactic and therapeutic vaccines for infectious
diseases of AIDS. The present invention encompasses the preparation
and purification of immunogenic antibody-antigen complexes which
are formulated into the vaccines of the present invention.
Inventors: |
Koff; Wayne C.; (Stony
Brook, NY) ; Phogat; Sanjay; (Frederick, MD) |
Family ID: |
40623925 |
Appl. No.: |
13/363313 |
Filed: |
January 31, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12261359 |
Oct 30, 2008 |
8105600 |
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13363313 |
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11929015 |
Oct 30, 2007 |
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12261359 |
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PCT/US07/83006 |
Oct 30, 2007 |
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11929015 |
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60855625 |
Oct 30, 2006 |
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61035653 |
Mar 11, 2008 |
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Current U.S.
Class: |
424/178.1 |
Current CPC
Class: |
A61K 2039/6056 20130101;
A61K 39/12 20130101; A61K 2039/55566 20130101; A61P 37/04 20180101;
C07K 16/1063 20130101; A61P 37/02 20180101; A61K 2039/505 20130101;
C12N 2740/16134 20130101; A61K 39/21 20130101; A61P 31/18 20180101;
A61K 2039/55555 20130101; A61K 2039/54 20130101; A61K 2039/545
20130101; A61K 2039/55516 20130101 |
Class at
Publication: |
424/178.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 37/04 20060101 A61P037/04; A61P 31/18 20060101
A61P031/18 |
Claims
1. A method of producing an immune response comprising
administering to a mammal a purified antibody-antigen complex.
2. The method of claim 1, wherein the purified antibody-antigen
complex is dissociated from polyclonal anti-HIV sera bound to
glycoprotein spikes on HIV envelopes.
3. The method of claim 1, wherein the purified antibody-antigen
complex dissociated from a mixture of broadly neutralizing
antibodies and HIV, wherein the mixture is bound to glycoprotein
spikes on HIV envelopes.
4. The method of claim 3 wherein the antibodies are monoclonal
antibodies.
5. The method of claim 4 wherein the monoclonal antibodies are b12,
2F5, 2G12, 4E10, M2909 or any combination thereof.
5. The method of claim 3 wherein the HIV is a HIV clade viral
isolate.
6. The method of claim 1 wherein the purified antibody-antigen
complex is administered in a pharmaceutically acceptable
carrier.
7. The method of claim 1 wherein the administering further
comprises a prime-boost regimen.
8. A method of producing an immune response comprising
administering to a mammal an antibody-antigen complex, wherein the
antigen is an HIV envelope protein.
9. The method of claim 8 wherein the HIV envelope protein is gp120,
gp140 or a membrane associated envelope trimer.
10. The method of claim 8 wherein the antibody is a broad
neutralizing antibody.
11. The method of claim 10 wherein the broad neutralizing antibody
is antibody b12.
12. The method of claim 8 wherein the antibody is a
non-neutralizing antibody.
13. The method of claim 12 wherein the non-neutralizing antibody is
a V3 specific antibody.
14. The method of claim 13 wherein the non-neutralizing antibody is
antibody 39F.
15. The method of claim 8 wherein the HIV envelope protein is a
membrane associated envelope trimer and the antibody is a trimer
specific antibody 2909.
16. The method of claim 8 wherein the antibody-antigen complex is
administered in a pharmaceutically acceptable carrier.
17. The method of claim 8 further comprising an adjuvant.
18. The method of claim 17 wherein the adjuvant is Adjuplex LAP.
Description
INCORPORATION BY REFERENCE
[0001] This application is a continuation in part of U.S.
non-provisional application Ser. No. 11/929,015 filed on 30 Oct.
2007 and PCT International Application No. PCT/US2007/083006 filed
on 30 Oct. 2007, which claims priority to U.S. provisional
application Ser. No. 60/855,625, filed on 30 Oct. 2006. This
application also claims priority to U.S. provisional application
Ser. No. 61/035,653, filed on 11 Mar. 2008.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced in the appln cited documents, and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention.
FIELD OF THE INVENTION
[0003] The present invention relates to antigen-antibody complexes
for use as prophylactic and therapeutic vaccines for infectious
diseases of AIDS.
BACKGROUND OF THE INVENTION
[0004] AIDS, or Acquired Immunodeficiency Syndrome, is caused by
human immunodeficiency virus (HIV) and is characterized by several
clinical features including wasting syndromes, central nervous
system degeneration and profound immunosuppression that results in
opportunistic infections and malignancies. HIV is a member of the
lentivirus family of animal retroviruses, which include the visna
virus of sheep and the bovine, feline, and simian immunodeficiency
viruses (SIV). Two closely related types of HIV, designated HIV-1
and HIV-2, have been identified thus far, of which HIV-1 is by far
the most common cause of AIDS. However, HIV-2, which differs in
genomic structure and antigenicity, causes a similar clinical
syndrome.
[0005] An infectious HIV particle consists of two identical strands
of RNA, each approximately 9.2 kb long, packaged within a core of
viral proteins. This core structure is surrounded by a phospholipid
bilayer envelope derived from the host cell membrane that also
includes virally-encoded membrane proteins (Abbas et al., Cellular
and Molecular Immunology, 4th edition, W.B. Saunders Company, 2000,
p. 454). The HIV genome has the characteristic
5'-LTR-Gag-Pol-Env-LTR-3' organization of the retrovirus family.
Long terminal repeats (LTRs) at each end of the viral genome serve
as binding sites for transcriptional regulatory proteins from the
host and regulate viral integration into the host genome, viral
gene expression, and viral replication.
[0006] The HIV genome encodes several structural proteins. The Gag
gene encodes core structural proteins of the nucleocapsid core and
matrix. The Pol gene encodes reverse transcriptase (RT), integrase
(Int), and viral protease enzymes required for viral replication.
The tat gene encodes a protein that is required for elongation of
viral transcripts. The rev gene encodes a protein that promotes the
nuclear export of incompletely spliced or unspliced viral RNAs. The
Vif gene product enhances the infectivity of viral particles. The
vpr gene product promotes the nuclear import of viral DNA and
regulates G2 cell cycle arrest. The vpu and nef genes encode
proteins that down regulate host cell CD4 expression and enhance
release of virus from infected cells. The Env gene encodes the
viral envelope glycoprotein that is translated as a 160-kilodalton
(kDa) precursor (gp160) and cleaved by a cellular protease to yield
the external 120-kDa envelope glycoprotein (gp120) and the
transmembrane 41-kDa envelope glycoprotein (gp41), which are
required for the infection of cells (Abbas, pp. 454-456). Gp140 is
a modified form of the env glycoprotein which contains the external
120-kDa envelope glycoprotein portion and a part of the gp41
portion of env and has characteristics of both gp120 and gp41. The
Nef gene is conserved among primate lentiviruses and is one of the
first viral genes that is transcribed following infection. In
vitro, several functions have been described, including down
regulation of CD4 and MHC class I surface expression, altered
T-cell signaling and activation, and enhanced viral
infectivity.
[0007] HIV infection initiates with gp120 on the viral particle
binding to the CD4 and chemokine receptor molecules (e.g., CXCR4,
CCR5) on the cell membrane of target cells such as CD4+ T-cells,
macrophages and dendritic cells. The bound virus fuses with the
target cell and reverse transcribes the RNA genome. The resulting
viral DNA integrates into the cellular genome, where it directs the
production of new viral RNA, and thereby viral proteins and new
virions. These virions bud from the infected cell membrane and
establish productive infections in other cells. This process also
kills the originally infected cell. HIV can also kill cells
indirectly because the CD4 receptor on uninfected T-cells has a
strong affinity for gp120 expressed on the surface of infected
cells. In this case, the uninfected cells bind, via the CD4
receptor-gp120 interaction, to infected cells and fuse to form a
syncytium, which cannot survive. Destruction of CD4+ T-lymphocytes,
which are critical to immune defense, is a major cause of the
progressive immune dysfunction that is the hallmark of AIDS disease
progression. The loss of CD4+ T cells seriously impairs the body's
ability to fight most invaders, but it has a particularly severe
impact on the defenses against viruses, fungi, parasites and
certain bacteria, including mycobacteria.
[0008] Research on the Env glycoproteins have shown that the virus
has many effective protective mechanisms with few vulnerabilities
(Wyatt & Sodroski, Science. 1998 Jun. 19; 280(5371):1884-8).
For fusion with its target cells, HIV-1 uses a trimeric Env complex
containing gp120 and gp41 subunits (Burton et al., Nat. Immunol.
2004 March; 5(3):233-6). The fusion potential of the Env complex is
triggered by engagement of the CD4 receptor and a coreceptor,
usually CCR5 or CXCR4. Neutralizing antibodies seem to work either
by binding to the mature trimer on the virion surface and
preventing initial receptor engagement events or by binding after
virion attachment and inhibiting the fusion process (Parren &
Burton, Adv Immunol. 2001; 77:195-262). In the latter case,
neutralizing antibodies may bind to epitopes whose exposure is
enhanced or triggered by receptor binding. However, given the
potential antiviral effects of neutralizing antibodies, it is not
unexpected that HIV-1 has evolved multiple mechanisms to protect it
from antibody binding (Johnson & Desrosiers, Annu Rev Med.
2002; 53:499-518).
[0009] There remains a need to identify immunogens that elicit
broadly neutralizing antibodies. Strategies include producing
molecules that mimic the mature trimer on the virion surface,
producing Env molecules engineered to better present neutralizing
antibody epitopes than wild-type molecules, generating stable
intermediates of the entry process to expose conserved epitopes to
which antibodies could gain access during entry and producing
epitope mimics of the broadly neutralizing monoclonal antibodies
determined from structural studies of the antibody-antigen
complexes (Burton et al., Nat. Immunol. 2004 March; 5(3):233-6).
However, none of these approaches have yet efficiently elicited
neutralizing antibodies with broad specificity.
[0010] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present application.
SUMMARY OF THE INVENTION
[0011] The current invention is based, in part, on Applicant's
discovery that immunization with antigen-antibody complexes elicit
neutralizing antibody responses. Broadly neutralizing antibodies,
if passively administered to monkeys, protect against an HIV
equivalent virus (SIV/HIV chimera, e.g., SHIV). The identification
of antigens that bind the neutralizing antibodies remains
challenging, especially elucidating the preferred conformation of
the antigen.
[0012] The solution proposed by the present invention is
immunization with the antibody-antigen complex, wherein the antigen
is held in its preferred conformation by the antibody or its
equivalent polyclonal sera. One of skill in the art would not
expect this approach to work as the antigen are bound to the
antibody and the epitopes are covered. Without being bound by
theory, it is hypothesized that an antibody-antigen complex is
presented to the immune system in a novel form, is dissociated
within the antigen presenting cells and elicits the correct
antibody response.
[0013] In an advantageous embodiment, the antigen-antibody complex
is an envelope protein (such as, but not limited to, gp120, gp140
or membrane-associated envelope trimers) complexed with a CD4
binding site broad neutralizing antibody (such as, but not limited
to, b12, 2F5), a variable loop 3 specific antibody (such as, but
not limited to, 39F), a trimer-specific antibody (such as, but not
limited to 2909, if the antigen is a envelope trimer protein) or a
CD4 induced epitope specific antibody.
[0014] The present invention encompasses identification of
antibody-antigen complexes for use as a HIV vaccine. In one
embodiment, the invention relates to the identification of
immunogenic antibody-antigen complexes.
[0015] In one embodiment, mixing polyclonal anti-HIV sera which
demonstrate broad neutralizing activity with purified HIV enables
the antibodies to bind to the glycoprotein spikes on the viral
envelopes. The antibody-antigen complexes are dissociated,
advantageously chemically dissociated, from the virus. The
antibody-antigen complexes are purified and formulated into the
vaccines of the present invention.
[0016] In another embodiment, broadly neutralizing HIV monoclonal
antibodies such as, but not limited to, b12, 2F5, 2G12, 4E10, M2909
either alone or combination, are mixed with purified HIV enables
the antibodies to bind the glycoprotein spikes on the viral
envelopes. The antibody-antigen complexes may be dissociated,
advantageously chemically dissociated, from the virus. The
antibody-antigen complexes may be purified and formulated into the
vaccines of the present invention.
[0017] In yet another embodiment, new broadly neutralizing
antibodies to HIV are identified and mixed with purified HIV
enables the antibodies to bind the glycoprotein spikes on the viral
envelopes. The antibody-antigen complexes are dissociated,
advantageously chemically dissociated, from the virus. The
antibody-antigen complexes are purified and formulated into the
vaccines of the present invention.
[0018] In still another embodiment, the antibody-antigen complexes
may be identified from alternate viral isolates, such as different
HIV clades. In this embodiment, polyclonal anti-HIV sera, broadly
neutralizing HIV monoclonal antibodies such as, but not limited to,
b12, 2F5, 2G12, 4E10, M2909 either alone or combination, or newly
identified broadly neutralizing antibodies to HIV are mixed with
different HIV clade viral isolates to enable the antibodies to bind
to varying antigens, thereby forming antibody-antigen complexes.
The antibody-antigen complexes are dissociated, advantageously
chemically dissociated, from the virus. The antibody-antigen
complexes are purified and formulated into the vaccines of the
present invention.
[0019] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0020] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0022] FIG. 1 depicts immune complexes of YU2 gp120-B12 run on a
gel filtration column.
[0023] FIG. 2 depicts an analysis of immune complexes before and
after gel filtration.
[0024] FIG. 3 depicts gel-filtration purified complexes on reducing
SDS-PAGE where lane 1 is b12, lane 2 is YU2 gp120, lane 3 is YU2
gp120+b12 and lane 4 is YU2 gp120+39F.
[0025] FIG. 4 depicts an antibody response to immune-complex by
single prime-boost immunization.
[0026] FIG. 5 depicts a mean titer: anti-env gp120 response,
specifically an ELISA anti-gp120 titer in bleeds collected from
rabbits 2 weeks post immunization by Yu2 gp120 and the immune
complex groups at weeks 2, 6, 10 and 14.
[0027] FIG. 6 depicts Yu2 gp120 and Yu2gp120-IgG b12 immune
complexes captured on the ELISA plate which were probed with
biotinylated conformational anti-HIV antibodies [b12 (binds CD4
binding site), 39F (binds V3 loop), 2G12 (recognizes glycan on the
surface) and A32 (recognizes epitope on C1 and C5 region of YU2
gp120)]. Except for IgG b12 site which is occupied in the immune
complex all the other antibodies showed comparable binding to both
Yu2 gp120 and YU2 gp120-IgG b12 immune complex.
[0028] FIGS. 7A and 7B depicts generation of YU2 gp120 immune
complex with Fab and IgG b12 (left) and anti-gp120 titer in rabbits
for the two immune complex group at weeks 6, 10 and 14. To
determine the role of Fc, Yu2 gp120 with Fab b12 and IgG b12 immune
complexes were generated and characterized (run and coommassie
stained FIG. 7A). When immunized in rabbits the IgG b12 immune
complex faired better in eliciting anti-gp120 titer (graph in 7B)
than the Fab b12 immune complex. At week 6 and week 10 (post 2 and
3 boost respectively) there was a 2-4 fold difference in titer. At
week 10 both the groups had similar titer suggesting other factors
like stabilization of the env, increase in size, besides the
ability of FC to present to the antigen presenting cells.
[0029] FIG. 8 depicts ELISA anti-gp120 titer for JRCSF gp10 and
JRCSF-gp120-Fc fusion protein (see, e.g., Binley J M et al.,
Inhibition of HIV Env binding to cellular receptors by monoclonal
antibody 2G12 as probed by Fc-tagged gp120, Retrovirology. 2006
Jul. 3; 3:39 and Retrovirology. 2007; 4:23) at weeks 2 and 6.
[0030] FIG. 9 depicts gel-filtration purified YU2 gp140 immune
complexes where lane 1 is marker, lane 2 is IgG b12, lane 3 is IgG
39F, lane 4 is YU2 gp140, lane 5 is YU2 gp140+IgG b12, lane 6 is
YU2 gp140+39F and lane 7 is marker.
[0031] FIG. 10 depicts an antibody response to YU2 gp140 immune
complex after immunization in rabbits at weeks 4 and 10.
DETAILED DESCRIPTION
[0032] The present invention relates to vaccines for HIV comprising
antibody-antigen complexes. The current invention is based, in
part, on Applicant's surprising discovery that immunization with
antigen-antibody complexes elicit neutralizing antibody
responses.
[0033] Previous attempts to elicit an effective neutralizing
response from antigen-antibody complexes have failed, in particular
an Env gp120-antibody A32 complex (see, e.g., Liao et al., J.
Virol. 2004 May; 78(10):5270-8).
[0034] The present invention encompasses identification of
antibody-antigen complexes for use as a HIV vaccine. In one
embodiment, the invention relates to the identification of
immunogenic antibody-antigen complexes.
[0035] In an advantageous embodiment, the antigen-antibody complex
is an envelope protein (such as, but not limited to, gp120, gp140
or membrane-associated envelope trimers) complexed with a CD4
binding site broad neutralizing antibody (such as, but not limited
to, b12, 2F5), a variable loop 3 specific antibody (such as, but
not limited to, 39F), a trimer-specific antibody (such as, but not
limited to 2909, if the antigen is a envelope trimer protein) or a
CD4 induced epitope specific antibody.
[0036] The invention encompasses mixing HIV antibodies, such as but
not limited to, polyclonal anti-HIV sera, broadly neutralizing HIV
monoclonal antibodies such as, but not limited to, b12, 2F5, 2G12,
4E10, M2909 either alone or combination or novel broadly
neutralizing antibodies to HIV with purified HIV to enables the
antibodies to bind to HIV antigens, such as but not limited to, the
glycoprotein spikes on the viral envelopes, to form
antibody-antigen complexes.
[0037] In an advantageous embodiment, the antigen-antibody complex
comprises any form of HIV envelope glycoprotein or derivative
thereof and any anti-HIV antibody or derivative thereof. The
antigen-antibody complex advantageously improves the immunogenic
response of an existing antigen.
[0038] The antigen-antibody complex of the present invention may
potentiate an immune response via Fc receptor activity, increase
germinal center formation to increase production of neutralizing
antibodies, expose cryptic epitope on the immunogen due to better
presentation of the antigen or protect and present a known epitope
for neutralization (such as an antigen-antibody complex comprising
neutralizing antibody b12).
[0039] Any HIV antigen may be used to form antibody-antigen
complexes. Advantageously, the HIV antigen is an HIV antigen, HIV
epitope or an HIV immunogen, such as, but not limited to, the HIV
antigens, HIV epitopes or HIV immunogens of U.S. Pat. Nos.
7,341,731; 7,335,364; 7,329,807; 7,323,553; 7,320,859; 7,311,920;
7,306,798; 7,285,646; 7,285,289; 7,285,271; 7,282,364; 7,273,695;
7,270,997; 7,262,270; 7,244,819; 7,244,575; 7,232,567; 7,232,566;
7,223,844; 7,223,739; 7,223,534; 7,223,368; 7,220,554; 7,214,530;
7,211,659; 7,211,432; 7,205,159; 7,198,934; 7,195,768; 7,192,555;
7,189,826; 7,189,522; 7,186,507; 7,179,645; 7,175,843; 7,172,761;
7,169,550; 7,157,083; 7,153,509; 7,147,862; 7,141,550; 7,129,219;
7,122,188; 7,118,859; 7,118,855; 7,118,751; 7,118,742; 7,105,655;
7,101,552; 7,097,971 7,097,842; 7,094,405; 7,091,049; 7,090,648;
7,087,377; 7,083,787; 7,070,787; 7,070,781; 7,060,273; 7,056,521;
7,056,519; 7,049,136; 7,048,929; 7,033,593; 7,030,094; 7,022,326;
7,009,037; 7,008,622; 7,001,759; 6,997,863; 6,995,008; 6,979,535;
6,974,574; 6,972,126; 6,969,609; 6,964,769; 6,964,762; 6,958,158;
6,956,059; 6,953,689; 6,951,648; 6,946,075; 6,927,031; 6,919,319;
6,919,318; 6,919,077; 6,913,752; 6,911,315; 6,908,617; 6,908,612;
6,902,743; 6,900,010; 6,893,869; 6,884,785; 6,884,435; 6,875,435;
6,867,005; 6,861,234; 6,855,539; 6,841,381 6,841,345; 6,838,477;
6,821,955; 6,818,392; 6,818,222; 6,815,217; 6,815,201; 6,812,026;
6,812,025; 6,812,024; 6,808,923; 6,806,055; 6,803,231; 6,800,613;
6,800,288; 6,797,811; 6,780,967; 6,780,598; 6,773,920; 6,764,682;
6,761,893; 6,753,015; 6,750,005; 6,737,239; 6,737,067; 6,730,304;
6,720,310; 6,716,823; 6,713,301; 6,713,070; 6,706,859; 6,699,722;
6,699,656; 6,696,291; 6,692,745; 6,670,181; 6,670,115; 6,664,406;
6,657,055; 6,657,050; 6,656,471; 6,653,066; 6,649,409; 6,649,372;
6,645,732; 6,641,816; 6,635,469; 6,613,530; 6,605,427; 6,602,709
6,602,705; 6,600,023; 6,596,477; 6,596,172; 6,593,103; 6,593,079;
6,579,673; 6,576,758; 6,573,245; 6,573,040; 6,569,418; 6,569,340;
6,562,800; 6,558,961; 6,551,828; 6,551,824; 6,548,275; 6,544,780;
6,544,752; 6,544,728; 6,534,482; 6,534,312; 6,534,064; 6,531,572;
6,531,313; 6,525,179; 6,525,028; 6,524,582; 6,521,449; 6,518,030;
6,518,015; 6,514,691; 6,514,503; 6,511,845; 6,511,812; 6,511,801;
6,509,313; 6,506,384; 6,503,882; 6,495,676; 6,495,526; 6,495,347;
6,492,123; 6,489,131; 6,489,129; 6,482,614; 6,479,286; 6,479,284;
6,465,634; 6,461,615 6,458,560; 6,458,527; 6,458,370; 6,451,601;
6,451,592; 6,451,323; 6,436,407; 6,432,633; 6,428,970; 6,428,952;
6,428,790; 6,420,139; 6,416,997; 6,410,318; 6,410,028; 6,410,014;
6,407,221; 6,406,710; 6,403,092; 6,399,295; 6,392,013; 6,391,657;
6,384,198; 6,380,170; 6,376,170; 6,372,426; 6,365,187; 6,358,739;
6,355,248; 6,355,247; 6,348,450; 6,342,372; 6,342,228; 6,338,952;
6,337,179; 6,335,183; 6,335,017; 6,331,404; 6,329,202; 6,329,173;
6,328,976; 6,322,964; 6,319,666; 6,319,665; 6,319,500; 6,319,494;
6,316,205; 6,316,003; 6,309,633; 6,306,625 6,296,807; 6,294,322;
6,291,239; 6,291,157; 6,287,568; 6,284,456; 6,284,194; 6,274,337;
6,270,956; 6,270,769; 6,268,484; 6,265,562; 6,265,149; 6,262,029;
6,261,762; 6,261,571; 6,261,569; 6,258,599; 6,258,358; 6,248,332;
6,245,331; 6,242,461; 6,241,986; 6,235,526; 6,235,466; 6,232,120;
6,228,361; 6,221,579; 6,214,862; 6,214,804; 6,210,963; 6,210,873;
6,207,185; 6,203,974; 6,197,755; 6,197,531; 6,197,496; 6,194,142;
6,190,871; 6,190,666; 6,168,923; 6,156,302; 6,153,408; 6,153,393;
6,153,392; 6,153,378; 6,153,377; 6,146,635; 6,146,614; 6,143,876
6,140,059; 6,140,043; 6,139,746; 6,132,992; 6,124,306; 6,124,132;
6,121,006; 6,120,990; 6,114,507; 6,114,143; 6,110,466; 6,107,020;
6,103,521; 6,100,234; 6,099,848; 6,099,847; 6,096,291; 6,093,405;
6,090,392; 6,087,476; 6,083,903; 6,080,846; 6,080,725; 6,074,650;
6,074,646; 6,070,126; 6,063,905; 6,063,564; 6,060,256; 6,060,064;
6,048,530; 6,045,788; 6,043,347; 6,043,248; 6,042,831; 6,037,165;
6,033,672; 6,030,772; 6,030,770; 6,030,618; 6,025,141; 6,025,125;
6,020,468; 6,019,979; 6,017,543; 6,017,537; 6,015,694; 6,015,661;
6,013,484; 6,013,432 6,007,838; 6,004,811; 6,004,807; 6,004,763;
5,998,132; 5,993,819; 5,989,806; 5,985,926; 5,985,641; 5,985,545;
5,981,537; 5,981,505; 5,981,170; 5,976,551; 5,972,339; 5,965,371;
5,962,428; 5,962,318; 5,961,979; 5,961,970; 5,958,765; 5,958,422;
5,955,647; 5,955,342; 5,951,986; 5,951,975; 5,942,237; 5,939,277;
5,939,074; 5,935,580; 5,928,930; 5,928,913; 5,928,644; 5,928,642;
5,925,513; 5,922,550; 5,922,325; 5,919,458; 5,916,806; 5,916,563;
5,914,395; 5,914,109; 5,912,338; 5,912,176; 5,912,170; 5,906,936;
5,895,650; 5,891,623; 5,888,726; 5,885,580 5,885,578; 5,879,685;
5,876,731; 5,876,716; 5,874,226; 5,872,012; 5,871,747; 5,869,058;
5,866,694; 5,866,341; 5,866,320; 5,866,319; 5,866,137; 5,861,290;
5,858,740; 5,858,647; 5,858,646; 5,858,369; 5,858,368; 5,858,366;
5,856,185; 5,854,400; 5,853,736; 5,853,725; 5,853,724; 5,852,186;
5,851,829; 5,851,529; 5,849,475; 5,849,288; 5,843,728; 5,843,723;
5,843,640; 5,843,635; 5,840,480; 5,837,510; 5,837,250; 5,837,242;
5,834,599; 5,834,441; 5,834,429; 5,834,256; 5,830,876; 5,830,641;
5,830,475; 5,830,458; 5,830,457; 5,827,749; 5,827,723; 5,824,497
5,824,304; 5,821,047; 5,817,767; 5,817,754; 5,817,637; 5,817,470;
5,817,318; 5,814,482; 5,807,707; 5,804,604; 5,804,371; 5,800,822;
5,795,955; 5,795,743; 5,795,572; 5,789,388; 5,780,279; 5,780,038;
5,776,703; 5,773,260; 5,770,572; 5,766,844; 5,766,842; 5,766,625;
5,763,574; 5,763,190; 5,762,965; 5,759,769; 5,756,666; 5,753,258;
5,750,373; 5,747,641; 5,747,526; 5,747,028; 5,736,320; 5,736,146;
5,733,760; 5,731,189; 5,728,385; 5,721,095; 5,716,826; 5,716,637;
5,716,613; 5,714,374; 5,709,879; 5,709,860; 5,709,843; 5,705,331;
5,703,057; 5,702,707 5,698,178; 5,688,914; 5,686,078; 5,681,831;
5,679,784; 5,674,984; 5,672,472; 5,667,964; 5,667,783; 5,665,536;
5,665,355; 5,660,990; 5,658,745; 5,658,569; 5,643,756; 5,641,624;
5,639,854; 5,639,598; 5,637,677; 5,637,455; 5,633,234; 5,629,153;
5,627,025; 5,622,705; 5,614,413; 5,610,035; 5,607,831; 5,606,026;
5,601,819; 5,597,688; 5,593,972; 5,591,829; 5,591,823; 5,589,466;
5,587,285; 5,585,254; 5,585,250; 5,580,773; 5,580,739; 5,580,563;
5,573,916; 5,571,667; 5,569,468; 5,558,865; 5,556,745; 5,550,052;
5,543,328; 5,541,100; 5,541,057; 5,534,406 5,529,765; 5,523,232;
5,516,895; 5,514,541; 5,510,264; 5,500,161; 5,480,967; 5,480,966;
5,470,701; 5,468,606; 5,462,852; 5,459,127; 5,449,601; 5,447,838;
5,447,837; 5,439,809; 5,439,792; 5,418,136; 5,399,501; 5,397,695;
5,391,479; 5,384,240; 5,374,519; 5,374,518; 5,374,516; 5,364,933;
5,359,046; 5,356,772; 5,354,654; 5,344,755; 5,335,673; 5,332,567;
5,320,940; 5,317,009; 5,312,902; 5,304,466; 5,296,347; 5,286,852;
5,268,265; 5,264,356; 5,264,342; 5,260,308; 5,256,767; 5,256,561;
5,252,556; 5,230,998; 5,230,887; 5,227,159; 5,225,347; 5,221,610
5,217,861; 5,208,321; 5,206,136; 5,198,346; 5,185,147; 5,178,865;
5,173,400; 5,173,399; 5,166,050; 5,156,951; 5,135,864; 5,122,446;
5,120,662; 5,103,836; 5,100,777; 5,100,662; 5,093,230; 5,077,284;
5,070,010; 5,068,174; 5,066,782; 5,055,391; 5,043,262; 5,039,604;
5,039,522; 5,030,718; 5,030,555; 5,030,449; 5,019,387; 5,013,556;
5,008,183; 5,004,697; 4,997,772; 4,983,529; 4,983,387; 4,965,069;
4,945,082; 4,921,787; 4,918,166; 4,900,548; 4,888,290; 4,886,742;
4,885,235; 4,870,003; 4,869,903; 4,861,707; 4,853,326; 4,839,288;
4,833,072 and 4,795,739.
[0040] In a particularly advantageous embodiment, the HIV antigen
is a peptide immunogen, such as but not limited to, 4E10 or 2F5.
The peptide immunogen may comprise a tag, such as but not limited
to, an HA tag or a sequence from the C5 region of HIV g120.
Antibodies against the tag may be used to make the antigen-antibody
complex to present the epitope to the immune system.
[0041] In another embodiment, HIV, or immunogenic fragments
thereof, may be utilized as the HIV antigen may be used to form
antibody-antigen complexes. For example, the HIV nucleotides of
U.S. Pat. Nos. 7,393,949, 7,374,877, 7,306,901, 7,303,754,
7,173,014, 7,122,180, 7,078,516, 7,022,814, 6,974,866, 6,958,211,
6,949,337, 6,946,254, 6,896,900, 6,887,977, 6,870,045, 6,803,187,
6,794,129, 6,773,915, 6,768,004, 6,706,268, 6,696,291, 6,692,955,
6,656,706, 6,649,409, 6,627,442, 6,610,476, 6,602,705, 6,582,920,
6,557,296, 6,531,587, 6,531,137, 6,500,623, 6,448,078, 6,429,306,
6,420,545, 6,410,013, 6,407,077, 6,395,891, 6,355,789, 6,335,158,
6,323,185, 6,316,183, 6,303,293, 6,300,056, 6,277,561, 6,270,975,
6,261,564, 6,225,045, 6,222,024, 6,194,391, 6,194,142, 6,162,631,
6,114,167, 6,114,109, 6,090,392, 6,060,587, 6,057,102, 6,054,565,
6,043,081, 6,037,165, 6,034,233, 6,033,902, 6,030,769, 6,020,123,
6,015,661, 6,010,895, 6,001,555, 5,985,661, 5,980,900, 5,972,596,
5,939,538, 5,912,338, 5,869,339, 5,866,701, 5,866,694, 5,866,320,
5,866,137, 5,864,027, 5,861,242, 5,858,785, 5,858,651, 5,849,475,
5,843,638, 5,840,480, 5,821,046, 5,801,056, 5,786,177, 5,786,145,
5,773,247, 5,770,703, 5,756,674, 5,741,706, 5,705,612, 5,693,752,
5,688,637, 5,688,511, 5,684,147, 5,665,577, 5,585,263, 5,578,715,
5,571,712, 5,567,603, 5,554,528, 5,545,726, 5,527,895, 5,527,894,
5,223,423, 5,204,259, 5,144,019, 5,051,496 and 4,942,122 are useful
for the present invention.
[0042] Any HIV antibody may be used to form antibody-antigen
complexes. For example, the anti-HIV antibodies of U.S. Pat. Nos.
6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743, 6,534,312,
6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646, 6,063,564,
6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304, 5,773,247,
5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009, 4,983,529,
4,886,742, 4,870,003 and 4,795,739 are useful for the present
invention. Furthermore, monoclonal anti-HIV antibodies of U.S. Pat.
Nos. 7,074,556, 7,074,554, 7,070,787, 7,060,273, 7,045,130,
7,033,593, RE39,057, 7,008,622, 6,984,721, 6,972,126, 6,949,337,
6,946,465, 6,919,077, 6,916,475, 6,911,315, 6,905,680, 6,900,010,
6,825,217, 6,824,975, 6,818,392, 6,815,201, 6,812,026, 6,812,024,
6,797,811, 6,768,004, 6,703,019, 6,689,118, 6,657,050, 6,608,179,
6,600,023, 6,596,497, 6,589,748, 6,569,143, 6,548,275, 6,525,179,
6,524,582, 6,506,384, 6,498,006, 6,489,131, 6,465,173, 6,461,612,
6,458,933, 6,432,633, 6,410,318, 6,406,701, 6,395,275, 6,391,657,
6,391,635, 6,384,198, 6,376,170, 6,372,217, 6,344,545, 6,337,181,
6,329,202, 6,319,665, 6,319,500, 6,316,003, 6,312,931, 6,309,880,
6,296,807, 6,291,239, 6,261,558, 6,248,514, 6,245,331, 6,242,197,
6,241,986, 6,228,361, 6,221,580, 6,190,871, 6,177,253, 6,146,635,
6,146,627, 6,146,614, 6,143,876, 6,132,992, 6,124,132, RE36,866,
6,114,143, 6,103,238, 6,060,254, 6,039,684, 6,030,772, 6,020,468,
6,013,484, 6,008,044, 5,998,132, 5,994,515, 5,993,812, 5,985,545,
5,981,278, 5,958,765, 5,939,277, 5,928,930, 5,922,325, 5,919,457,
5,916,806, 5,914,109, 5,911,989, 5,906,936, 5,889,158, 5,876,716,
5,874,226, 5,872,012, 5,871,732, 5,866,694, 5,854,400, 5,849,583,
5,849,288, 5,840,480, 5,840,305, 5,834,599, 5,831,034, 5,827,723,
5,821,047, 5,817,767, 5,817,458, 5,804,440, 5,795,572, 5,783,670,
5,776,703, 5,773,225, 5,766,944, 5,753,503, 5,750,373, 5,747,641,
5,736,341, 5,731,189, 5,707,814, 5,702,707, 5,698,178, 5,695,927,
5,665,536, 5,658,745, 5,652,138, 5,645,836, 5,635,345, 5,618,922,
5,610,035, 5,607,847, 5,604,092, 5,601,819, 5,597,896, 5,597,688,
5,591,829, 5,558,865, 5,514,541, 5,510,264, 5,478,753, 5,374,518,
5,374,516, 5,344,755, 5,332,567, 5,300,433, 5,296,347, 5,286,852,
5,264,221, 5,260,308, 5,256,561, 5,254,457, 5,230,998, 5,227,159,
5,223,408, 5,217,895, 5,180,660, 5,173,399, 5,169,752, 5,166,050,
5,156,951, 5,140,105, 5,135,864, 5,120,640, 5,108,904, 5,104,790,
5,049,389, 5,030,718, 5,030,555, 5,004,697, 4,983,529, 4,888,290,
4,886,742 and 4,853,326, are also useful for the present
invention.
[0043] In another embodiment, the antibody-antigen complexes may be
identified from alternate viral isolates, such as different HIV
clades (see, e.g., U.S. Provisional Patent Application No.
60/810,816, filed Jun. 2, 2006, the disclosure of which is
incorporated by reference). In this embodiment, polyclonal anti-HIV
sera, broadly neutralizing HIV monoclonal antibodies such as, but
not limited to, b12, 2F5, 2G12, 4E10, M2909 either alone or
combination, or newly identified broadly neutralizing antibodies to
HIV are mixed with different HIV clade viral isolates to enable the
antibodies to bind to varying antigens, thereby forming
antibody-antigen complexes.
[0044] The antibody-antigen complexes are dissociated,
advantageously chemically dissociated, preferably by solubilizing
the HIV lipid bilayer, from the virus. In another embodiment, the
antibody-antigen complexes may be dissociated with an affinity
column, such as, but not limited to, C1q, Protein A or Protein G
affinity columns or secondary antibodies.
[0045] Gel filtration is advantageously used to purify
antibody-antigen complexes in the present invention. Gel filtration
is well known in the art and methods of U.S. Pat. Nos. 7,320,893;
7,276,355; 7,101,695; 7,098,026; 6,921,813; 6,812,015; 6,774,220;
6,753,185; 6,627,194; 6,613,564; 6,607,878; 6,600,022; 6,559,298;
6,541,217; 6,395,469; 6,352,723; 6,303,361; 6,274,709; 6,232,089;
6,210,708; 6,207,464; 6,197,297; 6,180,360; 6,156,519; 6,143,875;
6,103,234; 6,060,283; 6,025,165; 5,976,820; 5,942,411; 5,932,705;
5,932,700; 5,912,324; 5,871,936; 5,869,053; 5,856,113; 5,821,061;
5,817,769; 5,807,711; 5,798,445; 5,780,247; 5,721,342; 5,717,074;
5,707,819; 5,696,238; 5,665,864; 5,631,221; 5,629,165; 5,614,612;
5,606,027; 5,599,708; 5,583,199; 5,545,530; 5,523,210; 5,503,828;
5,502,163; 5,496,802; 5,436,319; 5,436,154; 5,338,832; 5,336,491;
5,324,822; 5,284,749; 5,258,324; 5,250,297; 5,234,911; 5,229,110;
5,208,021; 5,200,344; 5,151,266; 5,091,511; 5,089,262; 5,082,928;
5,071,759; 5,068,178; 5,047,503; 5,047,324; 5,037,958; 5,021,560;
5,000,953; RE33,405; 4,962,187; 4,959,320; 4,945,086; 4,916,055;
4,870,162; 4,843,004; 4,833,074; 4,814,433; 4,742,000; 4,686,284;
4,681,761; 4,661,348; 4,594,244; 4,544,640; 4,537,712; 4,532,207;
4,514,506; 4,514,505; 4,489,158; 4,476,093; 4,468,457; 4,446,240;
4,446,122; 4,431,582; 4,414,336; 4,343,734; 4,297,274; 4,232,001;
4,223,002; 4,195,073; 4,160,023; 4,132,769; 4,123,427 and 4,065,445
may be useful for the present invention.
[0046] To purify antibody-antigen complexes, Protein A, Protein G,
precipitating secondary antibodies or Protein A-bearing S. aureus
cells may be used. The affinity of an antibody for Protein A or G
is dependent on the subclass of the immunoglobulin and the species
from which it comes. For example, Protein A is exceptionally well
suited for immunoprecipitation of all rabbit primary antibodies,
but not for chicken antibodies. To use Protein A for
immunoprecipitation of mouse primary antibodies, it is advisable to
add 5 .mu.g of rabbit anti-mouse IgG (secondary precipitating
antibody) prior to the addition of Protein A/G (mix gently, and
incubate for an additional 30 minutes at 4.degree. C. prior to
adding Protein A/G). Following addition of Protein A/G agarose,
incubate with gentle agitation for 30 minutes at 4.degree. C., then
wash at least three times by centrifugation and resuspension in
immunoprecipitation buffer and collect antibody-antigen-Protein A/G
complexes by centrifugation. The purified immune complex may be
used for immunizations or other immunochemical techniques.
[0047] Antibody-antigen complexes as vaccine formulations are known
in the art and the disclosures of any one of Akagaki & Inai
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[0048] The terms "protein", "peptide", "polypeptide", and "amino
acid sequence" are used interchangeably herein to refer to polymers
of amino acid residues of any length. The polymer may be linear or
branched, it may comprise modified amino acids or amino acid
analogs, and it may be interrupted by chemical moieties other than
amino acids. The terms also encompass an amino acid polymer that
has been modified naturally or by intervention; for example
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling or bioactive component.
[0049] As used herein, the terms "antigen" or "immunogen" are used
interchangeably to refer to a substance, typically a protein, which
is capable of inducing an immune response in a subject. The term
also refers to proteins that are immunologically active in the
sense that once administered to a subject (either directly or by
administering to the subject a nucleotide sequence or vector that
encodes the protein) is able to evoke an immune response of the
humoral and/or cellular type directed against that protein.
[0050] The term "antibody" includes intact molecules as well as
fragments thereof, such as Fab, F(ab').sub.2, Fv and scFv which are
capable of binding the epitopic determinant. These antibody
fragments retain some ability to selectively bind with its antigen
or receptor and include, for example:
[0051] (i) Fab, the fragment which contains a monovalent
antigen-binding fragment of an antibody molecule can be produced by
digestion of whole antibody with the enzyme papain to yield an
intact light chain and a portion of one heavy chain;
[0052] (ii) Fab', the fragment of an antibody molecule can be
obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody
molecule;
[0053] (iii) F(ab').sub.2, the fragment of the antibody that can be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held
together by two disulfide bonds;
[0054] (iv) scFv, including a genetically engineered fragment
containing the variable region of a heavy and a light chain as a
fused single chain molecule.
[0055] General methods of making these fragments are known in the
art. (See for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1988), which is
incorporated herein by reference).
[0056] It should be understood that the proteins, including the
antibodies and/or antigens of the invention may differ from the
exact sequences illustrated and described herein. Thus, the
invention contemplates deletions, additions and substitutions to
the sequences shown, so long as the sequences function in
accordance with the methods of the invention. In this regard,
particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a
family of amino acids. For example, amino acids are generally
divided into four families: (1) acidic--aspartate and glutamate;
(2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cystine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. It is reasonably predictable that an isolated replacement of
leucine with isoleucine or valine, or vice versa; an aspartate with
a glutamate or vice versa; a threonine with a serine or vice versa;
or a similar conservative replacement of an amino acid with a
structurally related amino acid, will not have a major effect on
the biological activity. Proteins having substantially the same
amino acid sequence as the sequences illustrated and described but
possessing minor amino acid substitutions that do not substantially
affect the immunogenicity of the protein are, therefore, within the
scope of the invention.
[0057] As used herein the terms "nucleotide sequences" and "nucleic
acid sequences" refer to deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA) sequences, including, without limitation, messenger RNA
(mRNA), DNA/RNA hybrids, or synthetic nucleic acids. The nucleic
acid can be single-stranded, or partially or completely
double-stranded (duplex). Duplex nucleic acids can be homoduplex or
heteroduplex.
[0058] As used herein the term "transgene" may used to refer to
"recombinant" nucleotide sequences that may be derived from any of
the nucleotide sequences encoding the proteins of the present
invention. The term "recombinant" means a nucleotide sequence that
has been manipulated "by man" and which does not occur in nature,
or is linked to another nucleotide sequence or found in a different
arrangement in nature. It is understood that manipulated "by man"
means manipulated by some artificial means, including by use of
machines, codon optimization, restriction enzymes, etc.
[0059] For example, in one embodiment the nucleotide sequences may
be mutated such that the activity of the encoded proteins in vivo
is abrogated. In another embodiment the nucleotide sequences may be
codon optimized, for example the codons may be optimized for human
use. In preferred embodiments the nucleotide sequences of the
invention are both mutated to abrogate the normal in vivo function
of the encoded proteins, and codon optimized for human use. For
example, each of the Gag, Pol, Env, Nef, RT, and Int sequences of
the invention may be altered in these ways.
[0060] As regards codon optimization, the nucleic acid molecules of
the invention have a nucleotide sequence that encodes the antigens
of the invention and can be designed to employ codons that are used
in the genes of the subject in which the antigen is to be produced.
Many viruses, including HIV and other lentiviruses, use a large
number of rare codons and, by altering these codons to correspond
to codons commonly used in the desired subject, enhanced expression
of the antigens can be achieved. In a preferred embodiment, the
codons used are "humanized" codons, i.e., the codons are those that
appear frequently in highly expressed human genes (Andre et al., J.
Virol. 72:1497-1503, 1998) instead of those codons that are
frequently used by HIV. Such codon usage provides for efficient
expression of the transgenic HIV proteins in human cells. Any
suitable method of codon optimization may be used. Such methods,
and the selection of such methods, are well known to those of skill
in the art. In addition, there are several companies that will
optimize codons of sequences, such as Geneart (geneart.com). Thus,
the nucleotide sequences of the invention can readily be codon
optimized.
[0061] The invention further encompasses nucleotide sequences
encoding functionally and/or antigenically equivalent variants and
derivatives of the antigens of the invention and functionally
equivalent fragments thereof. These functionally equivalent
variants, derivatives, and fragments display the ability to retain
antigenic activity. For instance, changes in a DNA sequence that do
not change the encoded amino acid sequence, as well as those that
result in conservative substitutions of amino acid residues, one or
a few amino acid deletions or additions, and substitution of amino
acid residues by amino acid analogs are those which will not
significantly affect properties of the encoded polypeptide.
Conservative amino acid substitutions are glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine;
and phenylalanine/tyrosine/tryptophan. In one embodiment, the
variants have at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
homology or identity to the antigen, epitope, immunogen, peptide or
polypeptide of interest.
[0062] For the purposes of the present invention, sequence identity
or homology is determined by comparing the sequences when aligned
so as to maximize overlap and identity while minimizing sequence
gaps. In particular, sequence identity may be determined using any
of a number of mathematical algorithms. A nonlimiting example of a
mathematical algorithm used for comparison of two sequences is the
algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA
1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc.
Natl. Acad. Sci. USA 1993; 90: 5873-5877.
[0063] Another example of a mathematical algorithm used for
comparison of sequences is the algorithm of Myers & Miller,
CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:
2444-2448.
[0064] Advantageous for use according to the present invention is
the WU-BLAST (Washington University BLAST) version 2.0 software.
WU-BLAST version 2.0 executable programs for several UNIX platforms
can be downloaded from ftp://blast.wustl.edu/blast/executables.
This program is based on WU-BLAST version 1.4, which in turn is
based on the public domain NCBI-BLAST version 1.4 (Altschul &
Gish, 1996, Local alignment statistics, Doolittle cd., Methods in
Enzymology 266: 460-480; Altschul et al., Journal of Molecular
Biology 1990; 215: 403-410; Gish & States, 1993; Nature
Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl. Acad.
Sci. USA 90: 5873-5877; all of which are incorporated by reference
herein).
[0065] The various recombinant nucleotide sequences and antibodies
and/or antigens of the invention are made using standard
recombinant DNA and cloning techniques. Such techniques are well
known to those of skill in the art. See for example, "Molecular
Cloning: A Laboratory Manual", second edition (Sambrook et al.
1989).
[0066] The nucleotide sequences of the present invention may be
inserted into "vectors."The term "vector" is widely used and
understood by those of skill in the art, and as used herein the
term "vector" is used consistent with its meaning to those of skill
in the art. For example, the term "vector" is commonly used by
those skilled in the art to refer to a vehicle that allows or
facilitates the transfer of nucleic acid molecules from one
environment to another or that allows or facilitates the
manipulation of a nucleic acid molecule.
[0067] Any vector that allows expression of the antibodies and/or
antigens of the present invention may be used in accordance with
the present invention. In certain embodiments, the antigens and/or
antibodies of the present invention may be used in vitro (such as
using cell-free expression systems) and/or in cultured cells grown
in vitro in order to produce the encoded HIV-antigens and/or
antibodies which may then be used for various applications such as
in the production of proteinaceous vaccines. For such applications,
any vector that allows expression of the antigens and/or antibodies
in vitro and/or in cultured cells may be used.
[0068] For applications where it is desired that the antibodies
and/or antigens be expressed in vivo, for example when the
transgenes of the invention are used in DNA or DNA-containing
vaccines, any vector that allows for the expression of the
antibodies and/or antigens of the present invention and is safe for
use in vivo may be used. In preferred embodiments the vectors used
are safe for use in humans, mammals and/or laboratory animals.
[0069] For the antibodies and/or antigens of the present invention
to be expressed, the protein coding sequence should be "operably
linked" to regulatory or nucleic acid control sequences that direct
transcription and translation of the protein. As used herein, a
coding sequence and a nucleic acid control sequence or promoter are
said to be "operably linked" when they are covalently linked in
such a way as to place the expression or transcription and/or
translation of the coding sequence under the influence or control
of the nucleic acid control sequence. The "nucleic acid control
sequence" can be any nucleic acid element, such as, but not limited
to promoters, enhancers, IRES, introns, and other elements
described herein that direct the expression of a nucleic acid
sequence or coding sequence that is operably linked thereto. The
term "promoter" will be used herein to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II and that when operationally
linked to the protein coding sequences of the invention lead to the
expression of the encoded protein. The expression of the transgenes
of the present invention can be under the control of a constitutive
promoter or of an inducible promoter, which initiates transcription
only when exposed to some particular external stimulus, such as,
without limitation, antibiotics such as tetracycline, hormones such
as ecdysone, or heavy metals. The promoter can also be specific to
a particular cell-type, tissue or organ. Many suitable promoters
and enhancers are known in the art, and any such suitable promoter
or enhancer may be used for expression of the transgenes of the
invention. For example, suitable promoters and/or enhancers can be
selected from the Eukaryotic Promoter Database (EPDB).
[0070] The vectors used in accordance with the present invention
should typically be chosen such that they contain a suitable gene
regulatory region, such as a promoter or enhancer, such that the
antigens and/or antibodies of the invention can be expressed.
[0071] For example, when the aim is to express the antibodies
and/or antigens of the invention in vitro, or in cultured cells, or
in any prokaryotic or eukaryotic system for the purpose of
producing the protein(s) encoded by that antibody and/or antigen,
then any suitable vector can be used depending on the application.
For example, plasmids, viral vectors, bacterial vectors, protozoal
vectors, insect vectors, baculovirus expression vectors, yeast
vectors, mammalian cell vectors, and the like, can be used.
Suitable vectors can be selected by the skilled artisan taking into
consideration the characteristics of the vector and the
requirements for expressing the antibodies and/or antigens under
the identified circumstances.
[0072] When the aim is to express the antibodies and/or antigens of
the invention in vivo in a subject, for example in order to
generate an immune response against an HIV-1 antigen and/or
protective immunity against HIV-1, expression vectors that are
suitable for expression on that subject, and that are safe for use
in vivo, should be chosen. For example, in some embodiments it may
be desired to express the antibodies and/or antigens of the
invention in a laboratory animal, such as for pre-clinical testing
of the HIV-1 immunogenic compositions and vaccines of the
invention. In other embodiments, it will be desirable to express
the antibodies and/or antigens of the invention in human subjects,
such as in clinical trials and for actual clinical use of the
immunogenic compositions and vaccine of the invention. Any vectors
that are suitable for such uses can be employed, and it is well
within the capabilities of the skilled artisan to select a suitable
vector. In some embodiments it may be preferred that the vectors
used for these in vivo applications are attenuated to vector from
amplifying in the subject. For example, if plasmid vectors are
used, preferably they will lack an origin of replication that
functions in the subject so as to enhance safety for in vivo use in
the subject. If viral vectors are used, preferably they are
attenuated or replication-defective in the subject, again, so as to
enhance safety for in vivo use in the subject.
[0073] In preferred embodiments of the present invention viral
vectors are used. Viral expression vectors are well known to those
skilled in the art and include, for example, viruses such as
adenoviruses, adeno-associated viruses (AAV), alphaviruses,
herpesviruses, retroviruses and poxviruses, including avipox
viruses, attenuated poxviruses, vaccinia viruses, and particularly,
the modified vaccinia Ankara virus (MVA; ATCC Accession No.
VR-1566). Such viruses, when used as expression vectors are
innately non-pathogenic in the selected subjects such as humans or
have been modified to render them non-pathogenic in the selected
subjects. For example, replication-defective adenoviruses and
alphaviruses are well known and can be used as gene delivery
vectors.
[0074] In particularly preferred embodiments adenovirus vectors are
used. Many adenovirus vectors are known in the art and any such
suitable vector my be used. In preferred embodiments the adenovirus
vector used is selected from the group consisting of the Ad5, Ad35,
Ad11, C6, and C7 vectors.
[0075] The sequence of the Adenovirus 5 ("Ad5") genome has been
published. (Chroboezek, J., Bieber, F., and Jacrot, B. (1992) The
Sequence of the Genome of Adenovirus Type 5 and Its Comparison with
the Genome of Adenovirus Type 2, Virology 186, 280-285; the
contents if which is hereby incorporated by reference). Ad35
vectors are described in U.S. Pat. Nos. 6,974,695, 6,913,922, and
6,869,794. Ad11 vectors are described in U.S. Pat. No. 6,913,922.
C6 adenovirus vectors are described in U.S. Pat. Nos. 6,780,407;
6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,942,235
and 5,833,975. C7 vectors are described in U.S. Pat. No.
6,277,558.
[0076] Adenovirus vectors that are E1-defective or deleted,
E3-defective or deleted, and/or E4-defective or deleted may also be
used. Certain adenoviruses having mutations in the E1 region have
improved safety margin because E1-defective adenovirus mutants are
replication-defective in non-permissive cells, or, at the very
least, are highly attenuated. Adenoviruses having mutations in the
E3 region may have enhanced the immunogenicity by disrupting the
mechanism whereby adenovirus down-regulates MHC class I molecules.
Adenoviruses having E4 mutations may have reduced immunogenicity of
the adenovirus vector because of suppression of late gene
expression. Such vectors may be particularly useful when repeated
re-vaccination utilizing the same vector is desired. Adenovirus
vectors that are deleted or mutated in E1, E3, E4, E1 and E3, and
E1 and E4 can be used in accordance with the present invention.
[0077] Furthermore, "gutless" adenovirus vectors, in which all
viral genes are deleted, can also be used in accordance with the
present invention. Such vectors require a helper virus for their
replication and require a special human 293 cell line expressing
both E1a and Cre, a condition that does not exist in natural
environment. Such "gutless" vectors are non-immunogenic and thus
the vectors may be inoculated multiple times for re-vaccination.
The "gutless" adenovirus vectors can be used for insertion of
heterologous inserts/genes such as the transgenes of the present
invention, and can even be used for co-delivery of a large number
of heterologous inserts/genes.
[0078] The nucleotide sequences and vectors of the invention can be
delivered to cells, for example if aim is to express and the HIV-1
antigens in cells in order to produce and isolate the expressed
proteins, such as from cells grown in culture. For expressing the
antibodies and/or antigens in cells any suitable transfection,
transformation, or gene delivery methods can be used. Such methods
are well known by those skilled in the art, and one of skill in the
art would readily be able to select a suitable method depending on
the nature of the nucleotide sequences, vectors, and cell types
used. For example, transfection, transformation, microinjection,
infection, electroporation, lipofection, or liposome-mediated
delivery could be used. Expression of the antibodies and/or
antigens can be carried out in any suitable type of host cells,
such as bacterial cells, yeast, insect cells, and mammalian cells.
The antibodies and/or antigens of the invention can also be
expressed using including in vitro transcription/translation
systems. All of such methods are well known by those skilled in the
art, and one of skill in the art would readily be able to select a
suitable method depending on the nature of the nucleotide
sequences, vectors, and cell types used.
[0079] Following expression, the antibodies and/or antigens of the
invention can be isolated and/or purified or concentrated using any
suitable technique known in the art. For example, anion or cation
exchange chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography,
immuno-affinity chromatography, hydroxyapatite chromatography,
lectin chromatography, molecular sieve chromatography, isoelectric
focusing, gel electrophoresis, or any other suitable method or
combination of methods can be used.
[0080] In preferred embodiments, the nucleotide sequences,
antibodies and/or antigens of the invention are administered in
vivo, for example where the aim is to produce an immunogenic
response in a subject. A "subject" in the context of the present
invention may be any animal. For example, in some embodiments it
may be desired to express the transgenes of the invention in a
laboratory animal, such as for pre-clinical testing of the HIV-1
immunogenic compositions and vaccines of the invention. In other
embodiments, it will be desirable to express the antibodies and/or
antigens of the invention in human subjects, such as in clinical
trials and for actual clinical use of the immunogenic compositions
and vaccine of the invention. In preferred embodiments the subject
is a human, for example a human that is infected with, or is at
risk of infection with, HIV-1.
[0081] For such in vivo applications the nucleotide sequences,
antibodies and/or antigens of the invention are preferably
administered as a component of an immunogenic composition
comprising the nucleotide sequences and/or antigens of the
invention in admixture with a pharmaceutically acceptable carrier.
The immunogenic compositions of the invention are useful to
stimulate an immune response against HIV-1 and may be used as one
or more components of a prophylactic or therapeutic vaccine against
HIV-1 for the prevention, amelioration or treatment of AIDS. The
nucleic acids and vectors of the invention are particularly useful
for providing genetic vaccines, i.e. vaccines for delivering the
nucleic acids encoding the antibodies and/or antigens of the
invention to a subject, such as a human, such that the antibodies
and/or antigens are then expressed in the subject to elicit an
immune response.
[0082] The compositions of the invention may be injectable
suspensions, solutions, sprays, lyophilized powders, syrups,
elixirs and the like. Any suitable form of composition may be used.
To prepare such a composition, a nucleic acid or vector of the
invention, having the desired degree of purity, is mixed with one
or more pharmaceutically acceptable carriers and/or excipients. The
carriers and excipients must be "acceptable" in the sense of being
compatible with the other ingredients of the composition.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to, water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, or combinations thereof, buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as scrum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0083] An immunogenic or immunological composition can also be
formulated in the form of an oil-in-water emulsion. The
oil-in-water emulsion can be based, for example, on light liquid
paraffin oil (European Pharmacopea type); isoprenoid oil such as
squalane, squalene, EICOSANE.TM. or tetratetracontane; oil
resulting from the oligomerization of alkene(s), e.g., isobutene or
decene; esters of acids or of alcohols containing a linear alkyl
group, such as plant oils, ethyl oleate, propylene glycol
di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene
glycol dioleate; esters of branched fatty acids or alcohols, e.g.,
isostearic acid esters. The oil advantageously is used in
combination with emulsifiers to form the emulsion. The emulsifiers
can be nonionic surfactants, such as esters of sorbitan, mannide
(e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene
glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid,
which are optionally ethoxylated, and
polyoxypropylene-polyoxyethylene copolymer blocks, such as the
Pluronic.RTM. products, e.g., L121. The adjuvant can be a mixture
of emulsifier(s), micelle-forming agent, and oil such as that which
is commercially available under the name Provax.RTM. (IDEC
Pharmaceuticals, San Diego, Calif.).
[0084] The immunogenic compositions of the invention can contain
additional substances, such as wetting or emulsifying agents,
buffering agents, or adjuvants to enhance the effectiveness of the
vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Company, (ed.) 1980).
[0085] Adjuvants may also be included. Adjuvants include, but are
not limited to, mineral salts (e.g., AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH(SO.sub.4).sub.2, silica, alum,
Al(OH).sub.3, Ca.sub.3(PO.sub.4).sub.2, kaolin, or carbon),
polynucleotides with or without immune stimulating complexes
(ISCOMs) (e.g., CpG oligonucleotides, such as those described in
Chuang, T. H. et al, (2002) J. Leuk. Biol. 71(3): 538-44;
Ahmad-Nejad, P. et al (2002) Eur. J. Immunol. 32(7): 1958-68; poly
IC or poly AU acids, polyarginine with or without CpG (also known
in the art as IC.sub.31; see Schellack, C. et al (2003) Proceedings
of the 34.sup.th Annual Meeting of the German Society of
Immunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508),
JuvaVax.TM. (U.S. Pat. No. 6,693,086), certain natural substances
(e.g., wax D from Mycobacterium tuberculosis, substances found in
Cornyebacterium parvum, Bordetella pertussis, or members of the
genus Brucella), flagellin (Toll-like receptor 5 ligand; see
McSorley, S. J. et al (2002) J. Immunol. 169(7): 3914-9), saponins
such as QS21, QS17, and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398;
6,524,584; 6,645,495), monophosphoryl lipid A, in particular,
3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also
known in the art as TQM and commercially available as Aldara.RTM.;
U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, A. K. et al (2004)
22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.
S. et al (2003) J. Exp. Med. 198: 1551-1562).
[0086] Aluminum hydroxide or phosphate (alum) are commonly used at
0.05 to 0.1% solution in phosphate buffered saline. Other adjuvants
that can be used, especially with DNA vaccines, are cholera toxin,
especially CTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J.
Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, H. R. (1998)
App. Organometallic Chem. 12(10-11): 659-666; Payne, L. G. et al
(1995) Pharm. Biotechnol. 6: 473-93), cytokines such as, but not
limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN-.alpha.,
IFN-.beta., and IFN-.gamma. (Boyer et al., (2002) J. Liposome Res.
121:137-142; WO01/095919), immunoregulatory proteins such as CD40L
(ADX40; see, for example, WO03/063899), and the CD1a ligand of
natural killer cells (also known as CRONY or .alpha.-galactosyl
ceramide; see Green, T. D. et al, (2003) J. Virol. 77(3):
2046-2055), immunostimulatory fusion proteins such as IL-2 fused to
the Fc fragment of immunoglobulins (Barouch et al., Science
290:486-492, 2000) and co-stimulatory molecules B7.1 and B7.2
(Boyer), all of which can be administered either as proteins or in
the form of DNA, on the same expression vectors as those encoding
the antigens of the invention or on separate expression
vectors.
[0087] In an advantageous embodiment, the adjuvants may be lecithin
is combined with an acrylic polymer (Adjuplex-LAP), lecithin coated
oil droplets in an oil-in-water emulsion (Adjuplex-LE) or lecithin
and acrylic polymer in an oil-in-water emulsion (Adjuplex-LAO)
(Advanced BioAdjuvants (ABA)).
[0088] The immunogenic compositions can be designed to introduce
the antibodies, antigens, antibody-antigen complexes, nucleic acids
or expression vectors to a desired site of action and release it at
an appropriate and controllable rate. Methods of preparing
controlled-release formulations are known in the art. For example,
controlled release preparations can be produced by the use of
polymers to complex or absorb the immunogen and/or immunogenic
composition. A controlled-release formulations can be prepared
using appropriate macromolecules (for example, polyesters,
polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate)
known to provide the desired controlled release characteristics or
release profile. Another possible method to control the duration of
action by a controlled-release preparation is to incorporate the
active ingredients into particles of a polymeric material such as,
for example, polyesters, polyamino acids, hydrogels, polylactic
acid, polyglycolic acid, copolymers of these acids, or ethylene
vinylacctate copolymers. Alternatively, instead of incorporating
these active ingredients into polymeric particles, it is possible
to entrap these materials into microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsule and
poly-(methylmethacrylate) microcapsule, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in New Trends and
Developments in Vaccines, Voller et al. (eds.), University Park
Press, Baltimore, Md., 1978 and Remington's Pharmaceutical
Sciences, 16th edition.
[0089] Suitable dosages of the antibodies, antigens,
antibody-antigen complexes, nucleic acids and expression vectors of
the invention (collectively, the immunogens) in the immunogenic
composition of the invention can be readily determined by those of
skill in the art. For example, the dosage of the immunogens can
vary depending on the route of administration and the size of the
subject. Suitable doses can be determined by those of skill in the
art, for example by measuring the immune response of a subject,
such as a laboratory animal, using conventional immunological
techniques, and adjusting the dosages as appropriate. Such
techniques for measuring the immune response of the subject include
but are not limited to, chromium release assays, tetramer binding
assays, IFN-.gamma. ELISPOT assays, IL-2 ELISPOT assays,
intracellular cytokine assays, and other immunological detection
assays, e.g., as detailed in the text "Antibodies: A Laboratory
Manual" by Ed Harlow and David Lane.
[0090] Previous attempts to elicit an effective neutralizing
response from antigen-antibody complexes have failed, in particular
an Env gp120-antibody A32 complex (see, e.g., Liao et al., J.
Virol. 2004 May; 78(10):5270-8). One hypothesis as to the failure
is the high dose of env (100 to 200 .mu.g) in the gp12-32 complex.
Advantageously, a lower dose of env is contemplated, such as about
1 .mu.g to about 50 .mu.g, about 2.5 .mu.g to about 40 .mu.g, about
5 .mu.g to about 30 .mu.g, about 7.5 .mu.g to about 20 .mu.g,
preferably about 10 .mu.g to about 15 .mu.g of env when env is the
antigen in the antigen-antibody complex.
[0091] When provided prophylactically, the immunogenic compositions
of the invention are ideally administered to a subject in advance
of HIV infection, or evidence of HIV infection, or in advance of
any symptom due to AIDS, especially in high-risk subjects. The
prophylactic administration of the immunogenic compositions can
serve to provide protective immunity of a subject against HIV-1
infection or to prevent or attenuate the progression of AIDS in a
subject already infected with HIV-1. When provided therapeutically,
the immunogenic compositions can serve to ameliorate and treat AIDS
symptoms and are advantageously used as soon after infection as
possible, preferably before appearance of any symptoms of AIDS but
may also be used at (or after) the onset of the disease
symptoms.
[0092] The immunogenic compositions can be administered using any
suitable delivery method including, but not limited to,
intramuscular, intravenous, intradermal, mucosal, and topical
delivery. Such techniques are well known to those of skill in the
art. More specific examples of delivery methods are intramuscular
injection, intradermal injection, and subcutaneous injection.
However, delivery need not be limited to injection methods.
Further, delivery of DNA to animal tissue has been achieved by
cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev.
38:268-274; and WO 96/20013), direct injection of naked DNA into
animal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;
Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)
Virology 199: 132-140; Webster et al., (1994) Vaccine 12:
1495-1498; Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et
al., (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection
of DNA using "gene gun" technology (Johnston et al., (1994) Meth.
Cell Biol. 43:353-365). Alternatively, delivery routes can be oral,
intranasal or by any other suitable route. Delivery also be
accomplished via a mucosal surface such as the anal, vaginal or
oral mucosa.
[0093] Immunization schedules (or regimens) are well known for
animals (including humans) and can be readily determined for the
particular subject and immunogenic composition. Hence, the
immunogens can be administered one or more times to the subject.
Preferably, there is a set time interval between separate
administrations of the immunogenic composition. While this interval
varies for every subject, typically it ranges from 10 days to
several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the
interval is typically from 2 to 6 weeks. The immunization regimes
typically have from 1 to 6 administrations of the immunogenic
composition, but may have as few as one or two or four. The methods
of inducing an immune response can also include administration of
an adjuvant with the immunogens. In some instances, annual,
biannual or other long interval (5-10 years) booster immunization
can supplement the initial immunization protocol.
[0094] The present methods also include a variety of prime-boost
regimens, especially DNA prime-Adenovirus boost regimens. In these
methods, one or more priming immunizations are followed by one or
more boosting immunizations. The actual immunogenic composition can
be the same or different for each immunization and the type of
immunogenic composition (e.g., containing protein or expression
vector), the route, and formulation of the immunogens can also be
varied. For example, if an expression vector is used for the
priming and boosting steps, it can either be of the same or
different type (e.g., DNA or bacterial or viral expression vector).
One useful prime-boost regimen provides for two priming
immunizations, four weeks apart, followed by two boosting
immunizations at 4 and 8 weeks after the last priming immunization.
It should also be readily apparent to one of skill in the art that
there are several permutations and combinations that are
encompassed using the DNA, bacterial and viral expression vectors
of the invention to provide priming and boosting regimens.
[0095] A specific embodiment of the invention provides methods of
inducing an immune response against HIV in a subject by
administering an immunogenic composition of the invention,
preferably comprising an adenovirus vector containing DNA encoding
one or more of the antibodies, antigens, antibody-antigen complexes
of the invention, one or more times to a subject wherein the
antibodies, antigens, antibody-antigen complexes are expressed at a
level sufficient to induce a specific immune response in the
subject. Such immunizations can be repeated multiple times at time
intervals of at least 2, 4 or 6 weeks (or more) in accordance with
a desired immunization regime.
[0096] The immunogenic compositions of the invention can be
administered alone, or can be co-administered, or sequentially
administered, with other HIV immunogens and/or HIV immunogenic
compositions, e.g., with "other" immunological, antigenic or
vaccine or therapeutic compositions thereby providing multivalent
or "cocktail" or combination compositions of the invention and
methods of employing them. Again, the ingredients and manner
(sequential or co-administration) of administration, as well as
dosages can be determined taking into consideration such factors as
the age, sex, weight, species and condition of the particular
subject, and the route of administration.
[0097] When used in combination, the other HIV immunogens can be
administered at the same time or at different times as part of an
overall immunization regime, e.g., as part of a prime-boost regimen
or other immunization protocol. In an advantageous embodiment, the
other HIV immunogen is env, preferably the HIV env trimer.
[0098] Many other HIV immunogens are known in the art, one such
preferred immunogen is HIVA (described in WO 01/47955), which can
be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in
a viral vector (e.g., MVA.HIVA). Another such HIV immunogen is
RENTA (described in PCT/US2004/037699), which can also be
administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a
viral vector (e.g., MVA.RENTA).
[0099] For example, one method of inducing an immune response
against HIV in a human subject comprises administering at least one
priming dose of an HIV immunogen and at least one boosting dose of
an HIV immunogen, wherein the immunogen in each dose can be the
same or different, provided that at least one of the immunogens is
an antibody, antigen or antibody-antigen complex of the present
invention, a nucleic acid encoding an antibody, antigen or
antibody-antigen complex of the invention or an expression vector,
preferably an adenovirus vector, encoding an antibody, antigen or
antibody-antigen complex of the invention, and wherein the
immunogens are administered in an amount or expressed at a level
sufficient to induce an HIV-specific immune response in the
subject. The HIV-specific immune response can include an
HIV-specific T-cell immune response or an HIV-specific B-cell
immune response. Such immunizations can be done at intervals,
preferably of at least 2-6 or more weeks.
[0100] It is to be understood and expected that variations in the
principles of invention as described above may be made by one
skilled in the art and it is intended that such modifications,
changes, and substitutions are to be included within the scope of
the present invention.
[0101] The invention will now be further described by way of the
following non-limiting examples.
Example
HIV Envelope Immune Complexes as a Vaccine Candidate
[0102] Immune complexes (ICs) of protein antigen and specific
antibodies can markedly enhance the immunogenicity of the antigen
(Roosnek and Lanzavecchia J Exp Med 173:487-489) by presumably
providing the adjuvant effect and better presentation of the
antigen to the antigen presenting cells.
[0103] The advantages of immune complexes include, but are not
limited to:
[0104] 1. Immune complexes are efficiently taken up by specialized
antigen-presenting cells (dendritic cells (DC)) via Fc
receptors,
[0105] 2. Binding of ICs to Fc receptors can mediate dendritic cell
maturation serving as a natural adjuvant,
[0106] 3. Immune complex increase the germinal center formation and
thus could effect the quality of antibody formation,
[0107] 4. Antibody binding to the antigen could potentially present
the cryptic epitopes by masking the immunodominant epitopes and
[0108] 5. Alteration of proteolysis of the antigen by formation of
the immune complex could alter the presentation to CD4 T-cell,
[0109] In the veterinary immune complex vaccine follicular
dendritic cells and B-lymphocytes were rescued from depletion
either by protection of the lymphocyte against the lytic effect of
the virus or by maintaining a more intact microenvironment needed
by FDC. The veterinary immune complex vaccine also induced
formation of germinal centers containing the Immune complex in
spleen. Immunization with Immune complexes accelerated the
development of memory B-cells and affinity maturation of antibodies
compared to antigen alone. The repertoire of antigen reactive
B-cells in immune complex immunization showed presence of
heterogenous VH gene expression while in antigen alone immunization
only single variable gene was observed.
[0110] Initially gp120 and gp120 complexes were tested with one
broad neutralizing antibody b12 and one non neutralizing antibody
39F. This line of experimentation helps determine the baseline
activity upon immunization.
[0111] The experimental steps were as follows:
[0112] 1. Determining YU2 gp120 run profile through a gel
filtration chromatography,
[0113] 2. Making Yu2gp120 and monoclonal antibody complexes and
purify on Ag-Ab complex by gel filtration,
[0114] 3. Characterizing the stochiometry of Ag-Ab by blue native
and denaturing PAGE,
[0115] 4. Immunizing rabbits intramuscularly with gp120 and
gp120-Ab complexes at 13 ug Env dose with adjuplex adjuvant and
[0116] 5. Evaluating the sera for binding and neutralization
assay.
[0117] The present study was performed with immune complexes of HIV
envelope YU2 gp120 and antibodies b12, a CD4 binding site broad
neutralizing antibody and 39F, a variable loop 3 specific antibody
on gp120.
[0118] The rationale for using b12 antibody to make Yu2gp120-b12
immune complex was:
[0119] 1. B12 Immune complexes would be efficiently taken up by
specialized antigen-presenting cells (dendritic cells (DC)) via Fc
receptors.
[0120] 2. B12 antibody binding region is crucial for the viral
entry and preservation of this site by making a complex with the
antibody is desirable and
[0121] 3. Binding of b12 to gp120 does not lead to major
conformational changes as measured by isothermal calorimetry
potentially allowing stabilization of the Env gp120 in one fixed
state. Env gp120 is highly flexible molecule and fixing in one
state is presumed to be better for the immunogenic property.
[0122] The binding of Fc receptor but not complement to antibody
b12 was shown to be important for anti-HIV activity.
[0123] The rationale for using 39F antibody to make Yu2gp120-b12
immune complex was:
[0124] 1. 39F Immune complexes would be efficiently taken up by
specialized antigen-presenting cells (dendritic cells (DC)) via Fc
receptors,
[0125] 2. 39F antibody binding region (Variable loop 3) is
immunidominant, masking of the immunodominant and strain specific
epitope and would support the presentation of cryptic or
immunosilent epitope and
[0126] 3. Binding of 39F to gp120 potentially allows stabilization
of the Env gp120 in one fixed state. Env gp120 is highly flexible
molecule and fixing in one state is presumed to be better for the
immunogenic property.
TABLE-US-00001 TABLE 1 Soluble gp120 YU2 complexes Antigen Antibody
Binding YU2 gp120 39F Binds V3 loop YU2 gp120 b12 CD4 Binding
Site
[0127] Yu2 gp120+b12 and Yu2 gp120+39F immune complexes were
generated and purified at a molar ratio of 1:1 Env to antibody
molecules pushed all the envelope molecules into complex formation
(FIG. 1). The immune complexes were stable when stored at 4 C for
days and at -70 C for days. The b12 immune complexes were of one
type 1:1 of gp120-b12 molecule, where as the 39F immune complexes
showed presence of two forms presumably 1:1 and 1:2 of gp120-39F
molecules (FIG. 2). The immune complexes were analyzed on reducing
PAGE for stochiometry and purity and equal amounts of Env (gp120)
and antibody (heavy and light chains) were observed (FIG. 3).
Approximately 10-15 ug of gp120 equivalent of Env or Env-Ab Immune
complexes were immunized in rabbits at 0, 4, 8 and 12 weeks with
and with out adjuvant Adjuplex LAP. Bleeds were collected at 2, 6,
10 and 14 weeks and analyzed by ELISA and neutralization assay.
After a single priming the immune complex group with adjuvant
showed formation of anti-Env antibodies at a titer of 6400 (FIG.
4). Following one prime and one boost anti-Env titer reached 105
for most animals in the immune complex group with adjuvant whereas
all other groups had very low titer antibodies. These sera were
further tested for neutralization ability against 10 different
HIV-1 isolates. The env alone group with adjuvant showed low titer
neutralization of easy to neutralize SF162 and NL4-3 viruses,
whereas the immune complex group with showed neutralization of good
neutralization Bal, BX08, SF162 and NL4-3, a few animals in the
immune complex group also neutralized HT593, JRCSF and BRO20
viruses.
[0128] The use of adjuvant adjuplex helped the immune complex group
significant as is seen by comparing the immune complex with and
with out adjuvant group. This adjuvant helps generation of high
titer ELISA antibodies in quick and durable fashion as has been
seen in case of Env gp140 and env gp120. Env alone group with
adjuplex showed generation of good titer anti-Env antibodies only
after one prime and two or three boost but showed neutralizing
antibodies only against SF162 and NL4-3, suggesting the importance
of immune complex in generating high titer neutralizing antibodies
with breadth of neutralization.
[0129] Experiments with Soluble HIV Env trimer. The data and assay
conditions so obtained from the above experiment are used generate
Ag-Ab complexes with HIV Env soluble gp140 molecules. For soluble
HIV-1 gp140 molecules, broad neutralizing antibodies b12 and 2F5
and non neutralizing antibody 39F directed to variable loop 3 on
gp120 are used.
[0130] Experimental steps include:
[0131] 1. Determining YU2 gp140 or JRCSF gp140 run profile through
a gel filtration chromatography,
[0132] 2. Making Yu2/JRCSF gp140 and monoclonal antibody complexes
and purify on Ag-Ab complex by gel filtration,
[0133] 3. Characterizing the stochiometry of Ag-Ab by blue native
and denaturing PAGE,
[0134] 4. Immunizing rabbits intramuscularly with gp140 and
gp140-Ab complexes at different dose with adjuplex adjuvant and
[0135] 5. Evaluating the sera for binding and neutralization
assay.
[0136] Experiment with membrane associated Envelope Trimer. HIV-1
virion treated with aldrithiol-2 inactivates the virus by
covalently modifying the essential zinc finger motifs in the
nucleocapsid protein (NC7). Interestingly, such AT-2 inactivated
viruses preserve the conformational integrity of the virion surface
proteins. The functional viral spike is membrane associated and
presumably the best trimer; in this regard, inactivated HIV-1
virion is used to make membrane associated trimer to form ICs with
2909 (trimer specific), b12 (CD4 Binding site) and V3 specific
antibodies.
[0137] A low dose of Env is important as immune complexes have
various regulatory functions and a high dose of Env may lead to
immunopathogenesis.
[0138] Biophysical Characterization of Immune Complexes as HIV-1
Vaccine Candidate. A protective vaccine against HIV-1 remains
elusive and the conventional methods of live attenuated or
inactivated virus vaccine raise significant safety concerns.
Subunit vaccines containing HIV-1 envelope glycoprotein elicit
neutralizing antibodies limited to the virus strains from which the
immunogen is designed and is thus inadequate as a vaccine
candidate. Immune complexes may be utilized as a candidate HIV
vaccine immunogen. Soluble YU2 gp120 (Clade B Envelope protein)
complexed with b12, a broadly neutralizing antibody against the CD4
binding site, and separately with 39F, a non-neutralizing antibody
against the immunodominant V3 loop were generated. The complexes
were purified by size exclusion chromatography on Superdex 200
column and characterized by SDS PAGE and dynamic light scattering.
To determine the size of the complexes and individual components,
size exclusion chromatography on Superose 12 column coupled to AKTA
UV detector, miniDawn Treos with WyattQELS and OptiLab rEX was
used. All parameters were calculated by using Astra 5.3.2 software.
For highly glycosylated HIV Envelope proteins, a three-detector
method was used. Serum from rabbits immunized with ICs contained
significant anti-Envelope binding and neutralizing antibody titers.
Suboptimal concentrations of the envelope in the immune complex
elicited significant neutralizing antibody responses against a
panel of clad B HIV viruses tested. Immune complexes evoked
neutralizing activity significantly more potent than the control
envelope alone group which was mapped to the variable loop 3 of the
HIV envelope glycoprotein. On-going studies may determine the role
of the Fey portion of antibody and the size of the complex in the
immune potentiation.
[0139] SF162 Neutralization Titer: IC.sub.50 and 90 Values.
Neutralization IC.sub.50 and IC.sub.90 values of immune complex
derived rabbit sera against HIV-1 clade B SF162 virus. As shown in
Table 2, Immune complex group sera has significant IC.sub.90 titer
against SF162 following a single prime and boost immunization.
TABLE-US-00002 TABLE 2 SF162 Neutralization Titer: IC50 and 90
Values ##STR00001## ##STR00002## Responding animals ##STR00003##
##STR00004## ##STR00005##
[0140] Rabbit Sera Neutralization:IC.sub.50 value. Table 3 depicts
neutralization IC.sub.50 value of immune complex derived rabbit
sera against a panel of 10 clade B HIV-1 viruses. Immune complex
derived sera neutralizes six out of 10 viruses tested.
TABLE-US-00003 TABLE 3 Rabbit Sera Neutralization: IC50 value
##STR00006## Responding animals ##STR00007## ##STR00008##
##STR00009## ##STR00010##
[0141] Mapping of the sera for binding and neutralizing antibodies.
Table 4 depicts the variable loop 3 sequence of al the viruses
neutralized by immune complex rabbit sera.
TABLE-US-00004 TABLE 4 Mapping of the sera for binding and
neutralizing antibodies base/ Stem/ Tip /stem /base Virus V3
Sequence Immunogen YU2 CTRPNNNTRKSINI--GPGRALYTTGEIIGDIRQAHC SF162
CTRPNNNTRKSITI--GPGRAFYATGGIIGDIRQAHC Bx08
CTRPNNNTRKSIHI--GPGRAFYTTGDIIGDIRQAHC HT593
CTRPNNNTSKRISI--GPGRAFRAT-KIIGNIRQAHC Bal
CTRPNNNTRKSIHI--GPGRALYTTGEIIGDIRQAHC BR020
CTRPNNNTRKSIHI--GPGRAFYATGDIIGDIRQAHC NL4-3
CTRPNNNTRKSIRIQRGPGRAFVTIGKI-GNMRQAHC
[0142] YU2 V3 Peptide Absorption of Neutralizing Ability. Table 5
depicts absorption of neutralization activity by YU2 V3 peptide for
a) anti-V3 monoclonal antibody 447-52D, b) a control human sera
with broad neutralization specificity c) YU2 gp120 generated rabbit
sera and d) Immune complex generated sera.
TABLE-US-00005 TABLE 5 YU2 V3 Peptide Absorption of Neutralizing
Ability ##STR00011## ##STR00012## ##STR00013## 50 ug/ml V3 peptide
added along with the sera and neutralization performed in a 10
point dilution to determine IC50 value
[0143] Size of Yu2 gp120 and Immune complexes. Table 6 depicts the
size (hydrodynamic radius) as measured by dynamic light scatter for
Fab, IgG and Env immune complexes.
TABLE-US-00006 TABLE 6 Size of Yu2 gp120 and Immune complexes
Protein or Rh (nm) from Rf (ml) of the complex SEC-QELS Mw (kDa)
major peak b12 Fab 3.8 50.7 15.5 b12 5.5 144.6 13.0 YU2 gp120 5.2
9.99{circumflex over ( )} 12.6 (55.6 + 44.3) YU2 gp120 + 6.1 11.9
(complex) b12 Fab 15.5 (Fab) YU2 gp120 + 7.5 10.2 b12 IgG Rh =
Hydodynamic radius; SEC-QELS Size exclusion Chromatography--Quasi
Elastic Light scattering; Mw: = Molecular weight; Rf = retention
factor.
[0144] The invention is further described by the following numbered
paragraphs:
[0145] 1. A method of producing an immune response comprising
administering to a mammal a purified antibody-antigen complex
dissociated from polyclonal anti-HIV sera bound to glycoprotein
spikes on HIV envelopes.
[0146] 2. A method of producing an immune response comprising
administering to a mammal a purified antibody-antigen complex
dissociated from a mixture of broadly neutralizing antibodies and
HIV, wherein the mixture is bound to glycoprotein spikes on HIV
envelopes.
[0147] 3. The method of paragraph 2 wherein the antibodies are
monoclonal antibodies.
[0148] 4. The method of paragraph 3 wherein the monoclonal
antibodies are b12, 2F5, 2G12, 4E10, M2909 or any combination
thereof.
[0149] 5. The method of any one of paragraphs 2-4 wherein the HIV
is purified HIV.
[0150] 6. The method of any one of paragraphs 2-4 wherein the HIV
is a HIV viral isolate.
[0151] 7. The method of paragraph 6 wherein the HIV viral isolate
is a HIV clade viral isolate.
[0152] 8. The method of any one of paragraphs 1-7 wherein the
purified antibody-antigen complex is chemically dissociated from
the glycoprotein spikes.
[0153] 9. The method of any one of paragraphs 1-8 wherein the
purified antibody-antigen complex is dissociated from the
glycoprotein spikes by solubilizing a HIV lipid bilayer.
[0154] 10. The method of any one of paragraphs 1-7 wherein the
purified antibody-antigen complex is purified with Protein A,
protein G, precipitating secondary antibodies or Protein A-bearing
S. aureus cells.
[0155] 11. The method of any one of paragraphs 1-10 wherein the
mammal is a human.
[0156] 12. The method of any one of paragraphs 1-11 wherein the
purified antibody-antigen complex is administered in a
pharmaceutically acceptable carrier.
[0157] 13. The method of any one of paragraphs 1-12 wherein the
administering further comprises a prime-boost regimen.
[0158] 14. A method of producing an immune response comprising
administering to a mammal an antibody-antigen complex, wherein the
antigen is an HIV envelope protein.
[0159] 15. The method of paragraph 14 wherein the HIV envelope
protein is gp120, gp140 or a membrane associated envelope
trimer.
[0160] 16. The method of any one of paragraphs 14-15 wherein the
antibody is a broad neutralizing antibody.
[0161] 17. The method of paragraph 16 wherein the broad
neutralizing antibody is antibody b12.
[0162] 18. The method of any one of paragraphs 14-15 wherein the
antibody is a non-neutralizing antibody.
[0163] 19. The method of paragraph 18 wherein the non-neutralizing
antibody is a V3 specific antibody.
[0164] 20. The method of paragraph 19 wherein the non-neutralizing
antibody is antibody 39F.
[0165] 21. The method of paragraph 15 wherein the HIV envelope
protein is a membrane associated envelope trimer and the antibody
is a trimer specific antibody 2909.
[0166] 22. The method of any one of paragraphs 14-21 wherein the
antibody-antigen complex is purified by gel filtration.
[0167] 23. The method of any one of paragraphs 14-22 wherein the
mammal is a human.
[0168] 24. The method of any one of paragraphs 14-23 wherein the
antibody-antigen complex is administered in a pharmaceutically
acceptable carrier.
[0169] 25. The method of any one of paragraphs 14-24 further
comprising an adjuvant.
[0170] 26. The method of paragraph 25 wherein the adjuvant is
Adjuplex LAP.
[0171] 27. The method of any one of paragraphs 14-26 wherein the
dosage of the HIV envelope protein is about 10 .mu.g to about 15
.mu.g.
[0172] 28. The method of any one of paragraphs 14-27 wherein the
administering further comprises a prime-boost regimen.
[0173] 29. The method of any one of paragraphs 14-28 wherein the
antibody-antigen complex is expressed in a viral vector.
[0174] 30. The method of paragraph 29 wherein the antibody-antigen
complex is expressed in vivo.
[0175] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
7135PRTHuman immunodeficiency virus 1Cys Thr Arg Pro Asn Asn Asn
Thr Arg Lys Ser Ile Asn Ile Gly Pro1 5 10 15Gly Arg Ala Leu Tyr Thr
Thr Gly Glu Ile Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys
35235PRTHuman immunodeficiency virus 2Cys Thr Arg Pro Asn Asn Asn
Thr Arg Lys Ser Ile Thr Ile Gly Pro1 5 10 15Gly Arg Ala Phe Tyr Ala
Thr Gly Gly Ile Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys
35335PRTHuman immunodeficiency virus 3Cys Thr Arg Pro Asn Asn Asn
Thr Arg Lys Ser Ile His Ile Gly Pro1 5 10 15Gly Arg Ala Phe Tyr Thr
Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys
35434PRTHuman immunodeficiency virus 4Cys Thr Arg Pro Asn Asn Asn
Thr Ser Lys Arg Ile Ser Ile Gly Pro1 5 10 15Gly Arg Ala Phe Arg Ala
Thr Lys Ile Ile Gly Asn Ile Arg Gln Ala 20 25 30His Cys535PRTHuman
immunodeficiency virus 5Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser
Ile His Ile Gly Pro1 5 10 15Gly Arg Ala Leu Tyr Thr Thr Gly Glu Ile
Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys 35635PRTHuman
immunodeficiency virus 6Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser
Ile His Ile Gly Pro1 5 10 15Gly Arg Ala Phe Tyr Ala Thr Gly Asp Ile
Ile Gly Asp Ile Arg Gln 20 25 30Ala His Cys 35736PRTHuman
immunodeficiency virus 7Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser
Ile Arg Ile Gln Arg1 5 10 15Gly Pro Gly Arg Ala Phe Val Thr Ile Gly
Lys Ile Gly Asn Met Arg 20 25 30Gln Ala His Cys 35
* * * * *