Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
ANTIGEN - ANTIBODY COMPLEXES AS HIV-1 VACCINES
INCORPORATION BY REFERENCE
This application is a continuation in part of U.S. non-provisional application
serial no.
11/929,015 filed on 30 October 2007 and PCT International Application No.
PCT/US2007/083006 filed on 30 October 2007, which claims priority to U.S.
provisional
application serial no. 60/855,625, filed on 30 October 2006. This application
also claims
priority to U.S. provisional application serial no. 61/035,653, filed on 11
March 2008.
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
The present invention relates to antigen-antibody complexes for use as
prophylactic
and therapeutic vaccines for infectious diseases of AIDS.
BACKGROUND OF THE INVENTION
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 (SW). 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.
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
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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.
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 (gpl60) and cleaved by
a cellular
protease to yield the external 120-kDa envelope glycoprotein (gp 120) and the
transmembrane
41 -kDa envelope glycoprotein (gp4 1), 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 gpl20 and gp4l. 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.
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 gpl20 expressed on the surface of infected cells. In this case,
the uninfected cells
bind, via the CD4 receptor-gp 120 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
2
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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.
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 gpl20 and gp4l subunits (Burton et al., Nat Immunol. 2004
Mar;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).
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
Mar;5(3):233-
6). However, none of these approaches have yet efficiently elicited
neutralizing antibodies
with broad specificity.
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
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.
3
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WO 2009/058989 PCT/US2008/081769
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.
In an advantageous embodiment, the antigen-antibody complex is an envelope
protein
(such as, but not limited to, gp 120, gp 140 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.
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.
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.
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.
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.
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-
4
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HIV sera, broadly neutralizing HIV monoclonal antibodies such as, but not
limited to, b 12,
2F5, 2G12, 4E10, M2909 either alone or combination, or newly identified
broadly
neutralizing antibodies to HIV are mixed with different HIV Glade 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.
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.
These and other embodiments are disclosed or are obvious from and encompassed
by,
the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
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:
FIG. 1 depicts immune complexes of YU2 gp120 - B12 run on a gel filtration
column.
FIG. 2 depicts an analysis of immune complexes before and after gel
filtration.
FIG. 3 depicts gel-filtration purified complexes on reducing SDS-PAGE where
lane 1
is b12, lane 2 is YU2 gpl20, lane 3 is YU2 gpl20 + b12 and lane 4 is YU2 gpl20
+ 39F.
FIG. 4 depicts an antibody response to immune-complex by single prime-boost
immunization.
FIG. 5 depicts a mean titer: anti- env gp 120 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.
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 Cl and C5 region of YU2 gpl20)]. Except for IgG
b12 site
5
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WO 2009/058989 PCT/US2008/081769
which is occupied in the immune complex all the other antibodies showed
comparable
binding to both Yu2 gp 120 and YU2 gp 120-IgG b 12 immune complex.
FIGS. 7A and 7B depicts generation of YU2 gp120 immuen 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 gpl20 with Fab b12 and IgG b12
immune
complexes were generated and characterized (run and coommassie stained FIG.
7A). When
immunized in rabbits the IgG b 12 immune complex faired better in eliciting
anti-gp 120 titer
(graph in 7B) than the Fab b 12 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.
FIG. 8 depicts ELISA anti-gp120 titer for JRCSF gp10 and JRCSF-gpl20-Fc fusion
protein (see, e.g., Binley JM 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.
FIG. 9 depicts gel-filtration purified YU2 gpl40 immune complexes where lane 1
is
marker, lane 2 is IgG b12, lane 3 is IgG 39F, lane 4 is YU2 gpl40, lane 5 is
YU2 gpl40+IgG
b12, lane 6 is YU2 gpl40 + 39F and lane 7 is marker.
FIG 10 depicts an antibody response to YU2 gp140 immune complex after
immunization in rabbits at weeks 4 and 10.
DETAILED DESCRIPTION
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.
Previous attempts to elicit an effective neutralizing response from antigen-
antibody
complexes have failed, in particular an Env gpl20 - antibody A32 complex (see,
e.g., Liao et
al., J Virol. 2004 May;78(10):5270-8).
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.
In an advantageous embodiment, the antigen-antibody complex is an envelope
protein
(such as, but not limited to, gp 120, gp 140 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-
6
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WO 2009/058989 PCT/US2008/081769
specific antibody (such as , but not limited to 2909, if the antigen is a
envelope trimer
protein) or a CD4 induced epitope specific antibody.
The invention encompasses mixing H1V 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.
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.
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 b 12).
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. Patent
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;
7
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WO 2009/058989 PCT/US2008/081769
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;
8
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WO 2009/058989 PCT/US2008/081769
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.
9
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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. Patent 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.
Any HIV antibody may be used to form antibody-antigen complexes. For example,
the anti-HIV antibodies of U.S. Patent 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.
Patent 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,
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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 June 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 Glade 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, 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, C 1 q, Protein A or Protein G affinity columns or
secondary
antibodies.
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.
Patent 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;
11
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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 gg
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 C prior to adding
Protein A/G).
Following addition of Protein A/G agarose, incubate with gentle agitation for
30 minutes at
4 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.
Antibody-antigen complexes as vaccine formulations are known in the art and
the
disclosures of any one of Akagaki & Inai (1983) Mol Immunol 20(11): 1221-6,
Alber et al.
(2000) Vaccine 19(7-8): 895-901, Andersson et al. (1995) Scand J Immunol
42(4): 407-17,
Barr et al. (2003) Immunology 109(1): 87-92, Berger et al. (1996) Res Virol
147(2-3): 103-8,
Berlyn et al. (2001) Clin Immunol 101(3): 276-83, Bonneau et al. (1972) Prog
Immunobiol
Stand 5: 537-41, Bouige et al. (1996) FEMS Immunol Med Microbiol 13(1): 71-9,
Cannat et
al. (1983) Ann Immunol (Paris) 134C(1): 43-53, Cavacini et al. (1995) J
Immunol 155(7):
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CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
3638-44, Celis et al. (1987) Hepatology 7(3): 563-8, Chargelegue et al. (2005)
Infect Immun
73(9): 5915-22, Dekker et al. (2004) Mol Biochem Parasitol 137(1): 143-9,
Fenner (1972)
Adv Exp Med Biol 31(0): 7-17, Genin & Lesavre (1983) Mol Immunol 20(10): 1069-
72,
Gnjatic et al. (2002) Proc Natl Acad Sci U S A 99(18): 11813-8, Guo et al.
(2004) Avian Dis
48(1): 224-8, Habig et al. (1988) J Pediatr 112(1): 162-3, Haddad et al.
(1997) Avian Dis
41(4): 882-9, Hanke et al. (1992) J Gen Virol 73 (Pt 3): 653-60, Hsueh et al.
(1997) Cancer J
Sci Am 3(6): 364-70, Ivan et al. (2001) Vet Immunol Immunopathol 79(3-4): 235-
48, Ivan et
al. (2005) Can J Vet Res 69(2): 135-42, Jeurissen et al. (1998) Immunology
95(3): 494-500,
Kostiala & Kosunen (1972) Scand J Immunol 1(2): 143-51, Kraiselburd (1987) P R
Health
Sci J 6(1): 27-9, Kraiselburd et al. (1981) Infect Immun 33(2): 389-94, Kurul
et al. (2004)
Pediatr Nephrol 19(6): 621-6, Li et al. (2004) BMC Neurosci 5: 21, Lurhuma et
al. (1994)
East Afr Med J 71(8): 493-5, Marques et al. (2005) Clin Diagn Lab Immunol
12(9): 1036-40,
McCluskie et al. (1998) Viral Immunol 11(4): 245-52, Montefiori et al. (1994)
J Infect Dis
170(2): 429-32, Navol'nev (1983) Vestn Dermatol Venerol(11): 23-7, O'Lee et
al. (1987)
Arch Oral Biol 32(8): 539-43, Paccaud et al. (1987) Clin Exp Immunol 69(2):
468-76, Parish
(1972) Immunology 22(6): 1087-98, Pizarro et al. (2002) Acta Crystallogr D
Biol Crystallogr
58(Pt 7): 1246-8, Pokric et al. (1993) Vaccine 11(6): 655-9, Polack et al.
(2002) J Exp Med
196(6): 859-65, Rafiq et al. (2002) J Clin Invest 110(1): 71-9, Randall et al.
(1993) Vaccine
11(12): 1247-52, Randall et al. (1994) Vaccine 12(4): 351-8, Randall & Young
(1988) J Gen
Virol 69 (Pt 10): 2505-16, Randall & Young (1989) J Virol 63(4): 1808-10,
Randall et al.
(1988) J Gen Virol 69 (Pt 10): 2517-26, Ridley et al. (1982) J Pathol 136(1):
59-72, Roic et
al. (2006) J Vet Med B Infect Dis Vet Public Health 53(1): 17-23, Root-
Bernstein (2004) J
Clin Virol 31 Suppl 1: S16-25, Schifferli et al. (1988) J Immunol 140(3): 899-
904, Schnurr et
al. (2005) Blood 105(6): 2465-72, Schultes & Nicodemus (2004) Expert Opin Biol
Ther 4(8):
1265-84, Schuurhuis et al. (2006) J Immunol 176(8): 4573-80, Semkow &
Wilczynski (1979)
Acta Virol 23(1): 52-8, Speranskaia et al. (1998) Zh Mikrobiol Epidemiol
Immunobiol(2):
14-8, Stager et al. (2003) Nat Med 9(10): 1287-92, Stoner et al. (1975) J
Infect Dis 131(3):
230-8, Trkola et al. (1995) J Virol 69(11): 6609-17, van Rooijen (1975)
Immunology 28(6):
1155-63, Wen et al. (1994) Chin Med J (Engl) 107(10): 741-4, Wen et al. (1999)
Int Rev
Immunol 18(3): 251-8, Wen et al. (1995) Lancet 345(8964): 1575-6, Whipple et
al. (2006)
Mol Immunol. 2006 Apr 20; [Epub ahead of print]; Wyss-Coray et al. (1992) Cell
Immunol
139(1): 268-73, Yoshikawa et al. (2004) J Gen Virol 85(Pt 8): 2339-46 and
Zheng et al.
(2001) Vaccine 19(30): 4219-25 may be utilized for methods of the present
invention.
13
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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.
The term "antibody" includes intact molecules as well as fragments thereof,
such as
Fab, F(ab')z, 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:
(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;
(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;
(iii) F(ab')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;
(iv) scFv, including a genetically engineered fragment containing the variable
region
of a heavy and a light chain as a fused single chain molecule.
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).
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
14
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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.
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.
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.
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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.
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.
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
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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.
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
PAM 120 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.
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 ed., 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).
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).
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.
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
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WO 2009/058989 PCT/US2008/081769
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.
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.
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).
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.
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
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WO 2009/058989 PCT/US2008/081769
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.
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 selelct 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.
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.
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
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embodiments the adenovirus vector used is selected from the group consisting
of the Ad5,
Ad35, Adl 1, C6, and C7 vectors.
The sequence of the Adenovirus 5 ("M5") genome has been published.
(Chroboczek,
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. Patent
Nos. 6,974,695, 6,913,922, and 6,869,794. Adl 1 vectors are described in U.S.
Patent No.
6,913,922. C6 adenovirus vectors are described in U.S. Patent 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. Patent No. 6,277,558.
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 El
region have improved safety margin because El-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, El and E3, and El and E4 can be
used in
accordance with the present invention.
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 E
1 a 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.
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
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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.
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.
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
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WO 2009/058989 PCT/US2008/081769
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.
The compositions of the invention maybe 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
serum 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 TWEENTM, PLURONICSTM or polyethylene glycol (PEG).
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-
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polyoxyethylene copolymer blocks, such as the Pluronic 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 (IDEC Pharmaceuticals, San
Diego, CA).
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).
Adjuvants may also be included. Adjuvants include, but are not limited to,
mineral
salts (e.g., A1K(SO4)2, A1Na(SO4)2, A1NH(S04)2, silica, alum, Al(OH)3,
Ca3(P04)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
IC31; see
Schellack, C. et al (2003) Proceedings of the 34th Annual Meeting of the
German Society of
Immunology; Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508), JuvaVaxTM
(U.S.
Patent 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. Patent
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
IQM and
commercially available as Aldara ; U.S. Patent 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).
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-a, IFN-
J3, and IFN-y (Boyer et al., (2002) J. Liposome Res. 121:137-142;
WOO1/095919),
immunoregulatory proteins such as CD40L (ADX40; see, for example,
W003/063899), and
the CD 1 a ligand of natural killer cells (also known as CRONY or a-galactosyl
ceramide; see
Green, T.D. et al, (2003) J. Virol. 77(3): 2046-2055), immunostimulatory
fusion proteins
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WO 2009/058989 PCT/US2008/081769
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.
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)).
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 vinylacetate 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.
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
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WO 2009/058989 PCT/US2008/081769
in the art, for example by measuring the immune response of a subject, such as
a laboratry
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-y
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.
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 g) in the gpl2-32 complex. Advantageously, a lower dose of
env is
contemplated, such as about 1 pg to about 50 g, about 2.5 gg to about 40 g,
about 5 g to
about 30 gg , about 7.5 pg to about 20 g, preferably about 10 g to about 15
gg of env when
env is the antigen in the antigen-antibody complex.
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.
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
CA 02704059 2010-04-28
WO 2009/058989 PCT/US2008/081769
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.
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.
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.
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.
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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.
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.
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).
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.
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.
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The invention will now be further described by way of the following non-
limiting
examples.
EXAMPLE: HIV envelope Immune complexes as a Vaccine Candidate
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.
The advantages of immune complexes include, but are not limited to:
1. Immune complexes are efficiently taken up by specialized antigen-presenting
cells (dendritic cells (DC)) via Fc receptors,
2. Binding of ICs to Fc receptors can mediate dendritic cell maturation
serving as
a natural adjuvant,
3. Immune complex increase the germinal center formation and thus could effect
the quality of antibody formation,
4. Antibody binding to the antigen could potentially present the cryptic
epitopes
by masking the immunodominant epitopes and
5. Alteration of proteolysis of the antigen by formation of the immune complex
could alter the presentation to CD4 T-cell,
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.
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.
The experimental steps were as follows:
1. Determining YU2 gpl20 run profile through a gel filtration chromatography,
2. Making Yu2gp 120 and monoclonal antibody complexes and purify on Ag-Ab
complex by gel filtration,
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3. Characterizing the stochiometry of Ag-Ab by blue native and denaturing
PAGE,
4. Immunizing rabbits intramuscularly with gp120 and gp120-Ab complexes at
13 ug Env dose with adjuplex adjuvant and
5. Evaluating the sera for binding and neutralization assay.
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.
The rationale for using b 12 antibody to make Yu2gp 120-b 12 immune complex
was:
1. B12 Immune complexes would be efficiently taken up by specialized antigen-
presenting cells (dendritic cells (DC)) via Fc receptors.
2. B 12 antibody binding region is crucial for the viral entry and
preservation of
this site by making a complex with the antibody is desirable and
3. Binding of b12 to gp120 does not lead to major conformational changes as
measured by isothermal calorimetry potentially allowing stabilization of the
Env gp 120 in
one fixed state. Env gp 120 is highly flexible molecule and fixing in one
state is presumed to
be better for the immunogenic property.
The binding of Fc receptor but not complement to antibody b 12 was shown to be
important for anti-HIV activity.
The rationale for using 39F antibody to make Yu2gp120-b12 immune complex was:
1. 39F Immune complexes would be efficiently taken up by specialized antigen-
presenting cells (dendritic cells (DC)) via Fc receptors,
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
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 1: Soluble gp120 YU2 complexes
Antigen Antibody Binding
YU2 gpl20 39F Binds V3 loop
YU2 gpl20 b12 CD4 Binding Site
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
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into complex formation (FIG. 1). The immune complexes were stable when stored
at 4C for
days and at -70C for days. The b 12 immune complexes were of one type 1:1 of
gp 120-b 12
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 (gp 120)
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 BR020 viruses.
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.
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
gpl20 are used.
Experimental steps include:
1. Determining YU2 gp140 or JRCSF gp140 run profile through a gel filtration
chromatography,
2. Making Yu2/JRCSF gp140 and monoclonal antibody complexes and purify on
Ag-Ab complex by gel filtration,
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3. Characterizing the stochiometry of Ag-Ab by blue native and denaturing
PAGE,
4. Immunizing rabbits intramuscularly with gp140 and gp140-Ab complexes at
different dose with adjuplex adjuvant and
5. Evaluating the sera for binding and neutralization assay.
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.
A low dose of Env is important as immune complexes have various regulatory
functions and a high dose of Env may lead to immunopathogenesis.
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
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variable loop 3 of the HIV envelope glycoprotein. On-going studies may
determine the role
of the Fcy portion of antibody and the size of the complex in the immune
potentiation.
SF162 Neutralization Titer: IC50 and 9o Values. Neutralization IC50 and IC90
values of
immune complex derived rabbit sera against HIV-1 Glade B SF162 virus. As shown
in Table
2, Immune complex group sera has significant IC90 titer against SF 162
following a single
prime and boost immunization.
Table 2: SF162 Neutralization Titer: IC50 and 90 Values
IC50 IC90 IC50 IC90
WkO <10 <10 WkO 410 <10
Wk2 410 <10 a Wk2 <10 <10
WK6 410 <10 }.' WK6 <10
r. Wk10 <10 <10 *-+ Wk10 u <10
M Wk14 <10 <10 Wk14
WkO <10 <10 > WkO <10 <10
Wk2 <10 <10 Wk2
WK6 <10 WK6 .,
Wk10
Wk14 .~-- Wk14
Wk0 <10 <10 WkO 410 <10
Wk2 410 <10 Wk2 <10 <10
WK6 <10 WK6
Wk10 = 1 <10 Wk10
Wk14 <10 Wk14
Responding uudimals
Rabbit Sera Neutralization:IC50 value. Table 3 depicts neutralization IC50
value of
immune complex derived rabbit sera against a panel of 10 Glade B HIV-1
viruses. Immune
complex derived sera neutralizes six out of 10 viruses tested.
Table 3: Rabbit Sera Neutralization: IC5 0 value
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119 BR02 HT593 US712 Bat BX08 JRFL SF162 JRCS NL4-3 aML
6 0 F V
Wk <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10
; Wk2 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10
WK6 <10 <10 <10 <10 <10 <10 <10 <10 <10
k10 <10 <10 <10 <10 <10 <10 + <10 <1;0
W <10
Wk14 <10 <10 <10 <10 f <10 <10
YYKO <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10
<10 <10 <10 <10 <10 <10 <10 <i0 <10 <10
Wk2
WK6 <10 <10 <10 <10 <10 <iO <i0
WHO <10 <10 <10 M ..+ <10 <10 <10
Wk14 <10 <10 <10 <10 <10
WkO <10 <10 <10 <10 j<10 <10 <10 <10 1<10 <10 1<10
Wk2 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10 <10
WK6 <10 <10 <10 <10 <f0 <10 <10 <10
Wk10 <10 <10 <10 <10 <10 <10 <10
k14 <10 <10 <10 t <10 <10
ilf 0
Responding animals D
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 4: Mapping of the sera for binding and neutralizing antibodies
Virus base/ Stem / Tip /stem /base
V3 Sequence
Immunogen YU2 CTRPNNNTRKSINI- GPGRALYTTGEIIGDIRQAHC
SF162 CTRPNNNTRKSITI- GPGRAFYATGGIIGDIR AHC
Bx08 CTRPNNNTRKSIHI--GPGRAEYTTGDIIGDIRQAHC
HT593 CTRPNNNTSKRISI- GPGRAFRAT-KIIGNIRQAHC
Bal CTRPNNNTREfSIH3--GPGRALYTTGEI.IGDIRQAHC
BRO24' CTRPNNNTRKSIHI--GPGRAFYATGDIIGDIRQAHC
NL4-3 CTRPNNNTRKSIRIQRGPGRAFVTIGKI-GIiHC
YU2 V3 Peptide Absorption of Neutralizing Ability. Table 5 depicts absorption
of
neutralizaion activity by YU2 V3 peptide for a) anti-V3 monoclonal antibody
447-52D, b) a
control human sera with broad neutralization specificity c) YU2 gp 120
generated rabbit sera
and d) Immune complex generated sera.
Table 5: YU2 V3 Peptide Absorption of Neutralizing Ability
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a) MMMMMMMMM
52D
447- P i >50
52D Pe eptide
441- V3 > 50 > 50 > 50
52D peptide
b)
s723 No
ea Peptide MMMMMMM <100
Z23 V3 <10
sera peptide MMWMWW~t~
C) Env No <10 <10 <10 <10 M <10 ~ <10
Peptide
Env p3 <10 <10 <10 <10 <10 <10
peptide
d) v + Peptide .. . <1 Q
Ah
Env + peptide 10 <10 MW <10 <10
Ab pe50 uglml V3 peptide added along with the sera and neutralization
performed in a 10 point dilution curve to determine
IC50 value
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 6: Size of Yu2 gp120 and Immune complexes
Protein or Rh (nm) from Rf (ml) of the
complex SEC-QELS Mw ____ major peak
b12 Fab 3.8 50.7 15.5
b12 5.5 146.6 13.0
YU2 gp12O 5.2 99.9^ 12.6
55.6+44.3
YU2 gp120 6.1 11.9 (complex)
+ b12 Fab .1 1.5.5 Fab
YU2 g 1120 7.5 10.2
b12I gG
Rh = Hydodynamic radius: SEC-QELS Size exclusion Chromatography - Quasi
Elastic Light
scattering; Mw:=Molecular weight; Rf = retention factor.
Superose 12 10/30 column
The invention is further described by the following numbered paragraphs:
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.
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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.
3. The method of paragraph 2 wherein the antibodies are monoclonal antibodies.
4. The method of paragraph 3 wherein the monoclonal antibodies are b 12, 2F5,
2G12, 4E10, M2909 or any combination thereof.
5. The method of any one of paragraphs 2-4 wherein the HIV is purified HIV.
6. The method of any one of paragraphs 2-4 wherein the HIV is a HIV viral
isolate.
7. The method of paragraph 6 wherein the HIV viral isolate is a HIV Glade
viral
isolate.
8. The method of any one of paragraphs 1-7 wherein the purified antibody-
antigen complex is chemically dissociated from the glycoprotein spikes.
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.
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.
11. The method of any one of paragraphs 1-10 wherein the mammal is a human.
12. The method of any one of paragraphs 1-11 wherein the purified antibody-
antigen complex is administered in a pharmaceutically acceptable carrier.
13. The method of any one of paragraphs 1-12 wherein the administering further
comprises a prime-boost regimen.
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.
15. The method of paragraph 14 wherein the HIV envelope protein is gp 120,
gp140 or a membrane associated envelope trimer.
16. The method of any one of paragraphs 14-15 wherein the antibody is a broad
neutralizing antibody.
17. The method of paragraph 16 wherein the broad neutralizing antibody is
antibody b 12.
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18. The method of any one of paragraphs 14-15 wherein the antibody is a non-
neutralizing antibody.
19. The method of paragraph 18 wherein the non-neutralizing antibody is a V3
specific antibody.
20. The method of paragraph 19 wherein the non-neutralizing antibody is
antibody
39F.
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.
22. The method of any one of paragraphs 14-21 wherein the antibody-antigen
complex is purified by gel filtration.
23. The method of any one of paragraphs 14-22 wherein the mammal is a human.
24. The method of any one of paragraphs 14-23 wherein the antibody-antigen
complex is administered in a pharmaceutically acceptable carrier.
25. The method of any one of paragraphs 14-24 further comprising an adjuvant.
26. The method of paragraph 25 wherein the adjuvant is Adjuplex LAP.
27. The method of any one of paragraphs 14-26 wherein the dosage of the HIV
envelope protein is about 10 .tg to about 15 g.
28. The method of any one of paragraphs 14-27 wherein the administering
further
comprises a prime-boost regimen.
29. The method of any one of paragraphs 14-28 wherein the antibody-antigen
complex is expressed in a viral vector.
30. The method of paragraph 29 wherein the antibody-antigen complex is
expressed in vivo.
***
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.
36
