U.S. patent application number 09/905962 was filed with the patent office on 2003-02-27 for hiv immunogenic complexes.
Invention is credited to Devico, Anthony L., Pal, Ranajit, Sarngadharan, Mangalasseril G..
Application Number | 20030039663 09/905962 |
Document ID | / |
Family ID | 22032603 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030039663 |
Kind Code |
A1 |
Devico, Anthony L. ; et
al. |
February 27, 2003 |
HIV immunogenic complexes
Abstract
A vaccine and a method of raising neutralizing antibodies
against HIV infection. The vaccine comprises a complex of gp120
covalently bonded to CD4 or to succinyl concanavalin A. Also
disclosed are immunological tests using the complex or antibody
thereto for detection of HIV infection.
Inventors: |
Devico, Anthony L.;
(Alexandria, VA) ; Pal, Ranajit; (Gaithersburg,
MD) ; Sarngadharan, Mangalasseril G.; (McLean,
VA) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
22032603 |
Appl. No.: |
09/905962 |
Filed: |
July 17, 2001 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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09905962 |
Jul 17, 2001 |
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09479675 |
Jan 7, 2000 |
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6328973 |
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09479675 |
Jan 7, 2000 |
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09075544 |
May 11, 1998 |
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6030772 |
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09075544 |
May 11, 1998 |
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08464680 |
Dec 20, 1995 |
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5843454 |
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08464680 |
Dec 20, 1995 |
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PCT/US94/05020 |
May 6, 1994 |
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PCT/US94/05020 |
May 6, 1994 |
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08060926 |
May 7, 1993 |
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Current U.S.
Class: |
424/196.11 ;
435/334; 435/339.1; 435/5; 435/7.1; 435/974; 435/975; 530/388.1;
530/388.22; 530/388.35; 530/389.4 |
Current CPC
Class: |
C12N 2740/16122
20130101; A61K 39/00 20130101; C07K 14/70514 20130101; C07K 14/005
20130101; C07K 16/1063 20130101; A61P 31/12 20180101; Y10S 435/975
20130101; C07K 16/2812 20130101; Y10S 435/974 20130101; A61P 31/18
20180101 |
Class at
Publication: |
424/196.11 ;
435/5; 435/334; 435/339.1; 435/7.1; 435/974; 435/975; 530/388.1;
530/388.22; 530/388.35; 530/389.4 |
International
Class: |
A61K 039/385; C12Q
001/70; G01N 033/53; C12N 005/06; C12P 021/08; C07K 016/00 |
Claims
We claim:
1. A vaccine comprising an immunogenically effective amount of a
complex of gp120 covalently bonded to CD4 in a pharmaceutically
acceptable medium.
2. A method of raising neutralizing antibodies against HIV
infection in a human, comprising administering a vaccine according
to claim 1 comprising an immunogenically effective amount of a
complex of gp120 covalently bonded to CD4 in a pharmaceutically
acceptable carrier.
3. A vaccine comprising an immunogenically effective amount of a
complex of gp120 covalently bonded to succinyl concanavalin A in a
pharmaceutically acceptable medium.
4. A method of raising neutralizing antibodies against HIV-1
infection in a human, comprising administering a vaccine according
to claim 3, comprising an immunogenically effective amount of a
complex of gp120 covalently bonded to succinyl concanavalin A in a
pharmaceutically acceptable carrier.
5. An immunogenic complex comprising gp120 covalently bonded to
CD4.
6. An immunogenic complex comprising gp120 covalently bonded to
succinyl concanavalin A.
7. A composition comprising the immunogenic complex of claim 5.
8. The composition of claim 7, further comprising an adjuvant
composed of aluminum phosphate gel.
9. A composition comprising the immunogenic complex of claim 6.
10. The composition of claim 9, further comprising an adjuvant
composed of aluminum phosphate gel.
11. An antibody reactive with the immunogenic complex of claim
5.
12. The antibody of claim 11, which is a monoclonal antibody.
13. An antibody reactive with the immunogenic complex of claim
6.
14. The antibody of claim 13, which is a monoclonal antibody.
15. An immortalized cell line that produces an antibody as recited
in claim 12.
16. An immortalized cell line that produces an antibody as recited
in claim 14.
17. A method for the detection of anti-HIV antibodies in a test
fluid, comprising contacting an immunogenic complex of gp120
covalently attached to one of CD4 and succinyl concanavalin A, and
detecting the presence of immune complexes formed between said
antibodies and said immunogenic complex.
18. A method for the detection of HIV antigen in a test fluid,
comprising contacting the test fluid with an antibody raised
against an immunogenic complex of gp120 covalently bonded to one of
CD4 and succinyl concanavalin A, and detecting the presence of
immune complexes formed between antigen in the test fluid and said
antibody.
19. A test kit for conducting the method of claim 17, comprising
said immunogenic complex that is bound to a solid substrate or
labelled and instructions for performing the detection method.
20. A test kit for conducting the method of claim 18, comprising
said antibody that is bound to a solid substrate or labelled and
instructions for performing the detection method.
Description
DESCRIPTION OF THE INVENTION
[0001] We discovered that a gp120-CD4 covalently bonded complex
presents a specific subset of cryptic epitopes on gp120 and/or CD4
not present on the uncomplexed molecules. These complexes elicited
neutralizing antibodies with novel specificities and are thus
useful in vaccines and immunotherapy against HIV infection. In
addition, the complexes or antibodies thereto can be used in
immunological tests for HIV infection.
BACKGROUND OF THE INVENTION
[0002] Neutralizing antibodies are considered to be essential for
protection against many viral infections including those caused by
retroviruses. Since the initial reports of neutralizing antibodies
in HIV-infected individuals, it has become increasingly clear that
high levels of these antibodies in serum correlate with better
clinical outcome (3-5). These studies suggested that the
identification of epitopes that elicit high titer neutralizing
antibodies would be essential for vaccine development against HIV
infection.
[0003] The primary receptor for the human immunodeficiency virus
type 1 (HIV-1) is the CD4 molecule, found predominantly on the
surface of T-lymphocytes. The binding of HIV-1 to CD4 occurs via
the major viral envelope glycoprotein gp120 and initiates the viral
infection process.
[0004] Current strategies for developing vaccines against infection
by the human immunodeficiency virus have focused on eliciting
antibodies against the viral envelope glycoprotein gp120 or its
cell surface receptor CD4. Purified gp120 typically elicits type
specific neutralizing antibodies that are reactive against epitopes
that vary among virus isolates. This characteristic has hindered
the use of gp120 as a vaccine.
[0005] CD4 has also been considered as a major candidate for
development of a vaccine against HIV-1. Recent studies have
demonstrated that sCD4 elicits HIV neutralizing antibodies in
animals and prevents the spread of infection in SIV-infected rhesus
monkeys (1). However, autoantibodies to CD4 may themselves create
immune abnormalities in the immunized host if they interfere with
normal T-cell functions. Neutralizing antibodies against gp120 are
elicited in vivo in HIV-1-infected individuals and can be elicited
in vitro using purified envelope glycoprotein. However, gp120
contains five hypervariable regions one of which, the V3 domain, is
the principal neutralizing epitope. Hypervariability of this
epitope among strains is a major obstacle for the generation of
neutralizing antibodies effective against diverse strains of HIV-1.
For these reasons it has been believed that vaccine strategies
using either purified CD4 or gp120 present several
disadvantages.
[0006] We have overcome the shortcomings of type specific anti
gp120 antibodies and antibodies against CD4 by raising anti-HIV-1
neutralizing antibodies using as the immunogen a complex of gp120
chemically coupled to either soluble CD4 or to the mannose-specific
lectin, succinyl concanavalin A (SC). We have found that these
compounds induce similar conformational changes in gp120. The
complexed gp120 appears to undergo a conformational change that
presents an array of epitopes that were hidden on the uncomplexed
glycoprotein (2). A portion of such epitopes elicit group-specific
neutralizing antibodies, which are not strain limited like the type
specific antibodies discussed above. We have discovered that the
covalently bonded CD4-gp120 complexes are useful for raising
neutralizing antibodies against various isolates of HIV-1 and
against HIV-2.
[0007] The major research effort in the development of subunit
vaccines against HIV has been directed toward the envelope
glycoprotein of the virus. Inoculation of gp160 or gp120 into
animals elicits neutralizing antibodies against HIV (3, 4). The
principal neutralizing epitope on gp120 has been located between
amino acids 306 and 326 in the third variable domain (V3 loop) of
the protein (4). This epitope has been extensively studied by using
both polyclonal and monoclonal antibodies (3, 4). In most cases
antibodies directed to this region neutralize HIV-1 in an isolate
specific manner although there is evidence that a weakly
neutralizing species of anti-V3 loop antibodies can cross-react
with diverse isolates (8). The reason for type specificity of
anti-V3 loop antibodies is the extensive sequence variability among
various isolates. Prolonged culturing of HIV-infected cells with
type specific anti-V3 loop antibodies induces escape mutants
resistant to neutralization (9).
[0008] In addition to the V3 loop, other neutralizing epitopes
encompassing genetically conserved regions of the envelope have
been identified (10, 11). However, immunization against these
epitopes elicits polyclonal antisera with low neutralizing titers
(12). For example, the CD4 binding region of gp120, encompassing a
conserved region, elicits neutralizing antibodies against diverse
isolates (13). However, this region is not normally an
immunodominant epitope on the glycoprotein.
[0009] The interaction of gp120 with CD4 has been studied in
considerable detail and regions of the molecules involved in
complex formation have been determined (14-16). There are now
several lines of evidence that interactions with CD4 induce
conformational changes in gp120. First, recombinant soluble CD4
(sCD4) binding to gp120 increases the susceptibility of the V3 loop
to monoclonal antibody binding and to digestion by exogenous
proteinase (2). Second, sCD4 binding results in the dissociation of
gp120 from the virus (17, 18). These conformational changes in
gp120 are thought to facilitate the processes of virus attachment
and fusion with the host cell membrane (2). Immunization with
soluble CD4 and recombinant gp120, complexed by their natural
affinity but not covalently bonded, resulted in the production of
anti CD4 antibodies (31). Several murine monoclonal antibodies have
been developed by immunization with mixtures of recombinant gp120
and sCD4 (31, 32). Antibodies raised in these studies were not
strictly complex-specific and reacted with free gp120 or CD4; the
neutralizing antibodies reacted with free sCD4, although they
displayed various degrees of enhanced reactivity in the presence of
gp120. The complexes used in these studies were unstable and
comprised noncovalently bound gp120 and CD4.
[0010] A variety of N-linked carbohydrate structures of high
mannose, complex and hybrid types present on the gp120 molecule may
also play a role in the interaction of gp120 with host cell
membranes (19-21). Indeed, a carbohydrate-mediated reactivity of
gp120 has already been demonstrated with a serum lectin, known as
mannose-binding protein, which has also been shown to inhibit HIV-1
infection of CD4+ cells (22). An additional carbohydrate-mediated
interaction of gp120 has been shown with the endocytosis receptor
of human macrophage membranes (21). It has been postulated that
high affinity binding of accessible mannose residues on gp120 to
the macrophage membrane may lead to virus uptake by the macrophage
(21).
[0011] Recombinant soluble CD4 has been shown to inhibit HIV
infection in vitro, mainly by competing with cell surface CD4. This
observation has led to the possibility of using sCD4 for the
therapy of HIV-infected individuals (23, 24). In addition, sCD4 has
been used as an immunogen to block viral infection in animals.
Treatment of SIV.sub.MAC-infected rhesus monkeys with sCD4 elicited
not only an antibody response to human CD4 but also to monkey CD4.
Coincident with the generation of such immunological responses, SIV
could not be isolated from the PBL and bone marrow macrophages of
these animals (1). A recent study with chimpanzees also
demonstrated that human CD4 elicited anti-self CD4 antibody that
inhibited HIV infection in vitro (25). Although immunization with
sCD4 may be beneficial in blocking HIV infection, circulating
antibody that recognizes self antigen may induce immune abnormality
and dysfunction by binding to uninfected CD4+ cells. Nevertheless
in theory anti-CD4 antibodies could be effective in blocking HIV
infection provided they can disrupt virus attachment and entry
without interfering with normal CD4 function. Ideally these
antibodies should recognize CD4 epitopes that are present only
after interaction with gp120.
SUMMARY OF THE INVENTION
[0012] We discovered that gp120-CD4 complex formation induces a
specific subset of cryptic epitopes on gp120 and/or CD4 not present
on the uncomplexed molecules. These epitopes elicit neutralizing
antibodies with novel specificities and are thus useful in vaccines
and/or immunotherapy of patients infected with HIV. In addition,
the antibodies or the complexes can be used in immunological tests
for HIV infection. We have demonstrated that the lectin, SC,
mediates changes in the structure of gp120 in a manner similar to
that mediated by CD4. The binding of SC to gp120 is another
mechanism for inducing novel epitopes on the viral
glycoprotein.
[0013] We used chemically-coupled gp120-CD4 complexes as immunogens
for raising neutralizing antibodies. We found that gp120-CD4
complexes possess novel epitopes that elicit neutralizing
antibodies. Coupling with SC caused perturbation in the gp120
conformation which in turn unmasked cryptic neutralizing epitopes
on gp120.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows the dissociation of gp120 from HIV-1 in the
presence of sCD4 and SC. In FIG. 1A labeled cells were treated with
0 (lanes 1, 2) or 1.5 .mu.g/ml sCD4 (lanes 3, 4). Virus bound
(lanes 1, 3) or soluble (lanes 2, 4) gp120 was detected by
immunoprecipitation with HIV-1 antibody-positive human serum,
SDS-PAGE and autoradiography. In FIG. 1B labeled cells were treated
with 0 (lanes 1, 2), 5 .mu.g/ml (lanes 3, 4) or 10 .mu.g/ml SC
(lanes 5, 6). Virus bound (lanes 1, 3, 5) or soluble (lanes 2, 4,
6) gp120 was detected as in 1A.
[0015] FIG. 2 illustrates the susceptibility of gp120 to thrombin
digestion in the presence of SC and sCD4. Molt3/HIV-1.sub.IIIB
cells were labeled with .sup.35S-methionine for 4 hr, followed by a
3 hr incubation with medium containing 0.25% methionine. In FIG. 2A
an aliquot of labeled medium (1 ml) was digested with thrombin (7
.mu.g/ml) at 37.degree. C. for 90 min and then immunoprecipitated
with HIV-1 positive human serum and analyzed by SDS-PAGE. Lane 1
shows untreated medium and lane 2, medium treated with thrombin.
Prior to thrombin digestion, aliquots of the medium were pretreated
with SC at concentrations of 2.5 .mu.g/ml (lane 3), or 10 .mu.g/ml
(lane 4); or with sCD4 at concentrations of 2.5 .mu.g/ml (lane 5)
or 10 .mu.g/ml (lane 6). The gp120 fragments generated by thrombin
cleavage are marked with arrows. In FIG. 2B aliquots of labeled
medium were digested by thrombin as before with no pretreatment
(lane 1), after pretreatment with 5 .mu.g/ml SC (lane 2 or with a
mixture of 5 .mu.g/ml SC and 0.1 mM .alpha.-methylpyranoside (lane
3).
[0016] FIG. 3 shows the inhibition of HIV-1 induced syncytia
formation by murine antisera raised against gp120-sCD4. In FIG. 3A
murine antiserum raised against gp120-sCD4 was added to CEM cells
along with cells infected with HIV-1.sub.IIIB (.largecircle.),
HIV-1.sub.MN (.quadrature.) or HIV-2.sub.WAVZ (.diamond.). In FIG.
3B murine antisera raised against thrombin treated gp120-sCD4
complexes were tested. The assay conditions are described in the
Examples. For each experimental condition, the syncytia in three
separate fields were counted. The average value is given as
syncytia/field.
[0017] FIG. 4 shows Western blot assays of monoclonal antibodies
raised against gp120-CD4 complexes with gp120, sCD4 and complex.
Lane 1 is MoAb7E3, lane 2 is MoAb 8F10B, lane 3 is MoAb 8F10C, lane
4 is MoAb 8F10D, lane 5 is anti-gp120 MoAb, lane 6 is anti-p24 MoAb
(negative control), lane 7 is rabbit anti-CD4 hyperimmune serum,
and lane 8 is normal rabbit serum.
[0018] FIG. 5 is a graph showing the binding of complex-specific
monoclonal antibodies to gp120-lectin complex. MoAbs
A(.largecircle.) and B(.DELTA.) were tested in ELISA, with either
gp120-SC (open symbols) or gp120 (closed symbols).
[0019] FIG. 6 is a graph showing competitive ELISA with
complex-specific monoclonal antibodies and immune goat serum.
Limiting dilutions of purified MoAb 7E3 (.box-solid.), MoAb 8F10B
(.largecircle.), MoAb 8F10C (.circle-solid.) and MoAb
8F10D(.tangle-solidup.) were incubated with serial dilutions of
goat 69 serum and tested in go120-CD4 ELISA. Percent competition
was calculated as level of antibody binding in immune serum versus
binding in prebleed serum.
[0020] FIG. 7 is a photograph of a gel showing gp120-CD4 complexes
prepared according to Example III. FIG. 4 lane 1 is gp120, lane 2
is sCD4, Lane 3 has a gp120-CD4 complex and lane 4 has molecular
weight markers.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0021] We determined that it was necessary to unmask or create new
epitopes on gp120 and/or CD4 capable of eliciting a strong, broadly
neutralizing immune response. We used a covalently linked gp120-CD4
complex as an immunogen. gp120 molecules were covalently coupled to
soluble recombinant CD4 using bivalent cross-linking agents to
ensure that the integrity of the complexes was maintained during
any manipulations. The components of the complex were expected to
differ from the free glycoprotein in at least two ways: (I) some
epitopes on gp120 and CD4 would be masked by complex formation and
(II) cryptic epitopes would become exposed as a result of
conformational changes in gp120 and CD4 of the complex. Because
these epitopes could play a significant role in viral entry into
target cells, antibodies directed against them should inhibit some
aspects of the entry process. We believed these antibodies may not
inhibit gp120-CD4 interaction but may instead prevent post-binding
fusion events necessary for infection.
[0022] The application of this strategy toward anti-HIV vaccines
offered several other advantages. First, epitopes specific to
complexed gp120 are not expected to be normal targets for
neutralizing antibodies in vivo. HIV-1 binds and enters target
cells within 3 min at 37.degree. C. (26). Given the transient and
short-lived nature of the native gp120-CD4 complex, it is unlikely
that it is presented to the immune system in such a way as to
elicit complex-specific antibodies. Therefore, the absence of
immune selection in vivo should in turn be reflected in a minimal
degree of variation in the complex-specific epitopes of different
viral strains. Second, antibodies against complex-specific epitopes
on CD4 are not expected to elicit anti-self antibodies capable of
recognizing uncomplexed CD4 on the surface of normal cells. This is
especially important, since anti-CD4 antibodies can mediate
cytotoxic effects.
[0023] In the development of vaccines against HIV, the ability to
induce novel epitopes on gp120 in the absence of CD4 would be of
considerable advantage. We discovered this is possible. We have
bound a mannose-specific lectin, SC, with gp120, which induces a
conformational change on the glycoprotein that appears to be
similar to that observed with sCD4. The alterations include
exposure of the V3 loop to exogenous protease and dissociation of
gp120 from the virus membrane. Therefore, covalently linked
gp120-SC complexes are also useful as immunogens for exposing novel
epitopes and complex specific antibodies in the absence of CD4.
[0024] The vaccines of the present invention are composed of the
complex of gp120-CD4 or gp120-SC together with an acceptable
suspension known in the vaccine art. Preferably, an adjuvant may be
added. The only adjuvant acceptable for use in human vaccines is
aluminum phosphate (alum adjuvant), and therefore preferably the
vaccine of the present invention is formulated with an aluminum
phosphate gel. See Dolin et al., Ann Intern Med, 1991;114:119-27,
which is incorporated herein by reference. The dose of the
immunogenic complex for purposes of vaccination is between about 40
.mu.g to about 200 .mu.g per inoculation. An initial inoculatior
may be followed by one or more booster inoculations. Preferably,
the vaccination protocol will be the same as protocols now used in
clinical vaccination studies and disclosed in Dolin et al., supra,
and Reuben et al., J Acquired Immune Deficiency Syndrome,
1992;5:719-725, also incorporated herein by reference.
[0025] It is also contemplated that antibodies raised against the
immunogenic complexes of the present invention can be used for
passive immunization or immunotherapy. The dosage and number of
inoculations of these antibodies will follow those established in
the art for immunization or immunotherapy with immunoglobulins.
[0026] The complexes or antibodies thereto can also be used in a
method for the detection of HIV infection. For instance, the
complex, which is bound to a solid substrate or labelled, is
contacted with the test fluid and immune complexes formed between
the complex of the present invention and antibodies in the test
fluid are detected Preferably, antibodies raised against the
immunogenic complexes of the present invention are used in a method
for the detection of HIV infection. These antibodies may be bound
to a solid support or labelled in accordance with known methods in
the art. The detection method would comprise contacting the test
fluid with the antibody and immune complexes formed between the
antibody and antigen in the test fluid are detected and from this
the presence of HIV infection is determined. The immunochemical
reaction which takes place using these detection methods is
preferably a sandwich reaction, an agglutination reaction, a
competition reaction or an inhibition reaction.
[0027] A test kit for performing the methods mentioned in the
preceding paragraph must contain either the immunogenic complex
according to the present invention or one or more antibodies raised
thereto. In the kit, the immunogenic complex or the antibody(ies)
are either bound to a solid substrate or are labelled with
conventional labels. Solid substrates and labels, as well as
specific immunological testing methods are disclosed in Harlow and
Lane, "Antibodies, A Laboratory Manual", Cold Spring Harbor
Laboratory, 1988, incorporated herein by reference.
EXAMPLES
[0028] We conducted several studies to show that new epitopes could
be exposed on gp120 and CD4. These studies also demonstrated that
neutralizing antibodies could be raised against gp120 after
treatment that altered the conformation of the glycoprotein.
EXAMPLE I
[0029] a. Conformational changes in gp120 induced by complex
formation with CD4:
[0030] We analyzed the release of gp120 from the virus surface
under various conditions. Molt3/HIV-1.sub.IIIB cells were labeled
with 35S-methionine (150 .mu.Ci/ml) for 3 hours. The labeled cells
were then washed and resuspended in RPMI medium containing cold
methionine. The cells were then cultured for 4 hours in the
presence of recombinant sCD4 (DuPont). The cell-free supernatant
was collected and then passed through a Sephacryl S 1000 column in
order to separate virions from free viral proteins. Each of the
fractions was treated with detergent, immunoprecipitated with human
sera positive for anti-HIV-1 antibodies, and analyzed by SDS-PAGE
and autoradiography. The amount of gp120 present in the virus and
free viral protein fractions was quantitated by a densitometric
scan of the autoradiograph. In accordance with previous studies
(17, 18), we observed that treatment of virus with sCD4 clearly
resulted in an increased level of gp120 in the free protein
fraction and a coincident decrease in the virus fraction (FIG. 1A),
indicating that the conformation of gp120 was altered to dissociate
it from the virion.
[0031] To further investigate how sCD4 alters the conformation of
gp120, we conducted studies on thrombin-mediated cleavage of gp120.
Digestion of gp120 by thrombin generates 70 kD and 50 kD products
(FIG. 2A) This cleavage takes place at the V3 loop. A monoclonal
antibody directed against an epitope within the loop blocks the
cleavage completely. The thrombin-mediated cleavage at the V3 loop
of gp120 is enhanced after binding with sCD4. This indicates an
increased exposure of the V3 loop on the surface of the protein,
which renders it more susceptible to protease cleavage.
[0032] b. Conformational changes in gp120 induced by complex
formation with succinyl concanavalin A:
[0033] It was previously demonstrated that the incubation of HIV
with mannose-specific lectins, such as concanavalin A or succinyl
concanavalin A attenuates viral infectivity (27, 28). Incubation of
35S-methionine-labeled gp120 with SC resulted in the enhanced
susceptibility of the V3 loop to thrombin digestion (FIG. 2A) This
effect was specific, as preincubation of lectin with a-methyl
mannoside blocked the enhanced effect completely (FIG. 2B). In
addition to increasing the exposure of the V3 loop, interaction of
HIV-1 with SC resulted in a dissociation of gp120 from the viral
membrane (FIG. 1B). The degree of such shedding was somewhat less
than that observed with sCD4. Nevertheless, these studies clearly
indicated that sCD4 and SC alter the conformation of gp120, and in
a very similar manner.
[0034] c. Immunological properties of chemically coupled gp120-CD4
complexes:
[0035] We demonstrated that gp120-sCD4 complexes are immunogenic
and capable of eliciting HIV-1-neutralizing antibodies. An
immunoaffinity procedure was used to purify gp120 from
chronically-infected H9/HIV-1.sub.IIIB cells. The purified gp120
was then crosslinked to sCD4 (DuPont) using the noncleavable,
water-soluble crosslinker, bis(sulfosuccinimidyl) suberate (BS).
Mice were inoculated with the complexes and the immune sera
examined for any effect on HIV-induced syncytium formation.
Syncytium formation induced by HIV-1.sub.IIIB and HIV-1.sub.MN
infected cells was markedly inhibited by the immune sera. A
representative inhibition curve of one immune serum is shown in
FIG. 3A. Syncytium formation induced by cells infected with the
highly related HIV-2 was also inhibited in the presence of the
serum. These results demonstrate that gp120-sCD4 complexes are
capable of eliciting broadly neutralizing antisera.
[0036] We also inoculated mice with complexes comprised of
thrombin-digested gp120 and sCD4. In this case, the gp120 V3 loop
was expected to be modified by protease cleavage. Since V3 has been
reported to be the neutralizing epitope on gp120, it has been of
interest to determine how such cleavage would affect the ability of
the complex to elicit neutralizing antibodies. As shown in FIG. 3B,
inoculation of mice with thrombin-digested gp120-CD4 complexes
elicited antibody capable of blocking syncytium formation induced
by the HIV-1.sub.IIIB and HIV-1.sub.MN isolates. However, this
inhibiting effect was not observed with HIV-2 induced syincytium
formation.
[0037] Our preliminary experiments clearly demonstrated that the
covalently coupled gp120-CD4 complexes can elicit a broadly
neutralizing antibody response. We then undertook to determine
whether cryptic epitopes on the complex are recognized by the
neutralizing antibodies and to characterize the epitopes.
EXAMPLE II
[0038] Immunological Properties of gp120-CD4 Complex
[0039] The glycoprotein gp120 used in the preparation of gp120-CD4
complex was purified from H9/HIV-1.sub.IIIB cells by immunoaffinity
chromatography. The cells were lysed in a buffer containing 20 mM
Tris (pH 8.2), 0.15 M NaCl, 1.0% Triton X-100, and 0.1 mM PMSF. The
lysate was centrifuged at 100,000.times.g for 1 hr. The NaCl
concentration in the supernatant was adjusted to 1 M and the lysate
was then reacted with an affinity matrix prepared with human
anti-HIV immunoglobulins purified from serum of an HIV-antibody
positive subject. The bound antigens were eluted with 50 mM
diethylamine, pH 11.5, and the pH of the eluate was immediately
adjusted to 8.0 with Tris HCl. The eluate was extensively dialyzed
against 10 mM phosphate buffer (pH 6.5) containing 0.5 M NaCl, 0.1
mM CaCl.sub.2, 1 mM MgCl.sub.2, and 0.2 mM MnCl.sub.2, followed by
the addition of Triton X-100 to reach 0.2% by weight solution of
the detergent. The dialyzed material was then passed through a
lentil-lectin column. The glycoproteins were isolated from the
lentil-lectin column by elution with 0.4 M .alpha.-methylmannoside
and were then dialyzed against 20 mM Tris HCl (pH 8.2) containing 1
M NaCl and 0.2% Triton X-100. The dialyzed material was then
applied to an affinity matrix prepared with a mouse monoclonal
antibody SVM-25 (U.S. Pat. No. 4,843,011) reactive against gp41 to
absorb gp160 and any gp41 present. The flow-through from the
affinity column was dialyzed extensively against 10 mM BES (pH 6.5)
containing 1 mM EDTA and was loaded on a phosphocellulose column
equilibrated with the same buffer. The column was developed with a
linear gradient of 0-500 mM NaCl and fractions containing gp120
were pooled, concentrated, and dialyzed against PBS.
[0040] The purified glycoprotein was coupled to sCD4 (commercially
obtained from duPont) by using bis (sulfosuccinimidyl) suberate
(ES) (Pierce) as a crosslinker. For this gp120 and sCD4 were mixed
at 1:2 molar ratio in PBS and incubated at 37.degree. C. for 1 hr
followed by treatment with 0.5 mM BS at room temperature for 1 hr.
The complex was further incubated overnight at 4.degree. C. The
excess BS was blocked with 20 mM Tris-HCl (pH 8.0).
[0041] Development of gp120-CD4 Complex-Specific Monoclonal
Antibodies
[0042] Balb/C mice were subjected to six biweekly inoculations of
the gp120-CD4 complex. The initial inoculum (48 .mu.g per mouse)
was emulsified in Complete Freunds Adjuvant and administered by
subcutaneous injection. In subsequent inocula (24 .mu.g/mouse) were
emulsified in Incomplete Freunds Adjuvant and were administered by
intraperitoneal injection. Two weeks after the final inoculation
the animals were bled and the sera examined for HIV-1 neutralizing
antibodies by a syncytium blocking assay. Briefly, CEM cells
(1.times.10.sup.5) were cocultured with HIV-1-infected cells
(1.times.10.sup.4) in the presence of the test serum and the number
of giant cells were counted after 24-40 hr. Syncytium formation
induced by HIV-1.sub.IIIB- and HIV-1.sub.MN-infected cells was
markedly inhibited by the serum of the mice that was immunized with
gp120-CD4 complex. Syncytium formation induced by HIV-2-infected
cells was also inhibited by these sera indicating that gp120-CD4
complexes are capable of eliciting broadly neutralizing antibodies
in mice.
[0043] After detection of neutralizing antibodies in mice, the
animals received a final intraperitoneal form of gp120-CD4 complex
in PBS without adjuvant. On the fourth day, the animals were
sacrificed and the spleen extracted. Splenic lymphocytes were
flushed from the spleen with a syringe. The cells
(7.times.10.sup.7) were fused with 1.times.10.sup.7 NS-1 mouse
myeloma cells (ATCC, Rockville, Md.), overnight in super HT [DMEM
containing 20% fetal calf serum (Hyclone), 0.1 M glutamine, 10%
NCTC-.sup.109 lymphocyte conditioned medium, 0.5 mM Na-pyruvate,
0.2 U/ml insulin, 1 mM oxalacetic acid, and 100 U/ml
penicillin/streptomycin] (GIBCO) containing 40% PEG 1540. The cells
are then suspended in super HT containing 0.4 .mu.M aminopterin and
placed in 96-well plates.
[0044] Initially, hybridomas were selected for the production of
gp120-CD4 complex-specific antibodies. Pooled hybridoma
supernatants were tested in the ELISA using gp120, CD4 and
gp120-CD4 as antigens. Supernatants of pools containing
complex-specific antibodies were tested individually. Hybridomas of
interest were cloned by replating in super HT at a density of 1
cell/well. Supernatants from cloned hybridomas were further tested
by ELISA using gp120-CD4 complexes.
[0045] Four hybridomas were selected which secreted immunoglobulin
demonstrating a high level reactivity against gp120-CD4 complex and
negligible reactivity with either gp120 or sCD4 in ELISA (Table 1).
Notably, one of the monoclonal antibodies, MoAb 7E3, was of the IgA
isotype. Immunoglobulins were subsequently purified from the
ascites fluid of each hybridoma and further analyzed by Western
blot assay with gp120-CD4 complexes, free gp120, or sCD4. While
none of the antibodies reacted with free gp120 or sCD4, antibodies
7E3 and 8F10B displayed high levels of reactivity with the complex
(FIG. 4) and a low molecular weight fragment of complex. Although
antibodies 8F10C and 8F10D reacted strongly with the complex in
ELISA (Table 1) , reactivity with the complex in Western blot was
weak. These results suggest that MoAbs 8F10C and 8F10D are directed
against a set of highly conformation-dependent, complex-specific
epitopes that are distinct from the epitopes recognized by MoAbs
7E3 and 8F10B.
[0046] Purified 7E3, 8F10B, 8F10C, and 8F10D immunoglobulins were
tested in cell-free infection assays using PHA-stimulated
peripheral blood mononuclear cells (PBMCs) and a variety of HIV-1
isolates. As shown in Table 2, none of the antibodies had any
significant effect on the infection of PBMC by the
laboratory-adapted strain, HIV-1IIIB. However, antibodies 7E3,
8F10B, and 8F10C neutralized the infection of PBMC by a primary
isolate of HIV-1MN to a significant extent, whereas antibody 8F10D
had no effect. In contrast to these results, none of the antibodies
blocked syncytium formation induced by H9/HIV-1IIIB or H9/HIV-1MN
on CEM cells. Our preliminary experiments suggest that the extent
of cell-free neutralization by these complex-specific antibodies
may depend on the infection rate of the isolate. In general,
primary HIV-1 strains with lower infection rates tend to be
neutralized more effectively than more virulent lab-adapted strains
of HIV-1.
[0047] To determine whether the complex-specific antibodies bind to
the gp120 or the CD4 moiety of the complex, we took advantage of
our demonstration that the mannose-specific lectin, succinyl conA
(Sc), perturbs the conformation of the glycoprotein in a manner
similar to that induced by sCD4 (33). SC and gp120 were
cross-linked with BS3 and tested in ELISA. MoAbs 7E3 and 8F10B
reacted strongly with the gp120-SC complex (FIG. 5) but did not
react with free gp120 or SC. In contrast, antibodies 8F10C and
8F10D showed only weak binding to the complex. These results
suggest that antibodies 7E3 and 8F10B are directed towards cryptic
epitopes exposed on gp120 in response to sCD4 and SC binding.
[0048] Immunological Response Against gp120-CD4 Complex in
Goats
[0049] We have also analyzed the immunogenic response against
gp120-CD4 complex in a larger species of animals. An animal (goat
69) was repeatedly inoculated with 100 .mu.g gp120-CD4 complex in
Freund's adjuvant and after the fifth inoculation the serum was
examined by ELISA for reactivity with gp120, sCD4 and the complex.
Antibodies reactive against both free gp120 and sCD4 were detected
in the sera. To determine if complex-specific antibodies were also
elicited, the serum was tested in cross-competition assays with
MoAbs 7E3, 8F10B, 8F10C, and 8F10D. Two-fold serial dilutions of
goat 69 serum were incubated with limiting dilutions of each MoAb
and tested in gp120-CD4 complex ELISA. As shown in FIG. 6,
antibodies in the goat serum were able to block the binding of all
four monoclonal antibodies.
[0050] The goat serum was tested for neutralizing antibodies in
syncytium blocking and cell-free infection assays (Table 3). For
comparison, serum from another animal (goat 58) taken after five
inoculations with HIV-1IIIE viral gp120, was also tested. In
syncytium assays, goat 69 serum reduced syncytium formation
.gtoreq.80% at titers of 1:640 and 1:80 against HIV-1IIIB and
HIV-1MN, respectively; goat 58 serum was much less effective. Goat
69 serum neutralized cell-free infection of CEM cells by HIV-1IIIB
with a titer of 1:80. Again, this titer was significantly higher
than the titer (1:20) of goat 58 serum. Goat 69 serum also mediated
group-specific neutralization of cell-free infection by primary
isolates HIV-1MN and HIV-1JRFL (Table 3). The neutralizing titer
(1:80) was comparable to that of a broadly neutralizing human serum
(1:160) tested in parallel; goat 58 serum failed to block HIV-1MN
infection even at <1:20 dilution. Goat 69 serum was retested
after removal of anti-CD4 antibodies by preabsorption with CEM
cells. Removal of such antibodies was verified by flow cytometric
analysis with SupT1 cells which showed nearly 90% reduction in cell
surface binding. Despite this reduction, the neutralization titer
of the absorbed serum was only two-fold less (1:40) than unabsorbed
serum, indicating that neutralization is not entirely due to
anti-CD4 antibodies.
[0051] The results presented in this example indicate that
covalently cross-linked gp120-CD4 complexes possess a number of
immunogenic complex-specific epitopes. At least a portion of these
epitopes reside on the gp120 moiety of the complex. Moreover, some
complex-specific epitopes are targets for broadly neutralizing
antibodies specifically effective against cell-free infection by
diverse HIV-1 strains, including primary field isolates targeted
toward PBMC. Based on these findings, it is possible that the
complexes could serve as a protective vaccine or immunotherapeutic
reagent.
EXAMPLE III
[0052] Preparation of gp120-CD4 Complex (1:1 Molar Ratio) Free from
Any Uncomplexed CD4
[0053] In the immunization protocol described above gp120 and CD4
were complexed at a 1:2 molar ratio. As the immunization with this
material resulted in the isolation of anti-CD4 antibodies, we
wanted to prepare gp120-CD4 complex (1:1 molar ratio) free from any
uncomplexed receptor molecules to optimize the conditions for
eliciting anti-gp120 antibodies. gp120 and CD4 (1:1 molar ratio)
were bound at 37.degree. C. for 1 hr, reacted with BS for 1 hr at
room temperature and then overnight at 4.degree. C. After blocking
the free crosslinker with Tris buffer (pH 8.0), the solution was
treated with Sepharose coupled to anti-CD4 monoclonal antibody E
for 30 min at room temperature. As E binds to an epitope on CD4
involved in the interaction with gp120, this treatment removed any
uncomplexed CD4 present. A gel showing gp120-CD4 complex prepared
in this manner is shown in FIG. 4. It was clear that only the
complex with molecular weight 170 kD and .about.340 kD is evident
in the gel. There was no free gp120 or CD4 present in the
preparation.
EXAMPLE IV
[0054] In order to more accurately determine if the immune response
to gp120-CD4 complexes differs from the responses to the individual
complex components, the following experiment was conducted.
Separate groups of mice were inoculated with equal amounts of CD4,
gp120 or gp120-CD4 complexes. After five inoculations, sera were
taken from the animals and analyzed. As shown in Table 4, all three
of the CD4-immunized animals possessed syncytium blocking
seroantibodies effective against HIV-1.sub.IIIB and HIV-1.sub.MN.
All four sera from the complex-immunized animals blocked
HIV-1.sub.IIIB induced syncytia; two of the four also blocked
syncytia induced by HIV-1.sub.MN. Overall, neutralizing titers in
sera from complex-immunized animals was lower than sera from
CD4-immunized animals. Surprisingly, none of the gp120-immunized
animals displayed syncytium blocking seroantibodies.
[0055] Reactivity with CD4 in ELISA between the CD4-immunized and
complex-immunized groups was similar (Table 4). The one exception
was a complex-immunized animal (mouse 8) which possessed a titer of
anti-CD4 antibodies significantly lower than the other animals.
Among complex-immunized animals, the level of anti-CD4 reactivity
did not correlate with syncytium blocking activity; mouse 10 serum
was more effective in blocking syncytia than mouse 9 serum, even
though mouse 9 serum had a slightly higher level of anti-CD4
reactivity.
[0056] Overall, complex-immunized animals possessed lower titers of
anti-V3 loop antibodies; such antibodies were virtually absent from
mouse 9 serum.
EXAMPLE V
[0057] Sera from CD4-immunized and complex-immunized animals were
also tested for reactivity with a variety of synthetic peptides
derived from the CD4 sequence (Table 5). Although the overall level
of anti-CD4 reactivity between CD4-immunized and complex-immunized
groups was similar (Table 4), the patterns of reactivity with
linear epitopes differed. While sera from CD4-immunized animals
reacted with peptides derived from the N-terminal portion of CD4
(peptides A and B), such reactivity was absent in sera from
complex-immunized animals. This is in accordance with the fact that
the N-terminus of CD4 reacts with gp120. The prevalence of
reactivity with a peptide derived from domain 3 of CD4 (peptide D)
was also reduced among complex-immunized animals relative to
CD4-immunized animals. Notably, reactivity with a peptide derived
from domain 4 of CD4 (peptide F) was unique to complex-immunized
animals 10 and 11.
[0058] The data of Examples IV and V, taken together, indicate that
the immune response against gp120-CD4 complexes is unique and
different from responses to free CD4 and free gp120. Differences in
the anti-complex response are reflected in 1) a reduced response
against the gp120 V3 loop; 2) a reduced response against linear
epitopes in the CD4 N-terminus; 3) an increased response to linear
epitopes in CD4 domain 4. It should be noted that the latter
epitopes may be hidden in the free CD4 molecule.
[0059] According to the present invention, using gp120-sCD4
complexes as immunogens, we have been able to raise HIV-1
neutralizing antibodies that are complex specific. The results we
have obtained with these antibodies show that covalently coupled
gp120-CD4 complexes possess immunogenic epitopes that are not
normally functional in the unbound proteins.
1TABLE 1 Reactivity of Monoclonal Antibodies Raised Against
gp120-CD4 Complexes in ELISA (OD 450 nm) Antibody Isotype CD4 gp120
Complex 7E3 IgA .427 .340 >3.0 8F10B IgG.sub.1 .146 .175 1.5
8F10C IgG.sub.1 .119 .191 >3.0 8F10D IgG.sub.1 .208 .202 >3.0
anti-gp120 IgG.sub.1 .088 >2.0 >2.0 anti-CD4 IgG.sub.1
>3.0 .103 >3.0
[0060] The results shown are with hybridoma supernatants, the same
specificities were evident with purified immunoglobulin.
2TABLE 2 Neutralization of Cell-Free HIV-1IIIB and of HIV-1MN
Primary Isolate by gp120-CD4 Complex-Specific Monoclonal Antibodies
Antibody Concentration % Inhibition.sup.a .mu.g/ml HIV-1.sub.IIIB
HIV-1.sub.MN 7E3 100 37 88.7 50 55 69.8 25 0 29.8 8F10B 100 26.2
67.2 50 6.2 36.3 25 0 28 8F10C 100 0 75.8 50 0 29 25 0 0 8F10D 100
17 0 Anti-CD4 50 100 100 (control)
[0061] aPHA stimulated PBMC (2.times.105 cells) were infected with
either HIV-1IIIB or a primary isolate of HIV-1MN (50 TCID50) for 18
hr in the presence of the indicated amounts of purified antibodies.
Cells were then washed and cultured in fresh medium containing the
same quantities of antibodies. The p24 content of the supernatant
was determined on day 7 and the percent inhibition was calculated
relative to control assays carried out in the absence of the
antibodies.
3TABLE 3 Neutralizing Activity of Sera from Goats Immunized with
Either gp120-CD4 Complex or gp120 Syncytium Blocking.sup.a
Cell-Free Neutralization.sup.b HIV-1 Strain HIV-1 Strain/Target
Cell Serum Immunogen IIIB MN IIIB/CEM MN/PBMC JRFL/PBMC Goat 69
gp120-CD4 1:640 1:80 1:80 1:80 1:80 Complex Goat 58 gp120 1:20
<1:20 1:20 <1:20 Not tested Goat 69 gp120-CD4 <1:25
<1.25 Not tested 1:40 Not tested (Cell Complex Absorbed)
.sup.aHIV-1.sub.IIIB-infected H9 cells were incubated with
uninfected CEM cells in the presence of two-fold serial dilutions
of each serum. The number of syncytia were scored in 3 fields of
each well after 24 hr. .sup.bImmune and preimmune serum from each
goat was diluted 1:10 in culture media. The immune serum was then
diluted serially in preimmune serum, thus maintaining a constant
serum concentration in all assay wells. Preimmune goat sera and
normal human serum did not demonstrate neutralization relative to
control assays in which serum was omitted. The titers shown
produced .gtoreq. 80% reaction in syncytia or neutralization
relative to matched preimmune sera.
[0062]
4TABLE 4 Syncytia Blocking Titer.sup.a HIV- HIV-1.sub.B10.sup.V3
Peptide CD4 ELISA Mouse Immunogen 1.sub.IIIB/HIV-1.sub.MN ELISA
Titer.sup.b Titer.sup.c 1 CD4 1:1600/1:1600 Not Tested
>1:256,000 2 CD4 1:800/1:1600 Not Tested >1:256,000 3 CD4
1:800/1:400 Not Tested >1:256,000 4 gp120 <1:50/<1:50
1:3200 Not Tested 5 gp120 <1:50/<1:50 1:1600 Not Tested 6
gp120 <1:50/<1:50 1:200 Not Tested 7 gp120 <1:50/<1:50
1:3200 Not Tested 8 complex 1:100/<1:50 1:400 1:32,000 9 complex
1:100/<1:50 <1:25 1:256,000 10 complex 1:400/1:200 1:800
1:128,000 11 complex 1:400/1:100 1:800 1:128,000 .sup.aTiters are
given as the highest serum dilution producing 100% blocking of
syncytia formation. Preimmune sera did not reduce syncytia relative
to control experiments in which serum was absent. .sup.bSerial
two-fold dilutions of each serum was tested. ELISA valves
(absorbance at 450 nm) were converted by subtraction of valves
obtained with the same dilutions of preimmune serum. Titers are
given as the highest serum dilution having a corrected ELISA valve
of .ltoreq.0.5. .sup.cTiters are given as the highest serum
dilution having a converted ELISA valve of .ltoreq.0.5.
[0063]
5TABLE 5 CDR4 Peptide ELISA Valves (A.sub.450 nm).sup.a Mouse
Immunogen A B C D E F G H I J K 1 CD4 1.58 1.05 0.16 2.65 0.21 0.18
0.35 0.21 0.13 0.15 0.19 2 CD4 2.37 0.38 0.17 2.6 0.22 0.17 0.14
0.21 0.15 0.18 0.24 3 CD4 0.56 0.29 0.12 2.34 0.18 0.13 0.21 0.19
0.13 0.14 0.20 8 Complex 0.28 0.27 0.17 0.40 0.23 0.16 0.13 0.19
0.15 0.16 0.26 9 Complex 0.23 0.29 0.20 0.23 0.21 0.19 0.15 0.20
0.14 0.24 0.17 10 Complex 0.17 0.33 0.26 0.61 0.3 1.53 0.17 0.36
0.17 0.35 0.13 11 Complex 0.33 0.43 0.3 2.2 0.36 0.56 0.34 0.36
0.28 0.3 0.20 .sup.aSera were tested at a dilution of 1:1000 for
reactivity with peptides derived from the CD4 sequence. Peptide A,
residues 35-58; B, residues 37-53; C, Residues 318-335; D, residues
230-249; E, residues 297-314; F, residues 330-344; G, residues
350-369; H, residues 310-324; I, residues 81-92 (Benzylated); J,
residues 81-92; K, irrelevant peptide. #ELISA valves .gtoreq.
two-fold higher than valves with irrelevant peptide are shown in
Bold type. Reactivity of preimmune serum with the CD4 peptides was
the same as with the irrelevant peptide.
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