Note: Descriptions are shown in the official language in which they were submitted.
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2pg8029
RETROVIRUS-LIKE PARTICLES CONTAINING
MODIFIED ENVELOPE GLYCOPROTEINS
The present invention relates to the field of
immunology and specifically to the preparation of
retrovirus-like particles, specifically HIV-like
particles, which are immunogenic and non-infectious.
The etiologic agent of acquired immune deficiency
syndrome (AIDS) is a human retrovirus termed human
immunodeficiency virus (HIV) of which there are
presently two major subgroups, HIV-1 and HIV-2. These
viruses are responsible for an ever widening world-wide
epidemic of immune deficiency and central nervous system
(CNS) disorders characterized by a slow, yet
progressive, degeneration of immune and CNS functions.
The earliest symptoms of HIV infection include an
acute influenza-like syndrome which persists for 2 to 3
weeks. Several weeks to many months or years following
infection, lymphadenopathy and/or progressive depletion
in CD4+ T-helper lymphocytes becomes apparent and
disease evolves to the point where immune deficiency
becomes manifest. The diagnosis of HIV infection is
confirmed by laboratory tests which include the
detection of HIV-specific antibodies and/or HIV antigens
in patient sera, and the isolation of infectious virus
from patient body fluids or cells. A similar disease is
observed in rhesus macaques infected with the simian
immunodeficiency virus (SIV).
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Immune deficiency in HIV infection is characterized
by opportunistic infections with microbial agents which
are not normally associated with disease in otherwise
healthy individuals. The severity of these infections
is exacerbated by the loss of helper T-cell function,
which, when combined with other symptoms, such as
diarrhoea and weight loss, leads to a general wasting
syndrome. Death usually results from one or more
opportunistic infections. As mentioned above, CNS
involvement is another manifestation of AIDS and can be
the result of direct HIV-induced neurological disease as
well as that of opportunistic infections.
The predominant host cells for HIV in infected
individuals are the CD4+ T-helper cell and the cells of
the monocyte/macrophage lineage. However, increasing
evidence points to the fact that HIV can infect a wide
variety of cell types, CD4' and CD4-, both in vivo and
in vitro. These cell types include those of the
haematopoietic system, the central nervous system, the
gastrointestinal tract, and skin. This wide host cell
tropism most likely accounts for the plethora of
symptoms and the severity of disease associated with HIV
infection.
HIV-1 and 2 have been the subject of massive and
unprecedented research efforts in recent years in a
number of areas including vaccine strategies. The
development of an efficacious vaccine for prevention of
HIV infection, is of considerable importance as it can
be easily recognized that prevention of infection is the
best way to combat any infectious disease.
Various strategies are currently being used in
attempts to develop an effective vaccine against AIDS.
Current strategies to develop a safe and efficacious
AIDS vaccine include whole inactivated viruses, subunit
vaccines, recombinant viruses, genetically engineered
virus-like particles, synthetic peptides, and anti-
idiotypic antibodies.
Inactivated, whole-virus vaccines consist of a
purified preparation of intact particles from a given
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3
viral pathogen which has been rendered non-infectious by
chemical or physical means. The inherent advantages of
these vaccines are their relative ease of production and
the fact that all or most of the important immunological
epitopes of the virus are present. However, a major
disadvantage of these vaccines is that infectious virus
must be propagated on a large scale, thereby exposing
production workers to significant risks, depending on
the nature of the pathogen. Equally important is the
fact that the virus must be rendered completely
non-infectious. This poses ethical problems since it is
extremely difficult to demonstrate that all infectious
genetic material has been removed. Moreover, extensive
inactivation regimes to kill all infectious viruses are
likely to destroy or alter various immunological
epitopes, thereby compromising the immunogenicity of the
vaccine.
A subunit HIV vaccine consists of one or more
purified HIV immunogens, either obtained from disrupted
whole virus or produced in genetically engineered
eukaryotic or bacterial expression systems. An
important advantage of this type of vaccine is the
relative ease with which these products can be produced.
However, this advantage can be countered by the fact
that subunit vaccines only contain a subset of HIV
antigenic determinants, which in some cases can lead to
a less than optimal immune response. Moreover, viral
protein subunits may adopt different spatial
conformations when extracted from the context of the
whole-virus particle. This may affect the structure of
important conformational epitopes and result in
inefficient immune responses.
Live recombinant virus vaccines consist of a non
pathogenic virus, such as vaccinia or adenovirus, which
has one or more non-essential genes replaced by a
nucleotide sequence encoding one or more HIV antigens .
Live recombinant viruses can often induce efficient
immune responses to single subunits of a particular
pathogenic virus. However, as with subunit vaccines,
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recombinant virus vaccines express only a fraction of
the total antigens of a given virus which can be
disadvantageous when highly efficient immune responses
are required.
Future vaccines may consist of synthetic peptides
containing multiple epitopes of a given pathogen. These
peptides, coupled to a carrier protein and combined with
an appropriate adjuvant, are potentially capable of
eliciting good and lasting humoral and cellular immune
responses against multiple components of a pathogen.
The development of an efficacious synthetic peptide
vaccine for AIDS is likely to require the full
identification of all the functionally important
immunological determinants of HIV-1 and HIV-2, a task
which may not be completed in the very near future. An
important disadvantage of peptide vaccines is the
difficulty to produce synthetic molecules mimicking
conformational epitopes (immunological determinants
which are formed by distant amino acid residues brought
together in space by protein folding). If
conformational epitopes are important for protection
against a particular infectious agent, it is unlikely
that traditional peptide vaccine designs will prove
successful.
Vaccines composed of whole, inactivated simian
immunodeficiency virus (SIV) were shown either to
prevent the establishment of virus infection or to delay
the appearance of disease in macaques challenged with
infectious virus. These encouraging results suggest
that perhaps a protective immune response against HIV-1
can
CA 02098029 2000-OS-02
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can effectively be obtained by incorporating most of the
viral antigens into a candidate vaccine.
Genetically engineered non-infectious HIV virus
like particles have been expressed from mammalian and
5 insect cells. Since such particles contain either most
or all of the HIV structural antigens, they are
potential candidate immunogens for the development of
improved cross-protective AIDS vaccines.
Several studies have shown that the principal
neutralizing determinant of HIV-1 lies within the tip of
the loop forming the third variable region (V3) of
gp120. Since neutralizing antibodies essentially
recognize the hypervariable epitope(s) of the loop, it
is conceivable to design cross-protective chimeric
vaccines by inserting the V3 loop epitopes of the most
predominant and divergent viral isolates into a single
envelope.
Several HIV isolates have been identified and
neutralizing antibodies as raised against one isolate
may not neutralize the other isolates. An HIV virus
like particle that expresses on its surface the V3 loop
epitopes of more than one HIV isolate is desirable in an
immunogen to provide an immune response against
immunologically distinct HIV isolates. Additionally it
may be desirable to introduce into the surface protein
of the HIV virus like particle epitopes from other human
retrovirus, such as HTLV-1 and HTLV-2.
In accordance with one aspect of the present
invention, there is provided a self-assembled, non
replicating, non-infectious, retrovirus-like particle
encoded by a modified HIV genome devoid of long terminal
repeats containing a nucleotide sequence coding for a
ch.imeric envelope glycoprotein, the chimeric envelope
glycoprotein having a first retroviral amino acid
sequence and a second retroviral amino acid sequence,
wherein the first amino acid sequence contains the
HIV-1 gp120 conserved region 2 and the second amino acid
sequence contains a retroviral amino acid sequence of a
heterologous strain of HIV-1, HIV-2, HTLV-1 or HTLV-II
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inserted into the first amino acid sequence by inserting
a nucleotide sequence encoding the second retroviral
envelope amino acid sequence into a nucleotide sequence
encoding the first retroviral envelope amino acid
sequence at an endogenous conserved region 2 restriction
site selected from the group consisting of BglII and
StuI.
Preferably, the retrovirus-like particle elicits an
immune response to both the first and second amino acid
sequence. The first amino acid sequence may elicit
neutralizing antibodies to the native retrovirus while
the second amino acid sequence elicits neutralizing
antibodies to a second retrovirus containing the
heterologous retroviral amino acid sequence.
The retrovirus may be selected from HIV-1, HIV-2,
HTLV-1, HTLV-2 and SIV, particularly HIV-1 or HLTV-1.
The first and second amino acid sequences preferably
correspond to sequences of at least one portion of
retroviral surface glycoproteins.
The invention further comprises the nucleotide
sequence coding for the chimeric envelope glycoprotein
of the retrovirus-like particles of the invention, an
expression vector capable of expressing the retrovirus-
like particle in mammalian cells comprising the
nucleotides sequence, immunogenic compositions capable
of eliciting an immune response comprising the
retrovirus-like particle of the invention or an antibody
recognized thereby, and a diagnostic kit for an
immunoassay comprising the retrovirus-like particle
reactive to antibodies in a test sample.
The nucleotide sequence contained in the expression
vector preferably is deficient in at least one sequence
selected from those functionally defining long terminal
repeats (LTR's), primer binding site (PBS) and a viral
RNA packaging sequence, preferably all such sequences.
The invention is described further below with
reference to the accompanying drawings, wherein:
Figure 1 shows, in an embodiment of the present
invention, constructions and vectors to express HIV
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virus-like particles with modified envelope proteins. A
25-by DNA fragment (nucleotides 753 to 777 from HIV-li,p,i)
which contains viral RNA packaging sequences was deleted
from plasmid pMTHIV to generate the expression vector
pMTHIVd25. In this vector, transcription is driven by
the human metallothienen (MT)IIa promoter. Vectors
pMTHIVST and pMTHIVBG were constructed by inserting
synthetic oligonucleotide cassettes encoding the 23
gp120 amino acids YNKRKRIHIGPGRAFYTTKNIIG (residues 306
to 328) from the V3 loop of the MN isolate into the
depicted StuI and BG1II restriction sites, respectively.
The predicted amino acid sequence of inserted epitopes
is indicated for each individual construct. SD, splice
donor; pA, simian virus 40 polyadenylation site; MT, MT
promoter.
Figure 2 shows an immunoblot analysis of material
immunoprecipitated from supernatants of cells
transfected with control (pMTHIVd25) and mutated
(pMTHIVST and pMTHIVBG) expression vectors. Culture
supernatants of cells transfected with the various
recombinant plasmid constructs were first
immunoprecipitated in the absence of any detergent or
denaturing agents with the human monoclonal antibody
268-11D which specifically recognizes a neutralization
epitope within the V3 loop of an HIV-1~,,~, envelope.
Immunoprecipitates were then resolved by SDS-PAGE and
analyzed by immunoblotting using the mouse anti-gp120
(IIIH) monoclonal antibody 5023 (Dupont). Mock,
immunoprecipitate of mock-transfected cell supernatant.
Particular retrovirus-like particles which may be
provided herein comprise an HIV-1 or HTLV-1 insertion
within the conserved region (C2) of HIV-1 gp120. The
amino acid sequence of the insert preferably comprises
an epitopic sequence of a V3 loop of an HIV-1 isolate or
an epitopic sequence of an HTLV-1 isolate.
Such insertion conveniently may be effected by
inserting a nucleotide sequence including a sequence
coding from the second amino acid sequence into a
nucleotide sequence coding for the first amino acid
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sequence at an endogenous restriction site selected from
BglII and StuI site within the nucleotide sequence
coding for the C2 conserved region.
Such first amino acid sequence preferably comprises
the gp120 of HIV-1 LAI isolate and the second amino acid
sequence comprises an epitopic sequence of the V3 loop
of HIV-1 MN isolate or an epitopic sequence of an HTLV-1
isolate.
Referring to Figure 1, there is depicted a vector
for the expression of a human immunodefficiency virus
like particle containing modified envelope glycoproteins
in mammalian cells, in accordance with an embodiment of
the invention. The vectors include the inducible human
metallothionein IIA (~T) promoter and the simian virus
to polyadenylation site. An 8.3 kb SacI to XhoI DNA
fragment encoding the GAG, POL and ENV proteins of HIV-
LAI is under the transcriptional regulation of the Hu-
MTIIA promoter.
The modifications may include deletion of
nucleotides 753 to 777 to delete an RNA packaging_
sequence and insertion of nucleotides encoding epitopes
from heterologous retroviruses, such as a neutralizing
epitope from the V3 loop of the HIV-Ice,, isolate at the
StuI and BglII sites of the HIV-~,I ENV gene, which
insertion might include amino acid sequence
YNKRKRIHIGPGRAFYTTKNIIG. In addition, the insertion
might include amino acids, corresponding to the epitopic
resin of HTLV-1 or HTLV-2. These sequences might
include LLPHSNLDHILEPSIPWKSK or PHWTKKPNRNGGGYYSASYSDP.
The plasmid, containing the modified HIV genome can
be introduced into mammalian cells such as HeLa COS-7 or
Vero cell by transfection and transient or permanent
expression of the HIV-VLPs obtained. The HIV-VLPs can
be isolated from culture superactant by for example by
pelleting and sucrose gradient purification. The VLPs
obtained can be analyzed by immunoblotting and
measurement at TR activity.
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The VLPs having chimeric envelope glycoproteins can
be used to elicit an immune response against at least
the heterologous amino acid sequence, and preferably to
both the native and heterologous sequences. Preferably
antibodies are generated that neutralize the native and
hetrologous retroviruses.
These retrovirus-like particles are useful in
immunogenic compositions for eliciting an immune
response against multiple retroviruses, the generation
of immune siolate in passive immunization and as a
component at diagnostic kits.
These retrovirus-like particles are useful in
immunogenic compositions for eliciting an immune
response against multiple retroviruses, the generation
of immune sera useful in passive immunization and as a
component of diagnostic kits.
Certain biological materials are described and
referred to herein that have been deposited with the
American Type Culture Collection (ATCC) located at
Rockville, Maryland, USA, pursuant to the Budapest
Treaty and prior to the filing of this application. The
invention described and claimed herein is not to be
limited in scope by the plasmids deposited, since the
deposited embodiment is intended only as an illustration
of the invention. Any equivalent plasmids that can be
used to produce equivalent retrovirus-like particles as
described in this application are within the scope of
the invention.
The above disclosure generally describes the
present invention. A more complete understanding can be
obtained by reference to the following specific
Examples. These Examples are described solely for
puxposes of illustration and are not intended to limit
the scope of the invention. Changes in form and
substitution of equivalents are contemplated as
circumstances may suggest or render expedient. Although
specific terms have been employed herein, such terms are
intended in a descriptive sense and not for purposes of
limitations.
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Example 1:
This Example describes the construction of
expression vectors for the production of HIV virus like
particles having natural and modified envelope proteins.
Referring to Figure 1, the expression plasmid vector
pMTHIVd25 was constructed from pMTHIV (ATCC No. 409121)
by deleting a 25-by DNA fragment (nucleotides 753 to
777; LAI sequence) containing viral RNA packaging
sequences. In this vector, the transcription of the HIV-
1 coding sequences is regulated by the inducible human
metallothionein MT IIa promoter and a simian virus 40
polyadenylation sequence. Vectors pMTHIVST and pMTHIVBG
were constructed by inserting synthetic oligonucleotide
cassettes encoding amino acid residues 306 to 328 from
the V3(MN) loop sequence into the indicated StuI and
BglII restriction sites, respectively. For all
constructs, the synthetic DNA cassettes were designed to
encode additional amino acid residues to maintain the
reading frame and create unique restriction sites
flanking the heterologous V3(MN) loop DNA segment. The
nucleotide sequences of all constructs were confirmed by
DNA sequencing.
Example 2:
This Example describes cell culture and
.25 transfections.
Monkey COS-7 and Vero cells were grown and passaged
bi-weekly in Dulbecco's modified Eagle's medium (DMEM;
Flow Laboratories, McLean, VA) supplemented with 10°s
heat-inactivated fetal bovine serum, glutamine (2 mM),
penicillin (50 IU/ml), and streptomycin (50 ug/ml).
COS-7, HeLa and Vero cells were grown to 800
confluence and transfected with 20 ~g of plasmid DNA
either by LipofectinTM (BRL; Bethesda Research
Laboratories, Gaithersburg, MD) or by the TransfinityTM
(BRL) calcium phosphate procedure. Cells transfected
with plasmids containing the human metallothionein
promoter were induced 24-36 h after transfection with 5
~M CdCl2 for 12-16 h. Cells and culture supernatants
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were typically analyzed for protein expression 48 h
post-transfection.
Example 3:
This Example describes the isolation and
characteristization of HIV virus like particles.
Culture media from transfected cells were collected and
clarified by centrifugation at 2,000 x g (Sorvall RT
6000B; Dupont Company, Wilmington, DE) for 15 min at
4°C. Virus-like particles were isolated by
ultracentrifugation as previously described.
To purify HIV-like particles for immunogenicity
studies, pelleted particles obtained by ultra-
centrifugation of cell culture supernatants were
resuspended in 200 ~1 of THE buffer (10 mM Tris-HC1, pH
8.0, 100 mM NaCl, and 1 mM EDTA), overlaid onto a
continuous sucrose gradient (20-60% w/v), and sedimented
at 100,000 x g in a Beckman SW40 rotor for 1.5 h at 4°C.
The gradient fractions were collected from the bottom in
500 ~1 aliquots. Reverse transcriptase activity was
measured in each fraction. The pellet was resuspended
in 30 ~1 of Tritons X-100 lysis buffer (50 mM Tris-HC1,
100 mM NaCl, 1 mm dithiothreitol, 0.1% TritonTM X-100,
pH 7.8) for subsequent reverse transcriptase activity
analysis.
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One third of the resuspended sample was added to a 90 ~l
reaction mixture containing 40 mM Tris-HC1, 4 mM
dithiothreitol, 45 mM KC1, 10 mM MgCl2, 20 uCi 3H-dTTP
(80 Ci/mmol), 50 ~g poly rA, and 1 ~g oligo dT at pH
7.8. This mixture was incubated at 37°C for 30 minutes
Radioactive incorporation into trichloroacetic acid-
precipitable nucleic acids indicated the presence of
reverse transcriptase activity.
To establish that the gp120 subunits produced by
pMTHIVST and pMTHIVBG contained the heterologous V3(MN)
loop epitope(s), the envelope proteins were
immunoprecipitated in the absence of any detergents or
denaturing agents with the human monoclonal antibody
268-11D directed against a neutralization epitope of the
V3(MN) loop. Immunoprecipitates were then resolved by
SDS-PAGE and analyzed by immunoblotting with the mouse
anti-gp120 (IIIB) monoclonal antibody 5023. The
immunoblot analysis (Fig. 2) revealed that antibody 5023
specifically recognized a single band corresponding to
gp120 in immunoprecipitates from the supernatants of
cells transfected with the pMTHIVST and pMTHIVBG
constructs. These results clearly indicate that the
processed env glycoprotein expressed from these vectors
contain the heterologous V3(MN) loop segment.
Example 4:
This Example describes the immunogenicity of HIV
virus like particles.
Fully-assembled, envelope-containing particles were
isolated from the supernatants of stably engineered Vero
cells transfected with plasmids pMTHIVd25, pMTHIVST, and
pMTHIVBG, by ultracentrifugation through a glycerol
cushion and purified by sucrose gradient fractionation.
The p24 content of the various particle species was
determined by a p24-specific enzyme immunoassay (Coulter
Immunology, Hialeah, FL). All stable cell lines secreted
approximately 500 ~g of p24 per liter.
CA 02098029 2000-OS-02
13 208029
Female SJL/J mice (Charles River, Montreal, Quebec)
between 6 and 8 weeks of age were immunized
subcutaneously with doses of purified particles
corresponding to 10 ~g of p24 antigen emulsified in
Freund's complete adjuvant (FCA). A booster injection
equivalent to 5 ~,g of p24 antigen was given 3 weeks
later in Freund's incomplete adjuvant (FIA). Mice were
sacrificed 9 days after the second immunization, and
sera were collected and heat-inactivated at 56°C for 30
minutes. The presence of antibodies to HIV-1 antigens
was determined by antigen-specific enzyme-linked
immunosorbent assay (ELISA). ELISA plates (LKELKAY
plates; Lab Systems, Shrewsbury, MA) were coated at 20°C
for 18 h with 100 ~1 of a solution containing either
gp120 or p24 at 0.1 ~g/ml or peptides at 10 ~g/ml in 50
mM carbonate buffer, pH 9.6. The recombinant gp120 was
obtained from American Bio-Technologies, Inc.
(Cambridge, MA), and p24 from Dupont Canada, Inc.
(Markham, ON). Synthetic peptides corresponding to the
neutralizing determinant found in the V3 loops of gp120
from HIV-1 strains HXB2, MN, and ELI (Table 1) were
purchased from American Bio-Technologies, Inc.
(Cambridge, MA). Plates were blocked at room temperature
for 1 h with 200 ~1 of 2% gelatin in PBS, and washed
three times with PBS containing 0.05% Tween-20. Serum
samples were serially diluted in PBS/Tween-20 and added
to individual wells for 1.5 h at room temperature. The
plates were then washed three times with PBS/Tween-20,
and a goat anti-mouse IgG-horseradish peroxidase enzyme
conjugate (Amersham Canada Ltd, Oakville, ON) diluted
1:5000 in PBS/TweenT"'-20 was added for 30 min at 37°C.
After an additional washing step , the color was
developed using 0.1% tetramethylbenzidine and 0.004%
hydrogen peroxide (ADI Diagnostics, Willowdale, ON).
Optical density was read at 450 nm using a Titertek
Multiskan MCC/340 plate reader (Flow Laboratories,
McLean, VA). Endpoint titers were
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14
defined as the highest serum dilution which resulted in
optical density readings at least two-fold greater than
the baseline absorbance established for normal mouse
serum controls.
The antibody response to envelope and core antigens
to HIV virus-like particles with modified env proteins
was analyzed by antigen-specific ELISA, and the results
obtained are presented in Table 1 below.
The antisera were further tested for their
reactivities with synthetic epitopes from the V3 loops
of three different HIV-1 isolates. These peptides
consist of amino acid residues 302-322, 307-325, and
303-323 of gp120 from the HXB2, MN, and ELI viral
strains, respectively. The three expression constructs
used to produce the retrovirus-like particles contain
the env coding sequences of the HIV-1~,I isolate, and the
reactivity of the antisera generated was tested with a
peptide containing most of the V3(HXB2) loop residues
but differing from the corresponding V3(LAI) sequence by
only one amino acid at position 306 (Table 1). HXB2
peptide-specific titers were similar in all three groups
of mice suggesting that the introduction of the
heterologous V3(MN) loop segment did not affect the
humoral response against the endogenous V3(LAI) loop.
The particles produced by cells transfected with
pMTHIVd25 that lacked the V3(MN) domain also elicited
cross-reacting anti-V3(MN) antibodies (1/2500),
suggesting that these antibodies recognize an epitope
shared by the two V3 loop peptide sequences which are
65°s similar. However, the expression vectors pMTHIVST
and pMTHIVBG which contained the V3(MN) loop coding
sequences produced virus-like particles which induced a
markedly enhanced (1/12500) and specific antibody
response against the V3(MN) loop peptide (Table 1). The
lack of antibody response to the divergent V3(ELI) loop
peptide served as a control for the specificity of the
antibody response.
CA 02098029 2000-OS-02
15
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CA 02098029 2000-OS-02
16
To determine whether immunization with HIV-like
particles containing envelope proteins with the
immunodominant V3 loop domains of HIV-l~"I and HIV-l,,B,,
induce neutralizing antibodies against both viral
strains, guinea pigs were immunized with pMTHIVd25,
pMTHIVST and pMTHIVBG. The immune sera were assayed
for their ability to prevent fusion of uninfected CD4-
expressing cells with cells chronically infected with
either HIV-l~"I or HIV-1,,Q,,. Shown are the results with
serum samples obtained two weeks after the fourth
booster immunization (Table 2). Animals immunized with
virus-like particles containing only the V3(LAI) domain ,
(pMTHIVd25) responded with antibodies that were
effective in blocking syncytia induced by HIV-1~"I.
Immune sera from guinea pigs immunized with particles
containing the V3 loop domains of HIV-l~,i and HIV-1~,,
(pMTHIVST and pMTHIVBG) also blocked fusion of CD4-
expressing cells with cells chronically infected with
HIV-1,,AI. In addition, immunization with HIV virus-like
particles containing chimeric envelopes was very
effective in inducing cross-neutralizing antibody
responses since six of seven samples from immunized
animals were able to block syncytia induced by either
HIV-1~~,"I or HIV-1~,,. Cross-neutralizing activity was also
observed in the serum of 1 of 3 guinea pigs immunized
with virus-like particles containing only the V3(LAI)
loop domain. Some of the sera were also checked for
their ability to blockade HIV-1~,F or HIV-2Z, and none of
the tested samples prevented syncytia induced by these
viral strains.
CA 02098029 2000-OS-02
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TABLE 2. Cell fusion blockade
Fusion inhibition"
Serum Sample Antigen HIV-1~I HIV-1~
11 pMTHIVd25 + -
12 pMTHIVd25 - -
13 pMTHIVd25 + +
pMTHIVST - +
10 16 pMTHIVST + +
17 pMTHIVST + +
19 pMTHIVBG + +
pMTHIVBG - -
21 pMTHIVBG + +
15 22 pMTHIVBG + +
"Adsorbed serum samples were tested at a final
dilution of 1/10 to block syncytium formation induced
by CEM-Bells chronically infected with either HIV-l~I
20 or HIV-l~,,r,. Numbers of syncytia in uninhibited wells
(preimmune or normal sera were greater than 50 for each
virus. A negative (-) score indicates no inhibition
and a positive score (+) indicates fusion inhibition by
the test serum, with five or less syncytia per well.
CA 02098029 2000-OS-02
