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J Virol, January 1998, p. 286-293, Vol. 72, No. 1
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Antibody-Dependent Cellular Cytotoxicity Directed against Cells
Expressing Human Immunodeficiency Virus Type 1 Envelope of Primary
or Laboratory-Adapted Strains by Human and Chimpanzee Monoclonal
Antibodies of Different Epitope Specificities
Osama
Alsmadi1,2 and
Shermaine A.
Tilley1,*
Public Health Research Institute, New York,
New York 10016,1 and
Department of
Biology, New York University, New York, New York
100032
Received 9 June 1997/Accepted 7 October 1997
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ABSTRACT |
The characteristics of antibody-dependent cellular cytotoxicity
(ADCC) directed by a panel of human and chimpanzee antienvelope (anti-Env) monoclonal antibodies (MAbs) of different epitope
specificities were studied; this was accomplished by using target
cells expressing human immunodeficiency virus type 1 (HIV-1) Envs of
either primary or laboratory-adapted strains. Human MAbs of similar
apparent affinities (1 × 109 to 2 × 109 liters/mol) against either a "cluster
II"-overlapping epitope of gp41 or against the CD4 binding site, V3
loop, or C5 domain of gp120 directed substantial and comparable levels
of specific lysis against targets infected with laboratory-adapted
strains of HIV-1. As expected, those MAbs specific for relatively
conserved regions of Env generally exhibited ADCC activity against a
broader range of HIV-1 strains than those directed against
variable epitopes. Significant ADCC activities of selected MAbs against
primary isolate Env-expressing cells were demonstrated. In
addition, a new ADCC epitope in the V2 domain of gp120 was defined.
CD56+ cells were demonstrated to be the
effector cells in these studies by fluorescence-activated cell sorting
followed by ADCC assays. Notably, all anti-Env MAbs tested in this
study, including MAbs directed against each of the known
neutralization epitope clusters in gp120, directed significant
levels of ADCC against targets expressing Env of one or more
HIV-1 strains. These results imply that many, if not most,
HIV-1-neutralizing human Abs of high affinity (
3 × 108 liters/mol in these studies) and of the immunoglobulin
G1 (IgG1) subclass (i.e., the predominate IgG subclass) are capable of
directing ADCC. Since neutralizing Abs have been associated with
long-term survival following HIV-1 infection, this suggests that ADCC
activity may be beneficial in vivo.
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INTRODUCTION |
The in vivo role(s) of antibodies
(Abs) that can direct antibody-dependent cellular cytotoxicity (ADCC)
against human immunodeficiency virus type 1 (HIV-1) Env-expressing
cells in vitro remains unclear. In ADCC, anti-Env Abs direct effector
cells to kill target cells bearing HIV-1 envelope on their surfaces;
this is accomplished via specific binding of the Abs' antigen-binding
sites to Envs and their Fc regions to Fc receptors on the effector
cells. Broadly strain reactive, ADCC-directing Abs arise early in
the immune response to HIV-1 infection in vivo (14) and may
be partially responsible for the initial clearance of viremia.
Earlier in the HIV-1 epidemic, concerns were raised that shed soluble
gp120 in HIV-1-infected individuals might bind to CD4+
cells, including uninfected ones, and could target these cells for
"innocent bystander" killing by ADCC (6). However,
effector cells armed with serum Abs able to direct ADCC in vitro
against either innocent bystanders or HIV-1-infected cells were found at highest frequency in asymptomatic, seropositive individuals; patients with AIDS-related complex and AIDS showed progressively diminished reactivities (20). Furthermore, in a recent study (1), the ability of monoclonal Abs (MAbs) against three
distinct gp120 epitopes to direct ADCC against uninfected
CD4+ cells to which rgp120SF2 had been
adsorbed (i.e., innocent bystanders) was demonstrated to be
less efficient by at least an order of magnitude than their ability to
direct ADCC against HIV-1-infected cells.
The existing data from in vivo studies (reviewed in reference
1) supports the efficacy, rather than the
pathogenicity, of ADCC-directing Abs against HIV-1. Consistent
with this data is our recent characterization of two MAbs, 42F and
43F, isolated from a long-term survivor of HIV-1 infection
(1); these MAbs directed significant levels of ADCC and
defined a new, conserved ADCC epitope in the C5 domain of HIV-1 gp120.
Preliminary evidence indicated that concentrations of 42F- and 43F-like
Abs in the serum of the donor were in the range required to direct high
levels of ADCC, and these MAbs were shown to bind both oligomeric
primary-isolate and laboratory-adapted Env efficiently (1).
Because of the potential importance of ADCC-directing Abs against
HIV-1, in this study we have evaluated ADCC directed against cells
expressing HIV-1 Envs of primary or laboratory-adapted strains by a
panel of human and chimpanzee anti-Env MAbs of different epitope
specificities. Significant ADCC activities of selected MAbs against
primary-isolate Env-expressing cells were demonstrated, and a new ADCC
epitope in the V2 domain of gp120 was defined. Finally, a MAb's
ability to direct ADCC against a specific target cell type was shown to
be dependent on additional factors beyond its ability to efficiently
bind antigen on the target cell and its possession of an Fc region of
the appropriate isotype to engage Fc
R on effector cells.
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MATERIALS AND METHODS |
Human and chimpanzee MAbs and fragments.
Human MAbs utilized
in this study were anti-CD4 binding site (bs) MAbs 1125H (10,
11) and 5145A (7), anti-V3 loop MAbs 4117C
(12) and 41148D (8), anti-C5 MAb 42F
(1), and anti-gp41 MAb 31710B (11). Chimpanzee
MAbs utilized were anti-V2 MAb C108G (17, 19, 21) and
anti-V3 loop MAb C311E (18). An F(ab')2 fragment
of MAb 1125H was produced by pepsin digestion as described previously
(8). All MAbs are of the immunoglobulin G1 (IgG1) isotype.
HIV-1 strains, HIV-1-infected cells, and recombinant HIV-1
envelope-expressing cells.
HIV-1 strains used in these studies and
the derivation of CEM.NKR cells chronically infected with these strains
have been described previously (1). Recombinant
Env-expressing vaccinia viruses and their use in similar studies have
also been described previously (1).
Purification and quantitation of MAbs.
Human and chimpanzee
MAbs were purified, concentrated, and quantitated as described
previously (7, 11).
Flow cytometric analyses.
Unless indicated otherwise (see
Table 1), these analyses were carried out as previously described
(1).
Preparation of specific human effector cell populations.
For
effector cell identification studies, aliquots of approximately
107 peripheral blood mononuclear cells (PBMC) were
incubated at 4°C for 30 min with cell type-specific, fluorescently
labeled MAbs in 1 ml of phosphate-buffered saline (PBS) containing 2%
heat-inactivated fetal calf serum (FCS). The MAbs were obtained from
Coulter Corp. and were used at a concentration of 60 µl of MAb per
ml; this concentration was determined in preliminary experiments to
give optimal staining of the appropriate cell populations. For specific staining of NK cells, B cells, and monocytes, MAbs NKH-1-RD1
(anti-CD56), B4-FITC (anti-CD19), and Mo2-FITC (anti-CD14),
respectively, were used. Following incubation with these MAbs, cells
were washed in PBS containing 2% FCS; then, positively and negatively
staining fractions of cells were sorted in an EPICS Elite cell sorter
(Coulter Corp.). Diagnostic flow cytometry analyses of the sorted cells confirmed that the positively selected cells were 85 to 90% pure and
that the PBMC which were depleted of specific cell types, i.e.,
negatively selected cells, retained 
3% of the positively stained
cells. Control PBMC for these experiments were incubated and washed
under the same conditions as the sorted cells but were not treated with
MAbs or sorted. When sorting was complete, all cells were pelleted and
resuspended in media for use in ADCC assays as described previously
(1).
Anti-HIV-1 ADCC assay.
This assay was carried out
essentially as described previously (1). In some assays,
normal chimpanzee PBMC rather than human PBMC were used as the source
of effector cells. Fresh blood from a young, healthy chimpanzee was
obtained for these experiments, and PBMC were isolated and prepared as
described previously (1).
Epitope mapping.
Synthetic peptides were used to coat
polyvinyl chloride enzyme-linked immunosorbent assay (ELISA) plates at
500 ng per well in Na2CO3-NaHCO3
buffer, pH 9.8, overnight at 4°C. Following a blocking step described
previously (11), cell supernatants containing approximately
1 µg of MAb per ml or purified MAb at 20 µg/ml were incubated with
each peptide. Diluted sera from seropositive individuals were also
separately incubated with the peptides on plates and served as positive
controls to ensure that each peptide had attached to the plate in
reactive form. A standard ELISA protocol (11) was used to
detect bound human and chimpanzee IgG Abs.
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RESULTS |
Binding and ADCC directed by MAbs against targets chronically
infected with various HIV-1 strains.
The abilities of a panel of
six human and two chimpanzee anti-Env MAbs of different epitope
specificities to bind and/or direct ADCC against target cells infected
with various HIV-1 strains were assessed in this study. Figure
1 shows the results of representative flow cytometry analyses of selected mAbs' binding to CEM.NKR cells chronically infected with the IIIB, MN, SF-2, or RF strain. MAbs 1125H
(anti-CD4 bs) and C311E (anti-V3 loop) efficiently stained cells that
were chronically infected with each of the four HIV-1 strains, while
C108G (anti-V2) specifically stained only the cells chronically
infected with the IIIB strain. These results correlated with the strain
specificities of these three MAbs previously observed in neutralization
assays (11, 18, 18a, 19).
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FIG. 1.
Results of flow cytometry analyses of uninfected or
HIV-1-infected CEM.NKR cells. MAbs 1125H, C108G, and C311E were used at
20 µg/ml. In this experiment, gate 2 was set to exclude 99% of the
background fluorescence observed with no first Ab added (only
fluorescein isothiocyanate conjugate was added) for each type of
infected or uninfected cells. The percentage of cells staining within
gate 2 (above background) is shown in each panel.
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While MAbs against the CD4 bs and V3 loop of gp120 and "cluster II"
of gp41 (reviewed in reference
13), as well as those
against the C5 domain of gp120 (
1), were previously observed
to direct ADCC, the relative activities of MAbs specific for these
different epitope clusters and their breadths of strain specificity
in
ADCC have not been compared prior to this study. Figure
2 shows
that the anti-gp41 MAb, 31710B,
and the two anti-CD4 bs MAbs,
5145A and 1125H, directed comparable
levels of specific lysis
against targets infected with each of the four
HIV-1 strains tested.
This is consistent with previous observations
that 31710B and
the anti-CD4 bs MAbs had similar apparent affinities
for monomeric
rgp160
LAI, i.e., 1 × 10
9 to
2 × 10
9 liters/mol, and that 1125H and 5145A had
comparable potencies,
within experimental error, for neutralizing the
strains involved
in this experiment (
7,
11). The anti-V3
MAb, 4117C, directed
specific lysis comparable to that directed by
31710B, 5145A, and
1125H against the MN- and SF2-infected cells, but
not against
the IIIB- or RF-infected cells. This correlates with
findings
that 4117C had a potency comparable to that of 1125H in the
neutralization
of the MN and SF2 strains but did not neutralize the
IIIB or RF
strains at
20 µg of mAb per ml (
12,
13a).
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FIG. 2.
Representative ADCC assay results obtained with a panel
of human anti-Env MAbs. CEM.NKR cells were chronically infected with
the HIV-1 strains indicated. The left side of each panel shows results
with the following controls: pooled seropositive serum at
10 3 dilution, irrelevant human (Hu) IgG1 (myeloma
protein; ICN Immunobiological, Costa Mesa, Calif.) at 20 µg/ml, and
rabbit antiserum against  2 microglobulin (Accurate Chemical,
Westbury, N.Y.), a positive control against both uninfected and
infected cells, at 33 µg/ml. Results with the latter control against
uninfected cells were comparable to those seen against the four
infected cell lines shown, while no specific lysis above background was
seen against uninfected cells for any of the other Abs and MAbs in this
experiment (data not shown). The right side of each panel shows results
with various concentrations of each human MAb. The error bars represent
standard deviations of duplicate points.
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Surprisingly, anti-V3 MAb 41148D directed substantial lysis against the
IIIB-, MN-, and SF2-infected cells (Fig.
2), though
in previous
neutralization assays this MAb was found to be less
potent by at least
an order of magnitude than MAb 4117C against
these HIV-1 strains
(
13a). Thus, viral neutralization potency
and ADCC activity
may often be correlated, as seen when comparing
the activities of
5145A, 1125H, and 4117C, but exceptions clearly
exist, as seen upon
comparing the results for 4117C and 41148D
in Fig.
2B and C to those of
previous neutralization assays.
As previously reported (
1), anti-C5 MAb 42F directed
significant lysis against the IIIB, SF-2, and RF strains, but not
against clone 7 of the MN-infected cells used in this experiment
(Fig.
2). However, in separate experiments, uncloned MN-infected
cells were
moderately lysed by 42F (
1). Since the apparent
affinity of
42F for rgp160
LAI was similar to that of 1125H (
1,
11), the level of specific lysis directed by 42F was slightly
lower than expected against IIIB-infected cells. Nevertheless,
the
level of specific lysis directed by 42F against SF2- and RF-infected
cells, especially at lower MAb concentrations, was notably high
(Fig.
2).
In all cases in Fig.
2 where cell lysis was observed with a given
MAb-target combination, the percent specific lysis was a
function of
the MAb concentration used in the assay. Furthermore,
the lysis was
dependent on intact Fc regions of the Abs and MAbs
used, since the
F(ab')
2 fragment of 1125H directed no specific
lysis
against IIIB-infected targets (Fig.
2A), though this fragment
bound
efficiently to antigen in ELISA assays (data not shown).
These findings
corroborate antibody-dependent cellular cytotoxicity
as the mechanism
responsible for the specific lysis observed.
In general, an excellent correlation was observed between the level of
staining of a given infected target cell type by a
MAb as seen by flow
cytometry and the level of specific lysis
directed against that target
by the MAb (
1) (e.g., results
with 1125H in Fig.
1 and
2).
Notable exceptions to this pattern
were seen, however, in the
interaction of anti-V3 MAbs 4117C and
41148D with RF-infected
cells. That is, these MAbs were observed
to bind the RF-infected
cells under our standard conditions (4°C,
30 min) as well as or
better than they bound SF2-infected cells,
for example (Table
1), but
to direct no significant ADCC against
the RF-infected targets (Fig.
2D).
To address this inconsistency, we varied the primary binding step to
include various periods of incubation at 37°C, reasoning
that this
would more closely mimic the conditions of the ADCC
assay itself. The
results, shown in Table
1, demonstrate
that
the binding of RF-infected cells by both 4117C and 41148D is,
indeed, slightly reduced at 37°C compared to that at 4°C.
Nevertheless,
the binding of RF-infected cells by both MAbs is under
all conditions
greater than or equal to the binding of SF2-infected
cells by
the MAbs. However, the MAbs direct substantial ADCC against
SF2-infected
cells but not against RF-infected cells (Fig.
2C and D).
We interpret
this conundrum to indicate that binding of 4117C or 41148D
to
RF-infected targets results in the MAbs' Fc regions becoming
sterically
or functionally inaccessible to effector cells for the
multivalent
engagement of Fc receptors. This interpretation is
corroborated
by results of the cold-target competition assays shown in
Fig.
3. While SF-2-infected, unlabeled
cells preincubated with 4117C
could effectively inhibit the specific
lysis of labeled, 4117C-coated
targets (SF-2-infected cells) by
effector cells, the RF-infected,
unlabeled cells preincubated with
4117C were not recognized by
the effector cells and did not inhibit the
effectors' lysis of
labeled targets (Fig.
3). In contrast,
RF-infected, unlabeled
cells preincubated with MAb 1125H could
effectively inhibit such
lysis (data not shown).
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TABLE 1.
Percentages of HIV-1-infected cells staining above
background by flow cytometry analysis following incubation with
ant-Env Abs and MAbs under different conditionsa
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FIG. 3.
ADCC assay results obtained by using
Na251CrO4-labeled SF2-infected
targets in the presence of various numbers of cold-target cells.
Various numbers of unlabeled cells were preincubated with 26 µg of
4117C MAb per ml, while the standard number of labeled targets
(104 cells) was separately preincubated with the same
amount of 4117C MAb. Following this preincubation, the labeled
target-MAb mixture was added to the unlabeled target-MAb (cold-target)
mixture, attaining the cold-target cell-per-milliliter concentrations
shown in the figure. The remainder of the ADCC assay was carried out as
previously described (1). The error bars represent standard
deviations of duplicate points.
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Figure
4 shows representative results of
ADCC assays using the two chimpanzee MAbs, C108G and C311E, to direct
lysis against
targets infected with each of the four laboratory-adapted
strains.
As expected from the strain specificities exhibited by these
MAbs
in neutralization assays (
18,
18a,
19) and in flow
cytometry
(Fig.
1), C108G directed ADCC only against the IIIB-infected
targets,
while C311E directed significant lysis against targets
infected
with all four HIV-1 strains. To our knowledge, this is the
first
observation of ADCC directed by a MAb (i.e., C108G) against an
epitope in the V2 domain of gp120.
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FIG. 4.
ADCC assay results obtained by using two chimpanzee
anti-Env MAbs. CEM.NKR cells were chronically infected with the HIV-1
strains indicated. The left side of each panel shows results with the
following controls: pooled seropositive serum at 103
dilution and human MAbs 5145A and 41148D, each at 20 µg/ml. The right
side of each panel shows results with various concentrations of each
chimpanzee MAb. No specific lysis above background was seen against
uninfected cells with any of the Abs or MAbs in this experiment (data
not shown). The error bars represent standard deviations of triplicate
points.
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The level of specific lysis directed by C108G against the IIIB-infected
targets (Fig.
4) was somewhat lower than that expected
based on its
level of staining of these cells (Fig.
1) and its
potency in
neutralization of the IIIB strain (
19). In addition,
the
specific lysis directed by C311E against IIIB-infected targets
and
targets infected by other strains was significantly less than
that
directed by C108G against IIIB-infected cells or by 5145A
against each
of the infected targets (Fig.
4), though the levels
of staining of
these cells by C311E were greater than or equal
to those seen with
1125H (Fig.
1) or 5145A (data not shown) and
the neutralization potency
of C311E against IIIB was similar to
that of C108G (
18,
18a).
Since human PBMC were used as effector cells in these ADCC assays (Fig.
4), we questioned whether the interaction of the chimpanzee
MAb Fc
regions and human Fc
receptors on effector cells was of
significantly lower avidity than the homologous human Fc-human
Fc
R
or chimpanzee Fc-chimpanzee Fc
R interactions; if so, this
might
account for the reduced efficacies of the chimpanzee MAbs
in these
experiments. To address this question, we performed ADCC
assays using
either human or chimpanzee PBMC as effectors in combination
with both
human and chimpanzee anti-Env sera and MAbs. In each
of two experiments
(data not shown), chimpanzee PBMC were significantly
(0.001 <
P < 0.01) less effective than human PBMC in mediating
ADCC; this was the case whether human or chimpanzee Abs or MAbs
were
directing the ADCC observed. Nevertheless, if one hypothesized
that the
chimpanzee Abs and MAbs were more effective than human
Abs and mAbs
when using chimpanzee PBMC as effectors, then the
ratio of specific
lysis directed by chimpanzee effectors to that
directed by human
effectors should be significantly higher for
the chimpanzee Abs and
MAbs than for the human Abs and MAbs. Calculation
of these ratios from
our data (data not shown) and performance
of Student's
t
test indicate that this hypothesis is improbable
(
P = 0.1 to 0.2). Thus, there was no detectable difference in
these
experiments between homologous versus heterologous Fc-Fc
R
interactions in ADCC.
ADCC directed by MAbs against primary-isolate Env-expressing
targets.
Previous studies (1) demonstrated the binding
of anti-C5 MAb 43F and anti-CD4 bs MAb IgG1b12 (2) to
CD4 T cells infected with recombinant vaccinia viruses
expressing laboratory-adapted and primary-isolate Envs from clades B
and E. Further binding studies of this type were done with additional MAbs of various epitope specificities (data not shown), and the results
of these confirmed our earlier impression that, of the three vaccinia
virus recombinant primary-isolate Envs tested, the clade E Env of vCB53
was most efficiently recognized by seropositive serum and a panel of
anti-Env MAbs. This appeared to be due to a higher level of Env
expression from the vCB53 recombinant virus (1). Thus, we
utilized cells infected with vCB53 as the prototypic targets for
primary-isolate Env-specific ADCC in these studies.
Figure
5D shows that three MAbs of
different epitope specificities, 5145A (anti-CD4 bs), 31710B
(anti-gp41), and 42F (anti-CD5),
directed substantial ADCC against the
vCB53-infected cells, while
two MAbs specific for variable domains in
gp120, 41148D (anti-V3)
and C108G (anti-V2), did not direct ADCC
against these targets.
The latter was expected, since these two MAbs
were raised against
a clade B isolate(s) and
variable-domain-specific MAbs are usually
clade specific.
MAbs 41148D and C108G do bind to cells infected
with certain
primary isolates of clade B (data not shown), and
C108G neutralizes
such isolates (
18). Figure
5C shows that each
of the MAbs
tested is capable of directing substantial ADCC against
vPE16-infected
cells; this recombinant virus encodes Env of the
BH8 clone of IIIB, a
clade B laboratory-adapted virus. The positive
controls, anti-vaccinia
virus serum and HIV immuneglobulin (HIVIG),
directed ADCC against cells
infected with wild-type vaccinia virus
(Fig.
5B) or recombinant
vaccinia virus (Fig.
5C and D), while
anti-
2 microglobulin directed
ADCC against both uninfected (Fig.
5A) and vaccinia virus-infected
(Fig.
5B to D) cells. Considering
the apparently lower level of
vaccinia virus protein (and, presumably,
recombinant Env) expression by
vCB53 versus that by vPE16, as
evidenced by lower levels of ADCC
directed by anti-vaccinia virus
serum and HIVIG in Fig.
5D compared to
those in Fig.
5C, the levels
of ADCC directed by MAbs 5145A, 31710B,
and 42F against primary-isolate
versus laboratory-adapted
Env-expressing cells are comparable
in these experiments. The insets in
Fig.
5C and D show that anti-C5
MAb 42F retains significant
ADCC-directing activity against either
the primary isolate or
laboratory-adapted Env-expressing targets
down to MAb concentrations as
low as 2.5 µg/ml.
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FIG. 5.
Results of ADCC assays using as targets A2.01 cells that
were uninfected (A), infected with wild-type vaccinia virus (WR) (B),
infected with vPE16, a vaccinia virus recombinant expressing Env of
laboratory-adapted strain BH8 (C), or infected with vCB53, a vaccinia
virus recombinant expressing a clade E primary isolate Env (D). The
left side of each panel shows results with the following controls:
irrelevant human (Hu) IgG1 (myeloma protein; ICN Immunobiological) at
40 µg/ml; rabbit antiserum against 2 microglobulin (Accurate
Chemical), a positive control against both uninfected and infected
cells, at 30 µg/ml; serum from a healthy individual recently
immunized against vaccinia virus (anti-vaccinia serum) at 1/200; and
HIVIG at 40 µg/ml. The right side of each panel shows results for two
or more concentrations of each human or chimpanzee MAb tested. The
error bars represent standard deviations of triplicate points.
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CD56+ cells are the effector cells in these ADCC
studies.
Previous studies (9, 15) addressing the
effector cell type in PBMC responsible for mediating ADCC against HIV-1
Env-expressing or -bearing cells indicated that primarily NK cells are
involved. However, the results of Posner et al. (9)
suggested that additional populations of effector cells functioned in
ADCC mediated by polyclonal sera as opposed to that mediated by
anti-Env MAb. These earlier studies involved the blocking of FcR
(CD16) on NK cells with Ab (9, 15) and/or the elimination of
NK cells by Ab and complement (15).
We revisited this issue via fluorescence-activated cell sorting of
specific cell populations from whole PBMC, followed by
ADCC assays
utilizing the positively selected or specifically
depleted populations
as effector cells. Table
2 shows that the
positively selected CD56
+ cells, but not the
CD14
+ or CD19
+ cells, were capable of mediating
ADCC against infected cells
in the presence of seropositive serum. The
CD56
+ cells were capable of mediating levels of specific
lysis comparable
to that mediated by total PBMC, but at lower
effector-to-target
(E/T) ratios than those for unseparated PBMC, as
expected if one
has enriched for the appropriate effector population.
The depletion
experiments (Table
3)
indicate that the CD56
+ cells are entirely responsible for
the ADCC mediated by total
PBMC, since PBMC depleted of these cells
mediate no significant
lysis, while those depleted of CD14
+
or CD19
+ cells show no significant reduction in specific
lysis. Similar
results were obtained when MAbs against HIV-1 Env were
utilized
to sensitize target cells (data not shown).
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TABLE 2.
Percent specific lysis of IIIB-infected CEM.NKR cells by
different positively selected human effector cell populations in
the presence of pooled, human seropositive sera (103
dilution)
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TABLE 3.
Percent specific lysis of IIIB-infected CEM.NKR cells
by total PBMC and PBMC depleted of different cell types in the presence
of pooled, human seropositive sera (103 dilution)
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Epitope of anti-gp41 MAb 31710B.
Having observed significant
and broad ADCC directed by the anti-gp41 MAb, 31710B (Fig. 2 and 5), we
sought to map its epitope as part of this study. Initially, this was
attempted using an epitope scanning kit (Cambridge Research
Biochemicals, Valley Stream, N.Y.) as previously described
(12). The kit contained 12-mer peptides with 10-amino-acid
overlaps that spanned the entire extracellular domain of
gp41LAI. Despite the reactivities of control antibodies and
peptides in these experiments, 31710B, at concentrations as high as 10 µg/ml, did not react with any of these gp41-derived peptides, though
it reacted with rgp160LAI (data not shown). These results
suggested that the 31710B epitope was conformational and/or required a
peptide greater than 12 residues in length for its formation. Thus,
truncation mutants of gp160 were utilized to localize the 31710B
epitope by radioimmunoprecipitation and sodium dodecyl sulfate gel
analysis essentially as described previously (5). Results of
these experiments (data not shown) demonstrated that 31710B could
precipitate the vPE17 (5) and vPE12B (4) truncation mutants but not the shorter vPE18 (5) truncation mutant, while the control seropositive serum could precipitate each of
these mutants. Since vPE12B contains 43 amino acids just N-terminal to
the transmembrane region of gp41 which are not contained in vPE18
(5), the 31710B epitope was localized to this region.
To map the 31710B epitope more precisely, we utilized an ELISA approach
involving a series of 20-mer peptides with 10-amino-acid
overlaps from
the 43-amino-acid region of gp41 described above.
Representative
results from these assays are shown in Table
4.
The 31710B MAb reacted with a single
peptide (2030) from this
region that contained amino acids 651 to 670 (Los Alamos numbering
for the MN strain). The fact that the MAb did not
react with peptides
2029 and 2031, which share 10 amino acids at the N
terminus and
C terminus, respectively, of peptide 2030 indicates that
the epitope
is centered near the middle of peptide 2030 and/or requires
additional
amino acids on either side of its core sequence for
recognition
by 31710B, as suggested by earlier experiments discussed
above.
The 31710B epitope overlaps, but does not coincide precisely
with,
the cluster II epitope of previously described ADCC-directing
human MAb 120-16 (
16).
| |
DISCUSSION |
The specific activities and breadths of HIV-1 strain recognition
in ADCC of MAbs against five different HIV-1 Env epitope clusters were
compared in this study. The results demonstrated that human MAbs of
similar apparent affinities (1 × 109 to 2 × 109 L/mol) against either a cluster II-overlapping epitope
of gp41 or against the CD4 bs, V3 loop, or C5 domain of gp120 directed substantial and comparable levels of specific lysis against targets infected with laboratory-adapted strains of HIV-1. Thus, these four
epitope clusters can elicit comparably potent, ADCC-directing Abs. A
new ADCC epitope in the V2 domain of HIV-1 gp120 was identified via the
observed specific lysis of IIIB-infected targets directed by anti-V2
chimpanzee MAb C108G (19).
As expected, the MAbs directed against relatively conserved regions of
Env, i.e., the CD4 bs and C5 domain of gp120 and the cluster
II-overlapping region of gp41, generally exhibited ADCC activity
against a broader range of HIV-1 strains than those directed against
variable epitopes, i.e., V3 loop and V2 domain. This extended to ADCC
studies employing cells expressing Env of a primary, clade E isolate;
these cells could be lysed only in the presence of the broadly
clade-reactive MAbs. Nevertheless, the MAbs against variable epitopes
were capable of binding to Envs of clade B primary isolates (1a,
18) and, as ADCC-directing MAbs, are likely to be able to direct
ADCC against cells expressing Env of such strains. To our knowledge,
these are the first observations of ADCC directed against
primary-isolate Env-expressing targets by anti-Env MAbs.
There was a precise correlation between the strain specificities of
neutralizing MAbs previously observed in neutralization assays and
those seen here in ADCC assays. However, attempts to correlate MAb
potencies seen in previous neutralization experiments with those
observed for the direction of ADCC in this study yielded disparate
results. Anti-V3 MAb 4117C and anti-CD4 bs MAbs 5145A and 1125H had
comparable potencies in neutralization (7, 11, 12), and
their specific ADCC activities were also similar. However, human
anti-V3 MAb 41148D was surprisingly efficient at directing ADCC against
IIIB-, MN-, and SF2-infected targets, considering its poor neutralizing
activity against the same strains (13a). Conversely,
chimpanzee anti-V3 MAb C311E was significantly less efficient at
directing ADCC than it was at neutralizing the same strains of virus
(18, 18a). These disparities between neutralizing and
ADCC-directing activities may reflect differences in oligomeric Env
structure as expressed on the surfaces of virions versus infected cells
and/or the fact that ADCC activity, unlike neutralizing activity,
depends on multivalent presentation of MAb Fc regions to FcR on
effector cells, in addition to the binding of MAb to viral antigens.
The lack of high-avidity, multivalent presentation of MAb Fc regions to
FcR on effector cells appears to be the explanation for our unusual
observations that anti-V3 MAbs 4117C and 41148D were not able to direct
ADCC against RF-infected targets, though they could bind to such cells
efficiently. These findings cannot be explained by a lack of
appropriate Fc regions in these MAbs, since the MAbs directed ADCC
efficiently against targets infected with other HIV-1 strains, e.g.,
the MN strain. Furthermore, MAbs against other epitopes, including a
MAb (C311E) against a distinct V3 epitope, could direct ADCC against
the RF-infected cells, so these targets were not generally resistant to
ADCC. Perhaps when the 4117C and 41148D MAbs engaged their epitopes on
RF Env, but not on the Env of other strains, their Fc regions became
sterically inaccessible to FcR on effector cells. Another
possibility is that, during the ADCC assay, the 4117C and 41148D MAbs
were rapidly internalized upon binding to the RF Env (but not upon
binding to Env of other strains) on target cells and, in this way,
their Fc regions became inaccessible to FcR. Our binding assays,
which were done in the presence of azide, would not have detected such an internalization if it were energy dependent. Whatever the
explanation, it is clear that, in this case, the binding of MAbs to
target cells and the MAbs' possession of Fc regions of the appropriate isotype did not result in their ability to direct ADCC against these
targets. Further research will be required in order to understand the
frequency with which such cases may occur and their potential biological significance.
Not all anti-Env human MAbs of the appropriate isotype mediated
significant ADCC in previous studies (16), indicating that some of these MAbs had insufficient affinities and/or were not directed
against epitopes that are accessible for ADCC. Given these
observations, it is noteworthy that all anti-Env MAbs tested here,
including MAbs directed against each of the known neutralization epitope clusters in gp120, directed significant levels of ADCC against
targets infected separately with one or more HIV-1 strains. These
results suggest that many, if not most, HIV-1-neutralizing Abs with
high levels of affinity (3 × 108 liters/mol in
these studies) and of the IgG1 subclass (i.e., the predominate
subclass) in humans are capable of directing ADCC. This finding
suggests that ADCC-directing Abs may be beneficial in vivo, given that
neutralizing Abs (now seen to be ADCC-directing Abs as well) have been
associated with long-term survival following HIV-1 infection
(3).
| |
ACKNOWLEDGMENTS |
We thank Douglas Cohn, Laboratory for Experimental Medicine and
Surgery in Primates, Tuxedo, N.Y., for normal chimpanzee blood samples;
P. Earl, C. Broder, and B. Moss, NIH, for recombinant vaccinia viruses
expressing truncation mutants of gp160 and complete primary-isolate
Envs; Ruth Herz, BMCC/CUNY, New York, N.Y., for sharing preliminary
results and technical expertise in ADCC assays; Carol Reiss, New York
University, and Abraham Pinter, Public Health Research Institute, New
York, N.Y., for critical reading of the manuscript; and M. Racho and R. Georgescu for valuable technical assistance. The A2.01 cells
(contributed by T. Folks), HIVIG (contributed by A. Prince), WR
vaccinia virus (contributed by B. Moss), vPE16 (contributed by P. Earl
and B. Moss), and 20-mer gp41 peptides were obtained from the NIH AIDS
Research and Reference Reagent Program.
This work was supported by Pediatric AIDS Foundation Summer Student
Research Awards to O.A., by NIH grant AI26081 and a Lucille P. Markey
Charitable Trust Award to S.A.T., and by NIH CFAR grant AI-72659.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Public Health
Research Institute, 455 First Ave., Room 1133, New York, NY 10016. Phone: (212) 578-0876. Fax: (212) 578-0804. E-mail:
tilley{at}phri.nyu.edu.
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J Virol, January 1998, p. 286-293, Vol. 72, No. 1
0022-538X/98/$04.00+0
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Top
Abstract
Introduction
